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Studies of Induction of Alpha-Synuclein Inclusion Pathology

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Title:
Studies of Induction of Alpha-Synuclein Inclusion Pathology
Creator:
Sacino, Amanda N
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
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Language:
english
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1 online resource (231 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Neuroscience (IDP)
Committee Chair:
GOLDE,TODD ELIOT
Committee Co-Chair:
GIASSON,BENOIT IVAN
Committee Members:
LEWIS,JADA M
BORCHELT,DAVID R
MCFARLAND,NIKOLAUS R
MUZYCZKA,NICHOLAS
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Brain ( jstor )
Cell aggregates ( jstor )
Diseases ( jstor )
Inclusion bodies ( jstor )
Mice ( jstor )
Parkinson disease ( jstor )
Pathology ( jstor )
Prions ( jstor )
Solar fibrils ( jstor )
Neuroscience (IDP) -- Dissertations, Academic -- UF
alpha-synuclein -- neurodegeneration -- prion
Genre:
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Medical Sciences thesis, Ph.D.

Notes

Abstract:
Alpha-Syucleinopathies are neurodegenerative disorders characterized by the accumulation of intracellular amyloidogenic alpha-synuclein (alpha-S) inclusions. Alpha-S is predominantly loosely localized around presynaptic vesicles of CNS neurons, where it is believed to assist in vesicular transport of neurotransmitters. It is not yet certain how alpha-S is triggered to misfold into amyloid and aggregate, how the amyloidogenic aS is transported, and if this contributes to the onset and progression of disease. Identification of modulators of alpha-S pathology may help to elucidate its role in disease, as well as provide molecular targets for therapeutic intervention. The focus of this dissertation is to characterize models of the induction ofalpha-S pathology in order to study contributing factors, which may also lead to elucidating mechanisms of disease progression. One of the leading hypotheses about neurodegenerative-associated pathology is that induction and spread is via a prionoid mechanism, where the misfolded protein is transmitted intercellularly and serves as a template for the conformational conversion of soluble intracellular protein into amyloid. Post-mortem studies completed on PD patients have shown i) that there may be a pattern of spread of alpha-S pathology from the peripheral nervous system to the central nervous system, and ii) that alpha-S pathology may have transmitted to therapeutic naive fetal dopaminergic neuron grafts. In conjunction with recent cell culture and in vivo studies showing that the addition of exogenous alpha-S fibrils can lead to alpha-S pathology induction, a hypothesis was proposed that aS pathology, as seen in Parkinson's disease, is behaving in a prionoid manner. Using a cell culture model and in vivo mouse models ofalpha-S pathology, we have extended these studies demonstrating that i) missense mutations of alpha-S differ in their pathology induction properties, ii) that there are significant barriers to widespread induction of alpha-S pathology in vivo, iii) that additional factors, such as neuroflammation and disruption of proteostasis, may play a role in induction and spread, and iv) that caution should be used when defining alpha-S pathology via its main antibody marker, pSer129. Collectively, these studies serve as a foundation for future mechanistic work on alpha-S pathogenesis. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: GOLDE,TODD ELIOT.
Local:
Co-adviser: GIASSON,BENOIT IVAN.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-05-31
Statement of Responsibility:
by Amanda N Sacino.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright by Amanda N. Sacino. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2016
Classification:
LD1780 2014 ( lcc )

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STUDIES OF INDUCTION OF ALPHASYNUCLEIN INCLUSION PATHOLOGY By AMANDA N. SACINO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2014 Amanda N. Sacino

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3 ACKNOWLEDGMENTS I would like to thank my parents and my brother for all of their support. I also would like to thank my mentors, Dr. Todd Golde and Dr. Benoit Giasson, and my committee (Dr. David Borchelt, Dr. Jada Lewis, Dr. Nikolaus McFarland, and Dr. Nicholas Muzyczka) for their gui dance during the course of my graduate work. Finally, I would like to thank the member s of the Center for Translational Research in Neurodegenerative Diseases the UF Neuroscience Department, and the UF College of Medicine for their kindness and willingness to help me on a daily basis. My graduate work was supported in part by the Wilder Family Fellowship.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 3 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 14 CHAPTER 1 THE ROLE OF S IN NEURODEGENERATIVE DISEASE ................................... 16 Introduction ............................................................................................................. 16 Why Study Parkinsonism? ...................................................................................... 17 S Genetics and Structure ...................................................................................... 18 S, the Conformational Chameleon ..................................................................... 20 Clearance of Misfolded S ..................................................................................... 22 A ........................................ 23 ............................................................................................. 24 Results Presented in this Dissertation .................................................................... 26 2 CONFORMATIONAL TEMPLATING OF SYNUCLEIN AGGREGATES IN NEURONAL GLIAL CULTURES ............................................................................ 33 Introduction ............................................................................................................. 33 Materials and Methods ............................................................................................ 35 Results .................................................................................................................... 40 Discussion .............................................................................................................. 46 3 INDUCTION OF CNS SYNUCLEIN PATHOLOGY BY FIBRILLAR AND NONAMYLOIDOGENIC RECOMBINANT SYNUCLEIN ............................................. 68 Introduction ............................................................................................................. 68 Materials and Methods ............................................................................................ 70 Results .................................................................................................................... 74 Discussion .............................................................................................................. 7 8 4 AMYLOIDOGENIC SYNUCLEIN SEEDS DO NOT INVARIABLY INDUCE RAPID, WIDESPEAD PATHOLOG Y .................................................................... 102 Introduction ........................................................................................................... 102 Materials and Methods .......................................................................................... 104

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5 Results .................................................................................................................. 111 Discussion ............................................................................................................ 119 5 PERIPHERAL INJECTION OF RECOMBINANT SYNUCLEI N INDUCES CNS SYNUCLEIN PATHOLOGY AND A RAPIDONSET, SYNCHRONIZED MOTOR PHENOTYPE ......................................................................................... 158 Introduction ........................................................................................................... 158 Materials and Methods .......................................................................................... 159 Re sults .................................................................................................................. 162 Discussion ............................................................................................................ 166 6 DISCUSSION ....................................................................................................... 182 Summary of Results Presented in this Dissertation .............................................. 182 Introduction ........................................................................................................... 182 Efficient Propagation and Strainlike Properties .................................................... 183 Resistance of Biological Clearance ...................................................................... 187 Bioavailability, Transport, and Spreading .............................................................. 189 Toxicity Induction and Transmission of Phenotypic Changes ............................... 194 ................................... 197 Is There Really a Prionoid Spread of S Pathology? ............................................ 199 LIST OF REFERENCES ............................................................................................. 203 BIOGRAPHICAL SKETCH .......................................................................................... 231

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6 LIST OF TABLES Table page 1 1 Parkinsonian disorders ....................................................................................... 31 1 2 Synucleinopathies ........................................................................................... 32 3 1 Summary of neonatal nonTg mice injected with S proteins .......................... 100 3 2 Summary of neonatal M20 human S Tg mice injected with S proteins ....... 101 5 1 Summary of M83 Tg mice injected with S proteins and LPS control ............. 180 5 2 Summary of M2 ............................................... 181 6 1 Summary of the prionoid characteristics of S ................................................. 202

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7 LIST OF FIGURES Figure page 1 1 Model of S misfolding and the development of Lewy bodies and Lewy neurites.. ............................................................................................................. 30 2 1 aggregate formation in AAV2/1neuronal glial cultures.. ...................................................................................... 53 2 2 Non amyloidogenic synuclein protei ... 55 2 3 1 ............................................................................ 56 2 4 .......................................... 58 2 5 inclusions in the mixed neuronal glial cultures colocalize with ubiquitin. ...... 59 2 6 glial cultures contain Thioflavin S positive structures. .............................................................................................. 61 2 7 Distinct morphological properties of inclusions in M83 (A53T) and M47 ................................................................... 62 2 8 mutant specific and dominant morphological induction of aggregates. ... 63 2 9 Three dimensional differences in morphology of inclusions. ......................... 65 2 10 seeded inclusion formation in astrocyte cultures. ......................................... 66 2 11 from cell lysates.. ................................................................................................ 67 3 1 Detection of injected human S in the needle track 2 days post neonatal injection. ............................................................................................................. 85 3 2 Schematic summary of the predominant cortical distribution of S pathology in nTg mice 8 months after brain neonatal injection of fibrillar 21140 S.. ........ 86 3 3 Increased postnatal expression of S in the brain of nTg and M20 Tg mice. ..... 87 3 4 Schematic representation of the distribution of S pathology at 8 months following brain neonatal injection of 21140 human S fibrils or 7182 human S in M20 S Tg mice. ........................................................................... 88 3 5 Induction of S pathology throughout the neuroaxis 8 months after neonatal brain injection of 25 g fibrillar 21 140 S in M20 human S Tg mice. .............. 89

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8 3 6 The majority of S inclusions in the substantia nigra of M20 Tg S neonatally injected with exogenous S are not in TH pos itive neurons. .............................. 91 3 7 IHC showing similar S pathology induced by neonatal brain injection of fibrillar human 21140 S and 7182 human S in M20 Tg mice compared to symptomatic M83 human S A53T Tg mouse.. .............................................. 92 3 8 Labeling of S inclusions in M20 Tg mice 8 months after PO brain injection of exogenous S with both aminoterminal S antibody SNL4 and pSer129.. ...... 93 3 9 Co localization of p62 with S inclusions in 8 monthold M20 Tg mice following neonatal brain injection of exogenous S.. .......................................... 94 3 10 Induction of S pathology throughout the neuroaxis 8 months after neonatal brain injection of 25 g 7182 S in M20 human S Tg mice. ....................... 95 3 11 Delayed induction of astrogliosis and microgliosis in mice neonatally injected with soluble 7182 S or fibrillar S at 8 months post injection. ....................... 96 3 12 Mass spectrometric analysis of the 71 82 S used for neonatal brain injection. ............................................................................................................. 98 3 13 Lack of induction of S pathology throughout the neuroaxis 8 months after neonatal brain injection of 25 g 7182 S in M20 human S Tg mice. ......... 99 4 1 Induction of S pathology at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice. ............................................................................. 127 4 2 Non specific staining by anti pSer129/81A antibody in the white matter tracts of M83 Tg mice 2 months post intrahippocampal injection of 21140 hfib S. .. 129 4 3 The ~70 kDa protein recognized by pSer129/81A antibody is phosphoserine 473 in NFL.. ...................................................................................................... 131 4 4 Immunocytochemical pSer129/81A staining of the white matter tracts in nTg/native mice is absent in NFL/ mice. ........................................................ 133 4 5 NF inclusions in NFHLacZ Tg mice are stained by pSer129/81A.. .................. 135 4 6 pSer129/81A detects NFL in white matter tracts from human nervous tissue sections. ........................................................................................................... 137 4 7 pSer129/81A detects phosphorylated NFL in mixed primary neuronal cultures.. ........................................................................................................... 138 4 8 Induction of S inclusions in nTg mice after intrahippocampal injection of 21140 hfib S or full length mfib S. .................................................................... 140

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9 4 9 Characterization and distribution of S inclusion pathology at 4 months post intrahippocampal injection of 21140 hfib S in M47 Tg mice. ......................... 142 4 10 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M8 3 Tg mice using antibody pSer129/81A. .................................................................................................... 144 4 11 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice using antibody Syn506. ............................................................................................................ 146 4 1 2 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice using an antibody to p62.. ............................................................................................... 148 4 13 Immunohistochemical analysis of pSer129/81A staining in M83 Tg mice 2 months post intrahippocampal injection with PBS and 71 82 S. ............... 149 4 14 Non S staining of white matter tracts by pSer129/81A antibody in nTg and SNCA / mice.. .................................................................................................. 150 4 15 Western blot analysis demonstrating the detection of a major ~70 kDa major nonS target reactive with pSer129/81A in mouse brain. ............................... 151 4 16 pSer129/81A detects phosphorylated NFL in the white matter tract s and cell bodies of nTg mice. ......................................................................................... 153 4 17 pSer129 antibody can intensely detect white matter tracts that is not due to S in human nervous tissue sections.. ............................................................. 154 4 18 Tau aggregate formation 4 months post intrahippocampal injection of 21140 hfib S in M47 Tg mice. .................................................................................... 155 4 19 Schematic summary of S pathology distribution at 4 months post intrahippocampal injection of hfib 21140, E46K, and A53T S in M47 Tg mice. ................................................................................................................. 156 4 20 Immunohistochemistry showing similar S pathology induced by intrahippocampal injection of 21140 WT, E46K, or A53T hfib S in M47 Tg mice at 4 months post injection.. ...................................................................... 157 5 1 S IM injection reduces survival in M83 Tg mice. ............................................ 169 5 2 S pathology in M83 Tg mice IM injected with S is similar to that seen in untreated, aged M83 Tg mice.. ......................................................................... 171

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10 5 3 Accelerated onset of astrogliosis and induction of microgliosis in M83 Tg mice IM injected with 7182 S and hfib S compared to untreated, aged M83 Tg mice. .................................................................................................. 173 5 4 Induction of S pathology at 7 months post IM injection of 10 g hfib S in M20 Tg mice. .................................................................................................... 175 5 5 Induction of S pathology throughout the CNS 2 months after IM injection of 10 g hfib S in M83 Tg mice. ........................................................................... 176 5 6 S inclusions in the substantia nigra of M83 Tg mice are not found in dopaminergic neurons.. .................................................................................... 177 5 7 S pathology post IM injection of 10 g hfib S or 10 g 7182 S shows similar IHC staining profile compared to mature inclusions seen in untreated, aged M83 Tg mice that developed motor impairments.. ................................... 178 5 8 S inclusions are not present in the sciatic nerve or skeletal muscle post IM injection of 10 g hfib S in M 83 Tg mice. ......................................................... 179

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11 LIST OF ABBREVIATIONS AAV Adeno associated virus A ABC Amyloid beta protein associated with extracellular plaques as seen in Alzheimers Disease Avidin/Biotin complex AMY synuclein protein Amygdala A30P A53T BCA Bicinchoninic acid assay S BSA Beta synuclein protein Bovine serum albumin CAG CC CMV early enhancer chicken beta actin promoter Corpus callosum CNS Central nervous system DAMPS DAPI DG DHSC Danger associated Molecular Pattern molecules 4,6 diamidino 2 phenylindole Dentate gyrus Dorsal hornspinal cord DIV Days in vitro DLB EGFP EC Dementia with Lewy Bodies Green Fluorescent Protein Entorhinal cortex E46K GAPDH Glyceraldehyde3 phosphate dehydrogenase

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12 GFAP Glial fibrillary acidic protein G51D HS HRP HYP Glycine51Aspartic Acid High salt Horseradish peroxidase Hypothalamus Hfib H50Q IHC Iba 1 Human synuclein derived fibrils Immunohistochemistry Ionized calcium binding adaptor molecule 1 LB Lewy Body LN Lewy Neurite LPS Lipopolysaccharide Mfib MC Mouse synuclein derived fibrils Motor cortex M20 Transgenic mouse over expressing human wild type synuclein M47 Transgenic mouse overexpressing human E46K synuclein M83 MSA Transgenic mouse overexpressing human A53T synuclein Multiple system atrophy NAC Non Abeta component of synuclein protein NFs Neurofilaments NFH Neurofilament heavy molecular mass subunit NFM Neurof ilament middle molecular mass subunit NFL Neurofilament light molecular mass subunit nTg OB Non tran s genic mouse Olfactory bulb

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13 PC Piriform cortex PD PNS Parkinsons Disease Peripheral nervous system P0 Neonatal days post birth PrP PBS Prion protein promoter Phosphate buffered saline PRR PMD ST Pattern recognition receptor Protein misfolding disorder Striatum SNpc TBS Substantia nigra pars compacta Tris buffered saline TH TSE VP VH SC Tyrosine hydroxylase thalamus Transmissible spongiform encephalopathy Ventral pons Ventral hornspinal cord WT Wild type

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STUDIES OF INDUCTION OF ALPHA SYNUCLEIN INCLUSION PATHOLOGY By Amanda N. Sacino May 2014 Chair: Todd E. Golde Cochair: Benoit I. Giasson Major: Neuroscience Sy n ucleinopathies are neurodegenerative disorders characterized by the accumulation of synuclein ( S) inclusions. T here are still unanswered questions regarding the role S plays in disease. How is S triggered to undergo a conformational change into amyloid and a ggregate? Does the induction and transmission of amyloidogenic S contribute to the onset and progression of disease? well as provide molecular targets for therapeutic interv ention. The focus of this dissertation is to characterize S proteinopathy models to study contributing factors to pathology induction, which may also lead to elucidating mechanisms of disease progression. It is thought that induction and spread of neurodegenerationassociated pathology is via a prionoid mechanism, where an initial misfolded protein that escapes degradation can serve as a seed to template the conformational conversion of soluble intracellular protein into amyloid. This pathologic S can be transmitted from cell to cell, and spread to different regions of the brain. Post mortem studies complet ed on PD patients have lead to the hypothesis that there

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15 m to the central nervous system. naive fetal dopaminergic neuron grafts in some PD patients suggested an intracellular transmission of pathology This data from human studies i n conjunction with recent cell culture and in vivo pathology induction, collectively lead many in this field to propose exploration of the hypothesis that as seen in PD is behaving in a prionoid manner. My studies s how induction properties, ii) pathology in vivo iii) additional factors, such as neuroflammation and disruption of proteostasis, may pl ay a role in induction and spread, and iv) caution should be used

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16 CHAPTER 1 THE ROLE OF S IN NEURODEGENERATIVE DISEASE Introduction Intracellular synuclein ( S) inclusions are a hallmark pathology of Parkinsons disease (PD) (Shults, 2006) PD, along with Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA), can be collectively grouped with other neurodegenerative diseases as synucleinopathies This term is a pathological descriptor for a set of clinical diseases characterized by the intracellular aggregation of conformationally abnormal system (Uversky, 2007) Although mutations in the gene encoding S have been linked to PD (Cookson, 2005) an etiological role for intracellular S inclusion pathology in disease has yet to be established. Indeed significant unanswered questions include how S pathology is induced and transmitted, and how S pathology contributes to the development and progression of disease. Based on autopsy data reporting peripheral S pathology PD patients [reviewed in (Lema Tome et al. 2012) ], it is thoug ht that S can conformationally convert to a pathogenic form that is transmitted intracellularly. I t is then able to serve as a template to induce the conversion of soluble, endogenous S to a pathogenic form This concept of a misfolded protein serving as an infectious agent of disease underlies the pathogenicity of prion disease (Prusiner, 1998) D ata has shown th at S does exhibit characteristics of a prion; however, there has been no conclusive evidence that pathologic S is communicable from an infectious individual to another (Dunning et al. 2013) Therefore, S is currently referred to as a prionlike or prionoid protein. In Chapters 1 a nd 6 of my dissertation will discuss S, prions, and the properties of S

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17 that identify it as a prionoi d protein. I will also discuss gaps in current studies along with data indicating that there may be additional mechanisms of induction and spread of S pathology. Why Study Parkinsonism? The National Institute of Neurological Disorders and Stroke has indicated that there are currently over 600 neurological disorders in the United States, which affect approximately 50 million Americans. The majority of these neurological disorders are agerelated neurodegenerative disorders (Brown et al.., 2005) Parkinsons disease (PD) is the second most common neurodegenerative disorder and is the most common movement disorder. It is characterized by progressive motor decline, including resting tremor, bradykinesia, cogwheel rigidity, and postural instability due to the death of dopaminergic neurons in the substantia nigra pars compacta (Hoehn & Yahr, 1967) More advanced stages of disease can also include sleep disturbances, autonomic instability, and depression (Arai et al.. 2000; Boeve et al.., 1998; Tandberg et al.. 1996) Approximately 1.5 million people in the United States suffer from PD, and the estimated cost of providing supportive care for PD patients is over $25 billion per year. There are 50,000 new cases diagnosed per year, and it is estimated that the incidence of PD will increase to 3 5 millio n by 2050 (Tanner & Goldman, 1996; Twelves et al.., 2003) As seen with AD, the overall cost for supportive care is high and is growing as the incidence of disease increases over the next 40 years. There are a large number of different neurological di sorders that have some or all of the clinical features of PD. Collectively, this clinical syndrome is termed parkinsonism, and disorders primarily composed of parkinsonism symptoms are termed

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18 parki nsonian disorders. Parkinsonian disorders can be inherited or sporadic, and caused by effects of environmental toxins, metabolic disturbances, or drugs [as reviewed in (Beitz, 2014) ]. Aside from PD, the two most common disorders on the parkinsonism spectrum are Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA). DLB is the sec ond most common neurodegenerative dementia after AD, and is characterized clinically by decreased executive function, visual hallucinations, and muscular rigidity (Gibb et al.., 1987) In MSA, patients predominantly present with autonomic dysfunction, but also show signs of cogwheel rigidity, bradykinesia, and postural instability (Wenning et al.., 1994) Other parkinsonian disorders are listed in Table 11. These divergent parkinsonian disorders are characterized by the loss of dopaminergic neur ons in the substantia nigra pars compacta and intracellular S inclusion pathology (to be described in later sections). S pathology found in these disorders can also be seen in other neurodegenerative disorders, including a Lewy body variant of Alzheimer s disease (LBVAD). D isorders containing S pathology are termed synucleinopathies (Table 12) [as reviewed in (Dickson, 2001; Goedert, 2001a; Goedert, 2001b; Trojanowski & Lee, 2003) ]. Therefore, the study of parkinsonian disorders not only gives insight into the pathogenesis behind PD, but can also help with the study of other emotionally and financially devastating neurodegenerative disorders, such as AD. S Genetic s and Structure S is a member of the synuclein family of proteins, which also includes synuclein and synuclein (Yuan & Zhao, 2013) Synuclein was first identified in the electric eel Torpedo california (Maroteaux et al.., 1988) A few years later, a rat

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19 homologue was cloned and named S (Jakes et al.. 1994) Under normal physiologic conditions, S in the soluble form is predominantly found in the presynaptic terminal of central nervous system neurons near synaptic vesicles, and has been found in lesser amounts in the nucleus and mitochondria of neurons in both free and membrane bound forms [ reviewed in (Auluck et al.., 2010; Waxman & Giasson, 2008a) ] S is highly conserved among vertebrates and is encoded by the SNCA gene on chromosome 4q21 (George et al. 1995; Jakes et al. 1994; Maroteaux & Scheller, 1991) The SNCA gene contains 6 exons, and v ia in frame deletions of exon sequences, 3 isoforms of S may be formed: S 140 (14.5kDa; the full length transcript), S 126 (13.1kDa; from splicing of exon 3 or amino acid residues 4154), and S 112 (11.4kDa; from splicing of exon 5 or amino acid residues 103130) [ reviewed in (Beyer & Ariza, 2013) ] The full length transcript of S is transcribe d at greater levels than the truncated isoforms (Campion et al. 1995) ; however, different expression levels of the S isoforms have been found in brain tissue in certain disease states. For example, in a subpopulation of DLB patients, transcripts for S140 and neuroprotective S126 are down regulated, and aggregate prone transcripts S112 and S98 are up regulated (Beyer et al. 2008) The S protein is highly charged and segregated into 3 main domains. The amino domain (res idues 1 60) and c entral domain (residues 6195 ) contain six imperfect KTKEGV repeats, which stabilize conformational changes of s into helices. These domains also include the site for the 5 currently known missence mutations associated with autosomal dominant, early onset PD. The central domain also contains the nonamyloid component (NAC) region (resides 61 95), which is the

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20 hydrophobic epitope necessary for the conformational conversion of S to a predominantly betasheet form. The highly negatively charged carboxy domain (101140) is thought to help resist the formation of amyldoidogenic S Studies completed with S C terminal truncation mutants have shown increased aggregation propensity compared to the wildtype S protein (Uversky, 2007) For example, 189, 1 102, and 1 110 S protein constructs exhibit faster rates of S amyloid formation in vitro (Murray et al. 2003) Furthermore, 15% of S found in inclusion pathology is composed of C terminally truncated S (Baba et al. 1998; Campbell et al. 2001) S, the Conformational Chameleon Changes in S structure are highly dynamic and contextually dependent upon association with lipid membranes (Cole & Murphy, 2002) In order to understand why S is prone to readily undergo conformational changes, one has to understand the structural plasticity required of the protein to perform its normal cellular functions. The evolution of the S conformational behavior from functional to p athologic forms is as follows: Natively unfolde d structure in aqueous solution: Under normal physiologic conditions, S exists in a natively unfolded state. Circular dichroism studies have shown that in this state, individual protein sequences do not form stable helical or sheet structures, nor do S proteins consistently associate with each other (Weinreb et al. 1996) H elical membranebound structure: The amino domain is responsible for the formation of amphipathic helices. This allows S to go from a random coiled

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21 structure in solution to a stable structure that is 80% helical when associating with ac idic phospholipids in membranes Five helices are formed, 4 of which help to associate S with membranes and the fifth to mediate other membrane associated protein interactions [ (Davidson et al. 1998; Perrin et al. 2000) reviewed in (Pfefferkorn et al. 2012) ] For example, the S helix robustly associates with membranes enriched phosphatidic acid (PA) to serve as an inhibitor of phospholipase D2 (PLD2) to regulate PA levels (Davidson et al. 1998; Jenco et al. 1998) Membrane bound S also modulates synaptic transmission via chaperoning the refolding of SNARE proteins responsible for membrane fusion during vesicular exocytosis (Chandra et al. 2005) Pr e molten globule structure: Under mildly denaturing conditions, such as low pH, high temperature, or exposure to herbicides/pesticides, S can assume a premolten globule state. In this structure, S can spontaneously oligomerize and facilitate aggregate formation (Uversky et al. 2001a) S multimeric species: In response to increased cellular stress, S can form dimer and oligomer intermediates (Uversky et al. 2001a) Both tyrosine nitration (at Tyr 125, 133, or 136) and methionine oxidation (at Met 5 ) modifications to S have been shown to result in crosslinking of the monomeric form into oligomers (Chavarria & Souza, 2013; Souza et al. 2000) S oligomers bearing spherical and crescent shapes have been shown to be protofibrils by atomic force microscopy (Ding et al. 2002) It still remains unclear whether these multimeric intermediate species are toxic or play a more prominent role in disease (Goldberg & Lansbury, 2000) Amyloidogenic, aggregated S: A beta sheet comprises the secondary structure of most of the S found in intracellular inclusions (Giasson et al. 2001b;

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22 Weinreb et al. 1996) These S inclusions are identified as amyloidogenic aggegrates by apple green birefringence when stained with Congo red dye and viewed under polarized light (El Agnaf & Irvine, 2000) Approximately 30100% of aggregated S seen in pathological inclusions is hyperphos phorylated at s erine 129 (Fujiwara et al. 2002; Okochi et al. 2000) Additionally, ubiquitinated, nitrated, and oxygenated forms of S have been found in S inclusions (Duda et al. 2000a; Giasson et al. 2000a; Giasson & Lee, 2003) As shown above, S is readily capable of undergoing conformational conversions into multiple structures. These conversions a re necessary for S to associate with the membrane Hence, with S dynamically changing structure to fit its environment, it can be referred to as a conformational chameleon. The spontaneous conversion of S into a betasheet amyloidogenic form may be ex plained by the great propensity of S to readily undergo conformational changes (Figure 11). Clearance of Misfolded S For example, SIRT1, a NAD dependent deacetylase, has been shown to activate heat (Donmez et al. 2012) Additionally, it has been found that Hsp9 0 can also associate with misfold oligomers in an ATP dependent manner (Daturpalli et al. 2013) via 3 cellular mechanisms: by the ubiquitinproteasome pathway (U PS) (Berke & Paulson, 2003; Ghavami et al. 2014; Webb et al. 2003) by chaperonemediated autophagy (Hsp70) (Cuervo et al. 2004; Ghavami et al. 2014; Webb et al. 2003) or by pro tease degradation (calpain I, matrix metalloproteinase 3, cathepsin D neurosin)

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23 [ reviewed in (Beyer & Ariza, 2013) ] Ebrahimi Fakhari et al. proposed a general model (Ebrahimi Fakhari et al. 2012) pathways. As disease progresses, the chaperone, UPS, and CMA pathways become overwh and are unable to degrade it (Figure 11) Macroautophagy is then used as a compensatory mechanism to clear the too bec omes overwhelmed, leading to cellular dysfunction and death. Intracellular inclusions in Parkinsonism patients have been found to include components of each systems may b e involved in aggregate formation (Crews et al. 2010; Kuusisto et al. 2003; Kuusisto et al. 2001; McLean et al. 2002; Shin et al. 2005) associated with neurodegenerative di sease in 1993 when a peptide corresponding to amino acid residues 6195 was purified from Alzheimers extracellular Abeta plaques. The peptide was termed the nonAbeta component or NAC component of th e plaques (Ueda et al. 1993) The NAC co mponent was later identified to be (Jakes et a l. 1994) linked to Parkinsons disease when it was discovered that kindred of Gre co Italian descent who were suffering from early onset, autosomal dominant PD carried a missense mutation (A53T) in the SNCA gene (Polymeropoulos et al. 1997) Additionally, two more missense mutations (A30P and E46K) along with duplications and triplications in SNCA were linked to other kindreds with PD and DLB (Kruger et al. 1998) (Chartier Harlin et al. 2004; Farrer et al. 2004;

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24 Singleton et al. 2003; Zarranz et al. 2004) More recently, it was shown that two new ly identified missense mutations (G51D ; H50 Q ) in SNCA also result in PD (Appel Cresswell et al. 2013; Kiely et al. 2013; Lesage et al. 2013; Proukakis et al. 2013) Brain nervous tissue obtained at autopsy from members of 3 kindreds (A53T, A30P, E36K) confirmed the presence of S inclusion pathology both in the cortex and substantia nigra (Cookson, 2005) H isto logical studies have s the predominant component of perinuclear, cytoplasmic inclusions (termed Lewy bodies LBs) and ribbonlike inclusions in cellular processes (term ed Lewy neuritesLNs) of PD patients (Baba et al. 1998; Gai et al. 2000; Spillantini et al. 1997) Addi also present in glia (GCIs) in MSA, and to a lesser extent in PD and DLB (Dickson et al. 1999; Gai et al. 1998; Tu et al. 1998) Electron microscopy of from diseased brains showed that amyloidogenic filaments (Crowther et al. 1998; Giasson et al. 1999) These discoveries lead to the in vitro conditions to support o amyloid were determined by multiple groups (Conway et al. 1998; Crowther et al. 1998; El Agnaf et al. 1998 a; Giasson et al. 1999; Han et al. 1995; Hashimoto et al. 1998; Iwai et al. 1995b; Narhi et al. 1999; Wood et al. 1999; Yoshimoto et al. 1995) The development of synthetic amyloidogoenic S fibrils has provided exciting progress with a molecular tool to study the etiologic relationship between S aggregation and disease. When misfolded S was linked to Parkinsonism, q u pathology forms and spreads, and its clinical significance to the onset and progression

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25 of disease S from cells and transmitted intercellularly, and that induction may occur in the peripheral nervous system with retrograde transport back to the central nervous system (Braak et al. 2006b; Braak et al. 2003a; Braak et al. 2003b) It was first reported released by cells by Borghi et al. via immunoprecipitation of PD patients (Borghi et al. 2000) S are increased levels of both oligom CSF of Parkinsonism patients (El Agnaf et al. 2003; Kasuga & Ikeuchi, 2012; Parnetti et al. 2013) Additionally, autopsy studies of PD patients who had received therapeutic intrastriatal embryonic neuronal tissue grafts have suggested intercellular transmission of S pathology At over a decade post transplant, the grafted neurons had developed (K ordower et al. 2008; Li et al. 2008; Li et al. 2010) Post mortem studies have reported that in about 5080% of PD cases there is (Braak et al. 2003a; Braak et al. 2003b) It has been proposed that the pathology follows two distinct neuroanatomical patterns projecting back to the brain and brain stem [ (Braak et al. 2003a; Braak et al. 2003b; Burke et al. 2008) reviewed in (Lema Tome et al. 2012) ] The first proposed scenario is via the olfactory bulb where a pathogen could lead to with retrograde transpor t back to the brain. The second is via pathogens or irritants to the intestinal epithelium that would promote an inflammatory or stressful environment in

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26 aggregated protein woul d then be transmitted retrogradely via the vagus nerve to the brain stem and into the brain (Braak et al. 2006b) Collectively, this data from PD patients that misfolded S may self propagate in association with a disease state pathogenesis is similar to that seen in prion disease. The term prion was coined in 1982 to describe a protein that was normally soluble but had acquired a conformational change into an infectious agent of disease (Prusiner, 1982; Prusiner, 1998) The idea that a misfolded protein could transmit disease was an anomaly until the search for the scrapie agent yielded a protease and UV/ionizing radiationres istant protein (Alper et al. 1967; Alper et al. 1966; Bolton et al. 1982; Latarjet et al. 1970) The protein was designated prion protein (PrPC) I t is present in most tissues, particularly nervous tissue, and shows extensive similarities in most animal species, especially the structured carboxy domain [ reviewed in (Grassmann et al. 2013; Kretzschmar & Tatzelt, 2013) ] T he misfolded form of PrPC (PrPSc) is responsible for cellular toxicity and the transmission of disease (Kim et al. 2010; Makarava et al. 2010; Prusiner, 1989; Wang et al. 2010) Eventually it was shown that prion disease was the cause of a number of neurodegenerative disorders in both animals and humans including Creutzfeldt Jakob disease (CJD), GertstmannStraussler Scheinker disease (GSS), and fatal familial insomnia (FFI) (Denkers et al. 2013; Saunders et a l. 2012; Tamguney et al. 2012; Wilson et al. 2012) Results Presented in this Dissertation The goal of my dissertation studies is to further understand the induction and spread of S inclusion pathology. My working hypothesis, supported by the previously

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27 mentioned human autopsy data, is that there is a prionoid spread of S inclusion pathology. To address this hypothesis and to understand its broader relevance to human PD, I charact erized three models of induction: i) addition of exogenous S fibrils to primary neuronal glial cultures overexpressing S, and ii) intracerebral injection and iii) intramuscular injection of exogenous S fibrils in non transgenic and S transgenic PD mous e models. When I began these experiments in September of 2011, there were only a handful of published studies showing that amyloidogenic S could induce intracellular S inclusion pathology (Luk et al. 2009; Nonaka et al. 2010) (Desplats et al. 2009; VolpicelliDaley et al. 2011; Waxman & Giasson, 2010) (VolpicelliDaley et al. 2011) These initial studi es showed that in different immortal cell lines overexpressing wildtype S, the addition of exogenous S fibrils leads to the formation of intracellular S inclusions (Luk et al. 2009; Nonaka et al. 2010) (Desplats et al. 2009; Volpicelli Daley et al. 2011; Waxman & Giasson, 2010) Subsequently, another study in primary neuronal cultures reported that exogenous S fibrils induce S pathology in the absence of protein overexpression (VolpicelliDaley et al. 2011) Building upon these studies, I have s hown in mixed primary neuronal glial cultures that i) wild type and missense mutations of S induce pathology in a strainlike fashion akin to that seen in prion disease, and ii) that S pathology can be passaged from infected cells to nave cells. This study is detailed in Chapter 2 of the dissertation. Initial i n vivo studies completed on S pathology induction and spread have focused on intracerebral injection of diseased brain lysate or synthetic S fibrils into nontransgenic or S transgenic mouse models (Luk et al. 2012a; Luk et al. 2012b;

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28 MasudaSuzukake et al. 2013; Mougenot et al. 2012) The collective results reported from these studies set the tone for the syncleinopathy field that i) intracerebral injection containing amyloidogenic S results in induction and spr ead of massive S pathology in both nontransgenic and S transgenic mice, and ii) that spread of S pathology throughout the CNS occurs via transport through white matter tracts. In an effort to reproduce and extend those studies, I have completed both neonatal and adult stereotaxic intracerebral injections of both amyoidogenic and soluble S protein into nontransgenic mice and S transgenic mouse models overexpressing human wildtype, A53T, and E46K S. The data from my in vivo studies provides 3 major contributions to the field. The first being that in both nontransgenic and S transgenic mice, there is induction of intracellular S pathology by amyloidogenic S at the injection site; however, spread of S pathology from the i njection site is variable depending upon the mouse line used. The second is that a soluble mutant construct of S is also capable of inducing S pathology, but less efficiently. The third is that the main antibody marker used to assess spread of S patholo gy (pSer129/81A) (Luk et al. 2012a; Luk et al. 2012b) crossreacts with low molecular weight neurofilament (NFL), a major component of white matter tracts. While this data does not refute a prionoid spread of S pathology, it implies that there may be additional biological mechanisms involved in disease progression. The studies on intracerebral injections are detailed in Chapters 3 and 4 of this dissertation. The results of the Braak staging studies lead to the hypothesis that there is transmission of peripherally induced S pathology to the CNS (Braak et al. 2003a; Braak et al. 2003b) The final portion of my graduate work was the development of a

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29 peripheral induction model of CNS S inclusion pathology. Intramuscular injection of either amyloidogenic or soluble S protein ca n result in the synchronous development of a motor phenotype and CNS S pathology in S transgenic mice overexpressing human wildtype or A53T S. Although this model provides evidence for induction, as of now there is no evidence to imply a prionoid transmission of S pathology. The ease of the injection procedure coupled with the rapid development of a phenotype and S pathology makes this model a valuable tool for future induction and spread studies. This study is detailed in Chapter 5 of this dissertation.

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30 Figure 11.Model of S misf olding an d the development of Lewy bodies and Lewy neurites. Under normal physiologic conditions, S exists in a natively unfolded state. Due to triggers such as environmental factors, age, and genetics, S can be induced to undergo a conformational change in to a predominantly sheet form. Misfolded S protein that evades degradati on by proteasomal and lysosomal structures can form amyloidogenic fibrils. These S fibrils are the major components of intracellular S inclusions known as Lewy bodies and Lewy neurites. This figure was adopted from (Irwin et al. 2013)

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31 Table 11 Parkinsonian disorders Disease t ypes Parkinson's disease Multiple system atrophy Dementia with Lewy bodies Progressive supranuclear palsy Corticobasal degeneration Guam parkinson dementia complex Chronic traumatic encephalopathy Frontotemporal lobar degeneration Vascular parkinsonism MPTP exposure Manganese poisoning Antipsychotic medications Postencephalic parkinsonism from influenza virus

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32 Table 12 Synucleinopathies D isease t ypes Parkinson's disease Lewy body variant of Alzheimer's disease Dementia with Lewy bodies Multiple system atrophy Neurodegeneration with brain iron accumulation 1 Down's syndrome Alzheimer's disease Autosomal recessive juvenile parkinsonism Parkinsonism dementia complex of Guam Progressive autonomic failure REM sleep behavior disorder

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33 CHAPTER 2 CONFORMATIONAL TEMPLATING OF SYNUCLEIN AGGREGATES IN NEURONAL GLIAL CULTURES Introduction Parkinsons disease (PD) is the most common movement neurodegenerative disorder (Dorsey et al. 2007) PD belongs to a spectrum of neurodegenerative synucleinopathies, unified by the presence of the brain accumulation of neuron (Goedert, 2001b; Spillantini et al. 1997) tely established; SNCA ) defects that cause autosomal dominant PD ( SNCA gene duplication/triplication and missense mutations A30P, E46K, G51D, H50Q, and A53T) (Appel Cresswell et al. 2013; Chartier Harlin et al. 2004; Farrer et al. 2004; Kiely et al. 2013; Kruger et al. 1998; Lesage et al. 2013; Polymeropoulos et al. 1997; Proukakis et al. 2013; Singleton et al. 2003; Zarranz et al. 2004) Differences in the clinical profiles and pathology of PD patients and mouse models with either the A53T or E46K mutation have been documented (Duda et al. 2002b; Emmer et al. 2011; Giasson et al. 2002; Polymeropoulos et al. 1997; Zarranz et al. 2004) In vitro biochemical studies on amyloid shown differences in nucleation and elongation of fibril polymerization, peptide structural order, and ultrastructural morphology (Brucale et al. 2009; Comellas et al. 2011; Giasson et al. 1999; Ono et al. 2011; Rospigliosi et al. 2009) Recent postmortem studies in PD patients suggest that the spread of pathology can occur intercell ularly (Braak et al. 2003a; Braak et al. 2003b; Kordower et al. 2008; Li et al. 2008; Mendez et al. 2008)

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34 that like mechanism (Luk et al. 2012a; Luk et al. 2012b) A key characteristic of prionlike transmission is permissive templating, in which the amyloidogenic form of the protein interacts with normal endogenous protein, and that interaction induces a conformational change in the endogenous protein to an amyloidogenic pleated sheet structure (Aguzzi & Falsig, 2012; Eisenberg & Jucker, 2012) A major difference between prionlike transmissi on and classical prion disease is paucity of inter organism transmission. For many amyloids this prionlike conversion of protein conformation can often have unique structural and morphological properties that can be transmitted and this phenomenon has been termed strainspecific (Aguzzi et al. 2007; Eisenberg & Jucker, 2012) Taking advantage of previous in vitro observations that wildtype, A53T, and structural properties (Brucale et al. 2009; Comellas et al. 2011; Giasson et al. 1999; Greenbaum et al. 2005; Ono et al. 2011; Rospigliosi et al. 2009) and that pathological inclusions in A53T and E46K transgenic mice are also distin ct (Emmer et al. 2011; Giasson et al. 2002) we u sed a novel AAV primary neuronal glial culture model to test the hypothesis that fibrillar A53T and E46K neuronal and astrocytic inclusions formed by fibrillar morphologically consistent with those in transgenic mice and that the seeding fibrils also find that inclusion pathology induced by exogenous fibrils in one astrocytes culture

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35 can be passaged to a second astrocytes culture. These data provides important support Materials and Methods Mixed neuronal glial primary cultures All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. SNCA null mice (Abeliovich et al. 2000) were obtained from The Jackson Laboratory (Bar Harbor, MA). Primary cultures were prepared from P0 C3HBL/6 mouse brains (Harlan Labs). Cerebral cortices were dissected from P0 mouse brains and were dissociated in 2mg/mL papain (Worthington) and 50 g/mL DNAase I (Sigma) in sterile Hanks Balanced Salt Solution (HBSS, Life Technologies) at 37C for 20 minutes. They were then washed t hree times in sterile HBSS to inactivate the papain and switched to 5% fetal bovine serum (HyClone) in Neurobasal A growth media (Gibco), which includes 0.5 mM Lglutamine (Gibco), 0.5 mM GlutaMax (Life Technologies), 0.01% antibiotic antimycotic (Gibco), and 0.02% SM1 supplement (Stemcell). The tissue mixture was then triturated three times using a 5 mL pipette followed by a Pasteur pipette, and strained through a 70 m cell strainer. The cell mixture was then centrifuged at 200 g for 3 minutes, and resuspended in fresh Neurobasal A media. They were then plated onto poly D lysine coated chamber slides (Life Technologies) or dishes at around 100,000200,000 cells/cm2. Cells were maintained in the Neurobasal A growth media mentioned above without fetal bovine serum at 37C in a humidified 5% CO2 chamber.

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36 AAV2/1 preparation and expression Adeno associated viruses serotype 2/1 expressing human wildenhancer/chicken actin (CAG) promoter, were generated as described previously (Kim et al. 2008) At 5 days in vitro (DIV), each virus was added to cultures at a final concentration of 1011 genome copies/mL. We have determined that at 12DIV, ~40% of cells in our cultures are transduced by AAV2 1. Recombinant, human synuclein and 21140, wild type, A53T 71described previously (Giasson et al. 2001b; Greenbaum et al. 2005; Waxman & Giasson, 2010; Waxman & Giasson, 2011b) For amyloid assembly 21140, wild type, buffered saline (PBS; Life Technologies, Carlsbad, CA, USA) at 37C with continuous shaking at 1050 rpm (Thermomixer R, Eppendorf, Westbury, NY, USA) and fibril formation was monitored by turbidity and K114 fluorometry (Waxman & Giasson, 2010) Fibrils were diluted to 1mg/mL in sterile PBS and sonicated for 2 hours, which resulting fragmentation into smaller fibrils of varying lengths (Luk et al. 2009; Waxman & Giasson, 2010) Cultures were treated with 1 M of fibril mix at 8 DIV. Fibrils pre labeled with ThioflavinS (ThS) were only used where indicated. A 1mg/mL fibril mix was incubated i n 0.05% ThS for 1 hour then spun at 16,000 g for 5 minutes, and washed with PBS three to five times and sonicated for 2 hours. Fibril mix was then added to cultures at a final concentration of 1 M. Biochemical Cellular Fractionation and Western Blotting Analysis Samples for biochemical analysis were harvested at 4 days post seeding. Cultures were washed

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37 in PBS and scraped in 1% Tx 100 TBS (50mM Tris, 150mM NaCl, pH 7.4) with protease and phosphatase inhibitors and placed on ice for 10 min. Lysates wer e then centrifuged at 100,000 g for 20 min at 4C. Supernatants were removed (Triton X 100 soluble fraction), and the pellet was washed with the TBS buffer and recentrifuged. The remaining pellet was then resuspended in 2% SDS, sonicated and heated to 100oC for 10 min (Triton X 100 insoluble fraction). 2% SDS was added to the Triton X 100 soluble fraction that was heated to 100oC. Equal amounts of protein were resolved by SDS PAGE on 15% polyacrylamide gels, followed by electrophoretic transfer onto ni trocellulose membranes. Membranes were blocked in Tris buffered saline (TBS) with 5% dry milk, and incubated overnight with Syn211, a mouse monoclonal antibody specific for amino acids 121(Giasson et al. 2000b) in TBS/ 5% dry milk or pSer129, a mouse monoclon Ser129 (Waxman et al. 2008) in TBS/ 5% bovine serum albumin (BSA). A total anti actin antibody (clone C4) (Millipore, Billerica, MA) was used as a loading control. Each incubation was followed by goat anti mou se conjugated horseradish peroxidase (HRP) (Amersham Biosciences, Piscataway, NJ ) or goat anti rabbit HRP (Cell Signaling Technology, Danvers, MA). Protein bands were detected using chemiluminescent reagent (NEN, Boston, MA) and a FluorChem E and M Imager (Proteinsimple, San Jose, California). Immunofluorescence Microscopy Analysis Samples for immunofluorescence analysis were taken at 4 days post seeding (and at 1 day post seeding where indicated). For double immunofluorescence analysis, cells were fixed with 4% paraformaldehyde/PBS. Following PBS washes, cells were blocked with 5% goat

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38 serum/PBS/0.3% Triton X 100 for 1 hour. Cultures were incubated in primary antibodies: pSer129 (1:500) and SNL4 (1:500), a rabbit polyclonal antibody raised against amino acids sequence 2(Giasson et al. 2000b) Oth er primary antibodies include rabbit polyclonal anti MAP2B (1:100), a neuronal marker (Millipore); rabbit polyclonal anti GFAP (1:1000), an astrocyte marker (Dako); and rabbit polyclonal anti ubiquitin (1:1000) (Abcam). This was followed by incubation in Alexa fluor 488 and 594 conjugated secondary antibodies (1:1000) (Invitrogen). Nuclei were counterstained with 4',6 diamidino2 phenylindole (DAPI; Invitrogen), and coverslips were mounted using Fluoromount G (Southern Biotech, Birmingham, AL). Thioflavi n S (Sigma Aldrich) immunostaining was performed after secondary antibody incubation at a concentration of 0.05% followed by three washes in 70% ethanol and three washes in water. Images were captured on a Leica TCS SP2 AOBS Spectral Confocal Scanner mounted on a Leica DM IRE2 inverted fluorescent microscope. All images were captured with either 20x or 63x water immersion objectives as projection images from a Z stack of <1.0 m per plane. Astrocyte cultures All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Astrocyte cultures were prepared from P2 C3HBL/6 mouse brains (Harlan Labs). Cerebral cortices were dissected from P2 mouse brains and were dissociated in 2mg/mL papain (Worthington) and 50mg/mL DNAase I (Sigma) in sterile Hanks Balanced Salt Solution (HBSS, Life Technologies) at 37C for 20 minutes. They were then washed three times in sterile HBSS to inac tivate the papain and switched to Dulbeccos Modified Eagle Medium (Life Technologies) with

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39 10% fetal bovine serum (Life Technologies) and 100U/mL Penicillin and 100 g/mL Streptomycin (Life Technologies). The tissue mixture was then triturated three times using a 5 mL pipette followed by a Pasteur pipette, and strained through a 70 m cell strainer. The cell mixture was then centrifuged at 200 g for 3 minutes, and resuspended in fresh DMEM media. They were then plated in chamber slides, 10 cm culture di shes, or T 75 flasks at 75,000 to 200,000 cells/cm2. Cells were maintained in the DMEM media mentioned above at 37C in a humidified 5% CO2 chamber. After two days, flasks were shaken for 30 seconds and supplied with fresh media. Upon reaching confluenc y, flasks were split using TrypLE Express (Life Technologies) for two generations per original culture. Cell lysate passaging in astrocyte cultures was seen in both neurons and astrocytes in mixed cultures, astrocyte cultures were used because they can be split which facilitated the passaging experiments. The first generation astrocyte cultures were split into 10 cm culture dishes at 75,000 cells/cm2. Adenoassociated viruses serotype 2/1 expressing human wildS under control of the CMV early enhancer/chicken actin (CAG) promoter, as described above, was added at 3 days following plating at a final concentration of 1011 genome copies/mL. At 5 days following plating, the media was changed and cultures were treated with 1 fibril mix for 5 days. Cultures were then washed three times in PBS and incubated in 0.25% TrypsinEDTA (Life Technologies) for 10 minutes to inac tivate residual minutes. Pellets were rinsed with PBS and then frozen. The second generation astrocyte cultures were again split and were transduced with adeno associate d virus

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40 serotype 2/1 expressing human wildthe cell pellets from the first generation cultures were thawed, resuspended in 1mL PBS, and lysated by sheer force with a 27g needle. 100 L of this lysate was t hen added to the second generation astrocyte cultures for 5 days. Quantitative Analysis. made by observer blinded, random field counting. Images were captured with an Olympus BX51 fluorescence microscope mounted with a DP71 digital camera (Olympus, Center Valley, PA) using 10x magnification and imaged were enlarged with Photoshop software and using a grid to allow for total field counting. For each seeding combination, 6 fields were counted. Criteri a for inclusion morphology were set according to that seen in the M83 and M47 mouse models (see Results Figure 2 7). Briefly, either M83like filling the somatodendritic compartment, or M47like with compact and rounded perinuclear inclusions. Results were expressed as the mean ratio of cells showing either type of morphology to the total number of inclusions per field SD. Comparisons for significance were made using oneway ANOVA with Bonferronis post hoc test in GraphPad Prism software (San Diego, C A). Res ults Induction of S aggregates in primary mixed neuronal glial cultures In previous studies, we and others (Luk et al. 2009; Waxman & Giasson, 2010; Waxman & Giasson, 2011b) have shown that under experimental conditions that promote the like inclusions can induce inclusion f ormation in primary mixed mouse neuronal glial cultures, we tested

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41 whether overexpression of wildaddition of aminoterminally truncated exogenous recombinant wildtype (21fibrils were sufficient to in formation using several previously established methods. First, we used anti this modification is an excell ent marker of aggregate formation (Anderson et al. 2006; Fujiwara et al. 2002; Luk et al. 2009; Waxman et al. 2008; Waxman & Giasson, 2010; Waxman & Giasson, 2011b) However, in primary neuronal cultures used here this antibody also recognizes a nonprocesses, as it stains these processes in cultures f rom wild type and SNCA null mice (Figure 2 1). Therefore, co 4, were used to track inclusion formation. Notably, the SNL4 antibody binds the extreme 1 140 and full we previously have shown that this antibody also stains the intracellular inclusions (Luk et al. 2009; Waxman & Giasson, 2011a) a nd therefore can be used to distinguish the nonspecific neuritic background staining seen with pSer129. In the primary mixed neuronal profile reflecting a presynaptic distribution ( Figure 2 1A), as previously demonstrated (Murphy et al. 2000; Withers et al. 1997) AAV2/1 the neuritic staining of the pSer129 non2 1B). Addition of preformed fibrils alone resulted in pSer129 staining of processes similar to that seen in blank controls, but this staining did not colocalize with SNL 4 (Figure 2 1C). Addition of

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42 formation (Figure 2 1D). When robust aggregate formation is present, the staining of these aggregates with pSer129 is much more robust then the nonthe weaker signal of the non formation, inclusion formation was rapid and efficient. Using AAV2 1 wild 0.4 0.1% and 29.1 3.8% of the transduced cells formed aggregates by 1 and 4 days following fibril addition, respectively. However, aggregate formati on was accelerated if the percentage of transduced cells with aggregates at 1 day following the addition of fibrils: AAV2 1 wild type 0.2%), AAV2 1 wild type (2.6 1.2%), AAV2 6.7%), AAV2 (7.9 2.4%), AAV2 1 type fibrils (11.9 1.9%), AAV2 fibrils (3.5 0.6%), AAV2 0.3%), AAV2 wild type fibrils (3.2 0.6%). for intracellular aggregate formation, we treated c synuclein or nonamyloidogenic 71 2 2) (Giasson et al. 2001b; Luk et al. 2009) Treatment with ei ther of these proteins did not result in intracellular aggregate formation, but nonspecific pSer129 staining in neuronal processes was still apparent.

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43 To further establish that these pSer129 and SNL4 immunostained aggregates were bonafide inclusions, we conducted biochemical fractionation and Western blot analysis previously used to validate the presence of Triton X 100 aggregates in culture (Waxman & Giasson, 2010) As shown in Figure 2 3A, AAVinduced the formation of pSer129 phosphorylated Triton X 100 insoluble aggregates. To further validate that extracellular, exogenous aggregates could enter cells and be S pre fibrils were added to cultures and shown to be present within intracellular aggrega tes (Figure 2 3B). formation; we performed double immunofluorescence with the neuronal specific marker MAP2B and the astrocyte specific marker GFAP (Figure 2 4). Inclusions formation was readily observed in both cell types. In many ex periments 4 days post seeding, more than 50% of both SNL4+ MAP2+ neurons and GFAP+ SNL4+ astrocytes demonstrated pSer129+ aggregates using the AAV2/1 wild and wildtype fibril combination. By 8 days post seeding nearly all cells that were transduced with the 4+ showed pSer129+ inclusions. As the rAAV2/1 vectors efficiently transduce greater than 50% of astrocytes and neurons in culture, these studies reveal that this system shows remarkable efficiency of inclusion formation. (George et al. 1995; Iwai et al. 1995a; Jakes et al. 1994; Withers et al. 1997) by using AAV

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44 those observed in human brains, we demonstr ated that the inclusions formed were partially ubiquitin and ThioflavinS (ThS) positive (Figure 2 5, 2 6). In cultures treated with AAV2/1 human wildnot overlap with endogenous ubiquitin (Fig ure 2 5A). The overexpression of human wild aggregates that colocalization with ubiquitin (Figure 2 5B). Co localization of ubiquitin both in neurons (Figure 2 5C) and astrocytes (Figure 2 5D). Similarly, pSer129+ aggregates show colocalization with ThS (Figure 2 6A). The ThS staining profile is seen in both neurons (Figure 2 6B) and astrocytes (Figure 2 6C). However, it appears that ThS only stains a fraction of the pSer129 immunoreactive inclusion, suggesting that even within individual cells the structure of the aggregate is not homogenous. glial cultures Transgenic mouse models for human A53T (line M83) and E46K (line (Emmer et al. 2011; Giasson et al. 2002) Both transgenic models express similar leve mouse prion protein promoter and develop similar agedependent motor phenotypes ly demonstrates a flamelike somatodendritic and neuritic profile; whereas, those seen in the M47 transgenic model have a rounded and compact profile (Figure 2 7). We therefore studied the effects that the A53T and E46K mutations would have on the morphol

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45 whether these differences were due predominantly to the type of protein being overexpressed, the type of fibril seeding pathology, or both. As seen in Figure 2 8A, wild hology. Regardless of the protein being overexpressed, seeding with A53T fibrils induced an overall a flame like somatodendritic inclusion that we will refer to as M83like pathology; whereas, seeding with E46K fibrils induced a more rounded and compact i nclusion that we will refer to as M47 like pathology (Figures 2 8 and 2 9). Quantification data also showed that the morphology of the inclusions was heavily dependent upon the type of fibril used to seed the culture and that the ratios of M83 to M47like pathologies were significantly different (all p<0.01). As state above, wildtype wild type showed slower aggregation kinetics relative to combinations that included a mutant protein, but it resulted mainly in M83like pathology; however, when wildtype was used to seed A53T or E46K, the predominant morphology depended upon the protein being expressed, suggesting that wild2 8B). Our astrocytes (Figure 2 (SNL 4, Figure 2 10A) and also lack the background neuritic staining of pSer129 (Figure 2 10A D). As with the primary mixed neuronal 2 10D). For passaging studies, cultures were treated with AAV2/1 hu man wild

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46 Cell lysates prepared as described in Material and Methods from the seeded cultures was added to astrocyte cultures overexpressing human wildAAV2/1). The cultures were left to incubate wi th the lysate for 5 days. Cultures exposed to aggregatecontaining cell lysate from the overexpression of human wildtype derived human fibrils, showed passaging of pSer129 positive aggregates (Figure 2 11A). Whereas, astrocyte c ultures overexpressing human wildderived fibrils (Figure 2 11B), AAV2/1 human wild2 11C), or nothing (Figure 2 11D), did not show any pSer129 and SNL4 positive aggregates. After the first passage, 2.9 0.7% of transduced cells contained pSer129+ aggregates. Discussion synucleinopathies. Many studies have indicated that aggreg neuronal demise (Cookson, 2005; Goldberg & Lansbury, 2000; Norris et al. 2004; Waxman et al. 2009) There fore it is important to better understand the mechanisms in vitro change into an amyloidogenic, fibrillar form and it is well established that in vitro aggregation into amyloid is a nucleation dependent process and can be greatly induced by the addition of a seed or nucleus of pre(Cookson, 2005; Norris et al. 2004; Uversky, 2007; Waxman et al. 2009; Wood et al. 1999) Cellular studies have shown that the entry fibrils into immortalized cell lines using reagents that promote the entry of these seeds

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47 across the plasma membrane can very efficiently induce the formation of large intracellular amyloid inclusions (Luk et al. 2009; Waxman & Giasson, 2010) In another ( VolpicelliDaley et al. 2011) However, in our primary neuronal glial culture system, the addition of only processes that did not colocalize with a marker for endogeno 4), and this (Abeliovich et al. 2000) indicating that it is primarily due to cross reactivity. Although it is possible that some of the pSer129 immunostaining seen in neuritic processes of the seeded cultures does represent bonafide inclusion formation, it is imperative that other markers of inclusion pathology show colocalization with the neuritic pSer129 staining to demonstrate true inclusion formation. Indeed, the primary reliance on pSer129 immunostaining in both previous neuronal culture studies and in vivo may confound the interpret mixed primary neuronal glial cultures. These aggregates are composed of endogenous and share the biochemical properties of LBs and LNs of being pSer129 hyperphosphorylated, ubiquitinated, ThioflavinS positive, and Triton X 100 insoluble. They occur in both neurons and astrocytes in a high percentage of both cell types. Although LB and LN pathology in brain tissue samples from PD patients is along with a massive astrogliosis surrounding dying neurons in the SNpc of PD patients

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48 (Halliday et al. 2011) Using a mixed culture model where pathology is seen in both cell types allows for further study of the interplay that may be occurring during the pathogenesis of PD. Although subtle differences in experimental paradigms may well explain the differences between our study and the VolpicelliDaley et al. study where seeded inclusion formation was reported in primary neuronal cultures not (VolpicelliDaley et al. 2011) this discrepancy will require further experimental investigation. Some post mortem the CNS and from the CNS onto grafted neurons (Braak et al. 2003a; Braak et al. 2003b; Kordower et al. 2008; Li et al. 2008; Mendez et al. 2008) but different concepts have been proposed to explain the mechanism of pathology propagation. These have included: chronic generalized neuroinflammation, which may promote the excitotoxicity, which causes post makes the protein more aggregate prone; loss of homeostasis from chronic cellular stress, which may lead to the failure of molecular chaperones and other machinery to like spread where misfolde inclusions (Brundin et al. 2008) intercellular release and reuptake

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49 that intracerebral injection of prease (Luk et al. 2012b; Mougenot et al. 2011) Injection of pretype mice was also recently injection (Luk et al. 2012a) These finding sugges mechanism mentioned above may also be involved can be excluded. To further investigate the permissive conformational templ aggregation, we took advantage of the unique properties of diseasecausing mutants A53T and E46K, which also have unique effects on aggregate formation. Both mutations are found in the amino terminal of the protein, proximal to the central hyd over the E46K mutation (Conway et al. 1998; Giasson et al. 1999; Greenbaum et al. 2005; Narhi et al. 1999) to alterations in and may make them more susceptible to aggregation (Comellas et al. 2011; Rospigliosi et al. 2009) In addition, in vitro (Comellas et al. 2011; Giasson et al. 1999; Greenbaum et al. 2005; Ono et al. 2011) and these differences are most likely directly responsible for the different observed morphological findings

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50 like profile, while similar treatment with E46K nd, compact in vivo It is not clear if the differences in inclusion morphologies result in any significant altered pathophysiological outcomes or if they simply reflect difference st ructure variants. Mice distinct morphological features, but both types of inclusions are associated with similar severe motor phenotype resulting in death (Emmer et al. 2011; Giasson et al. 2002) These data, along with other e (Kordower et al. 2008; Lee et al. 2010b; Li et al. 2008; Luk et al. 2012a; Luk et al. 2012b; Mendez et al. 2008) intercellularly by conformation templating of amyloid formation akin to a strainspecific prionlike mechanism that has been documented for other prionoid diseases (Aguzzi et al. 2007; Eisenberg & Jucker, 2012) One difference between our model and typical prion strainspecific properties (Aguzzi et al. 2007; Eisenberg & Jucker, 2012) is that causing mutants, while the same type of process can occur with wildtype prion proteins. Future studies will investigate whether unique post like conformational templating. Because both intrinsic perturbations of proteostasis and S inclusion formation it is currently challenging to definitively distinguish between true seeding of pathology and other factors that may contribute to inclusion formation in vivo (Brundin et al. 2008) Based on th e

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51 morphological seeded templating described here it should be possible to distinguish between true exogenous seeding and other mechanism of pathology induction in vivo allowing for studies of how the interplay between these cells may contribute to disease. Because of its efficiency, future studies examining both the prionlike spread and its effect on cellular function can be facilely carried out using this culture model. Further w ork to provide more support for the prionlike spread can be done using this model. One of the characteristics of a prionlike spread is that pathology can be continuously passaged from an infected to nave host and that different prion strains maintain their pathological characteristics (Collinge & Clarke, 2007) aggregation can be passaged from cell lysates derived from astrocytes cultures seeded currently expanding our studies to further investigate this phenomenon. These studies will help us to elucidate the mechanisms by which pathology spreads not only in PD but also potentially in other neurodegenerative disorders and help to study a means for therapeutic intervention. We report on a novel mixed primary glialneuronal culture system that can be of extracellular seeds added. Using this experimental system and diseaseshow that the addition of extracellular preformed fibrils can induce conformational templating of intracellular aggregates and that the nature of mutant fibrils has a dominant effect on inclusion formation. These studies provide further credence to the -

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52 templating (prionlike) mechanisms at least in terms of intercellular transmissi on and induction of change in protein conformation by direct protein interaction. If such conformational templating occurs in vivo then distinct inclusion morphology induced by d in vivo

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53 Figure 2mixed neuronal glial cultures. Double immunofluorescence with SNL4 (red) and pSer129 (green): A) In untreat predominantly a punctate staining profile reflecting a presynaptic distribution, as previously demonstrated (Murphy et al. 2000; Withers et al. 1997) and B) AAV2/1 fibrils alone resulted in pSer129 staining of processes also seen in blank controls, but not overlapping with SNL4. D) Both AAV2/1 mediated immunoreactive inclusions in both the cell body and processes that overlaps with SNL 4. In cultures derived from SNCA null mice, both E) untreated and F) addition of fibrils resulted in a neuritic staining profile with pSer129, in the absence of SNL4 staining. The addition of fibrils did not increase the amount of p Ser129 neuritic staining. Cultures were counter stained with DAPI (blue). Bar scale = 100 m; insets = 25 m.

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54

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55 Figure 22 Non Double immunofluorescence with SNL4 (red) and pSer129 (green): Mixed glialneuronal cultures with AAV2/1B) 71 8 C) synuclein. Only the treatment Cultures were counter stained with DAPI (blue). Bar scale = 100 m; insets = 25 m.

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56 Figure 2 ls and formation of intracellular Triton X 100 A) Formation of intracellular Triton X 100 were treated with either AAV2/1 wilduorescent protein control) alone or in combination with wildpost seeding, cultures were biochemically fractionated using Triton X 100 containing buffer as described in Materials and Methods. found in the Triton X 100 insoluble fraction. Syn211, which recognizes the AAV2/1 wild mediated overexpression fibrils i T riton X 100 insoluble fraction. pSer129, which recognizes the overexpressio 100 insoluble fraction. B) Z slice se ction analysis of inclusions showing incorporation of extracellular fibrils into aggregates. Thioflavin S pre labeled wildincorporated into pSer129 aggregates (red). Bar scale = 10 m.

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57

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58 Figure 2by double immunofluorescence with the neu pSer129 positive aggregates (green) induced by treatment with extracellular astrocytes as shown by double immunofluorescence with the astrocyte marker GFAP (red). Cultures were counter stained with DAPI (blue). Bar scale = 25 m.

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59 Figure 2 5. inclusions in the mixed neuronal glial cultures colocalize with ubiquitin. A) In cultures treated with AAV21 human wildbackground staining does not colocalize with endogenous ubiquitin (green). B) In cultures treated with AAV2/1 human wildhuman wildin these cultures are ubiquitinated (green). This ubiquitin staining profile (green) colocalizes with the neuronal marker MAP2B (red, C) and the astrocyte marker GFAP (red, D). Cultures were counter stained with DAPI (blue). Bar scale = 25 m.

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60

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61 Figure 2 ronal glial cultures contain Thioflavin S positive structures. A) In cultures treated with AAV2/1 human wildand human wildinclusions were also ThS positive (green). This ThS staining profile (green) co localizes with the neuronal marker MAP2B (red, B) and the astrocyte marker GFAP (red, C). Cultures were counter stained with DAPI (blue). Bar scale = 50 m.

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62 Figure 2 7. Distinct morphological properties of inclusions in M83 (A53T) and M47 4 (red) or pSer129 (red) show a flamelike SNL 4 (red) or pSe r129 (green) show a roundlike compact morphology. Sections were counter stained with DAPI. Bar scale = 25 m.

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63 Figure 2 8. mutant specific and dominant morphological induction of aggregates. challenge. Cultures were transduced with AAV2/1 expressing wildtype, A53T, or E46K as indicated in each resp ective panel and described in Materials and Methods. Cultures were then treated with preformed fibrils comprised of wild type, A53T, or E46K as indicated in each respective panel. At one day post seeding (and four days post seeding for wildtype w ild type), cultures were fixed and stained with pSer129 antibody (green) to assess for differences in morphology. Cells were counterstained with DAPI (blue). Bar scale = 25 m. B) Quantification of morphological differences for tical differences in inclusion morphology among seeding combinations. Oneway ANOVA with Bonferroni correction for comparison of all groups to each other for flamelike M83 pathology reveals significant differences in inclusion morphology between all A53T seeded (**) cultures, but not with WT and A53T combinations (*). Wild type wild type combination (*) was more likely to result in M83like inclusion morphology; however, when wildtype fibrils were used to seed ei ther intracellularly expressed mutant proteins, the mutant protein predominantly determined inclusion morphology further indicating the dominant effect of the mutant proteins. p<0.01; n=6 fields/seeding combination. Values are means SD.

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64

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65 Figure 2 9. Three dimensional differences in morphology of inclusions. Confocal Z A) M83 like inclusions derived from AAV2/1mediated overexpression of 3T distribution throughout the cell body, in contrast, B) M47like inclusions derived from AA2/1of E46K Bar scale = 10 m.

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66 Figure 2 seeded inclusion formation in astrocyte cultures. Double immunofluorescence with SNL4 (red) and pSer129 (green): A) In untreated 4, mediated overexpression of human wildtype S increases both the cell body and processes staining intensit mediate d overexpression of human wildcell body and processes that overlaps with SNL4. Cultures were counterstained with DAPI (blue). Bar scale = 100 m; insets = 25 m.

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67 Figure 2 passaged from cell lysates. A) Astrocyte cultures with AAV2/1mediated overexpression of human wilde seeding, this resulted in pSer129 aggregates in both the cell body and processes, which overlapped with SNL4. Th is was compared to astrocyte cultures with AAV2/1 mediated overexpression of human wildlysates from astrocyte cultures treated with human A53T AAV2/1 mediated overexpression of human wilduntreated cultures D). In the latter 3 conditions, at five days post seeding there was with DAPI (blue). Bar scale = 100 m; insets = 25 m.

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68 CHAPTER 3 INDUCTION OF CNS SYNUCLEIN PATHOLOGY BY FIBRILLAR AND NONAMYLOIDOGENIC RECOMBINANT SYNUCLEIN Introduction A characteristic of Parkinsons disease, the most common neurodegenerative movement disorder, is the presenc e of intraneuronal Lewy bodies (LBs) in neurons. These inclusions are formed from the amyloidogenic aggregation of the normally inclusions also are present in a synucleinopathies (Cookson, 2005; Goedert, 1997; Waxman & Giasson, 2008a) neurodegeneration is supported by missense mutations or increase copy number of the SNCA ) in some patients with Parkinsons disease and the related disorder dementia with Lewy bodies (Farrer et al. 2004; Kiely et al. 2013; Kruger et al. 1998; Lesage et al. 2013; Polymeropoulos et al. 1997; Proukakis et al. 2013; Singleton et al. 2003; Zarranz et al. 2004) Despite a large number of experimental studies, the (Goldber g & Lansbury, 2000; Waxman & Giasson, 2008a) Synucleinopathies are progressive diseases and in recent years there have been increasing efforts to identify the mechanisms involved in intracerebral spread of pathology, as it is reasoned that therapies that could slow or halt pathology spread would likely be disease modifying. Recently, several ex perimental and pathological studies have suggested that spreading of S pathology might occur via a seeded conformational templating protein aggregation mechanism. For example, LB formation was observed in fetal dopaminergic neurons of a subset of PD patients that received

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69 striatal transplants as an attempted therapeutic intervention (Kordower et al. 2008; Li et al. 2008; Li et al. 2010) A seeding mechanism would also generally be consistent with the proposed Braak staging of disease that appears to follow neuroanatomical pathways (Braak et al. 2006a) Experimentally it was reported that the intracerebral injection o f extracts from moribund A53T human S transgenic (Tg) mice (line M83) that develop a late onset severe motor phenotype associated with widespread formation of neuronal S inclusions into younger healthy M83 Tg mice could induce these cellular and phenotypic pathologies (Giasson et al. 2002; Luk et al. 2012b; Mougenot et al. 2012) Furthermore, brain injection of pre formed recombinant S fibrils into M83 mice can also induce S pathology within brain regions that are distant from the injection site (Luk e t al. 2012b) suggesting that these S species can initiate and perhaps lead to transmission of S pathology. Induction of brain S pathology was also reported in nonTg (nTg) mice following intrastriatal injection of murine fibrillar S (Luk et al. 2012a) More recently it was reported that the injection of either preformed human or mouse S fibrils in the substantia nigra of nTg mice could also induce neuronal S pathology, but this pathology could only be observed 3 months or more after exposure (Masuda Suzukake et al. 2013) Collectively these studies along with numerous in vitro and conformational templating mechanism. However, this mechanism of pathology induction remains to be formally proven in vivo as other possible mechanisms could innate immune activation (Brundin et al. 2008; Golde et al. 20 13; Lema Tome et al. 2012; Sacino & Giasson, 2012)

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70 To further elucidate the mechanisms associated with the induction of intraneuronal S inclusion pathology resulting from exogenous S challenge and to evaluate a potential higher throughput experimental paradigm, we injected amyloidogenic and nonamyloidogenic forms of S into the brain of neonatal nTg and M20 Tg mice expressing wildtype human S. Neonatal injection is a significantly easier and faster surgical procedure than stereotactic injection in the adult brain, mainly because cryoanesthesia can be utilized and the skull is still soft and flexible. These studies reveal that neonatal cerebral injection of amyloidogenic S results in limited neuronal S inclusions in nontransgenic mice that are observed predominantly 8 months after injection. Similar studies in M20 Tg mice also revealed a lag time in the formation of detectable S pathology, but pathology was more widespread throu ghout the neuroaxis and was induced by the injection of both amyloidogenic and nonamyloidogenic forms of S. Materials and Methods Antibodies phosphorylated at Ser129 (Waxman & Giasson, 2008b) Syn211 and LB509 are mouse (Baba et al. 1998; Giasson et al. 2000b) SNL 1 is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 104(Giasson et al. 2000b) SNL 4 is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 2(Giasson et al. 2000b) Syn506 is a conformational anti pathological inclusions (Duda et al. 2002a; Waxman et al. 2008) Anti p62 (SQSTM1; Proteintech; Chicago, IL), anti glial fibrillary acidic protein (GFAP) (Promega; Madison,

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71 WI), and anti ionized calcium binding adaptor molecule 1 (IBA 1) (DAKO; Glostrio, Denmark) are rabbit polyclonal antibodies. An anti glyceraldehyde3 phosphate dehydrogenase (GAPDH) mouse monoclonal antibody was obtained from Biodesign (Memphis, TN). nTg mice and M20 S Tg mice All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. BL6C3HF1 mice (Charles River Laborat ories International Inc, Wilmington, MA) have the same strain S Tg mice (line M20) and were used as nTg mice. The M20 Tg mice express human wildtype S under the control of the mouse PrP promoter and these mice do not develop any intrinsic phenotype or S pathology (Emmer et al. 2011; Giasson et al. 2002) Hemizygous M20 male mice were mated with female BL6C3HF1 mice and genotyped by PCR, but also confirmed by immunohistochemical (IHC) staining of mouse brain section with anti S antibody Syn211. All animals were housed three to fiv e to a cage and maintained on ad libitum food and water with a 12 h light/dark cycle. Brain S injection into neonatal mice Bilateral neonatal (P0) injection of S proteins was performed by inserting the needle about 0.5cm deep into the brain just lateral to the lateral ventricles (see Figure 3 1) using cryoanesthesia as described previously (Chakrabarty et al. 2010) In brief, P0 pups were cryoanesthetized on ice for up to 5 minutes. Each pup rec S proteins. Injections were made using a 10L Hamilton syringe with a 30gauge needle. Different syringes were

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72 used for each type of protein to prevent any contamination. Post injection, the pups were placed on a heating pad for recovery before being returned to their home cage. The pRK172 cDNA constructions expressing full length huma ith amino acid 7182 deletion ( 7182), and N terminal truncated 21amino acid 21) were previously described (Giasson et al. 2001b; Waxman et al. 2009) exclusion (Superdex 200 gel filtration) and ion exchanged (Mono Q) chromatographies as previously described (Giasson et al. 2001b; Greenbaum et al. 2005) . 21protein was assembled into filaments by incubation at 37oC at 5 mg/mL in sterile phosphate buffered saline (PBS, Invitrogen) with continuous shaking at 1050 rpm as previously described with K114 fluorometry (Crystal et al. 2003; Waxman et al. 2009) hours. These fibrils were tested for induction of intracellular amyloid inclusion formation as previously described (Waxman & Giasson, 2010; Waxman & Giasson, 2011a) Immunohistochemical analysis Mice were sacrificed with CO2 euthanization and perfused with PBS/heparin, followed by perfusion with either 70% ethanol/150mM NaCl or PBS buffered formalin. The brain and spinal cord were then removed and fixed for at least 24 hours in the respective fixatives used for perfusion. As previously described, tissues were dehydrated at room temperature through a series of ethanol solutions, followed by xylene and then were infiltrated with paraffin at 60C (Duda et al.

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73 2000b) sections. Immunostaining of the sections was performed using previously described methods (Murphy et al. 2000) with the avidinbiotin complex (ABC) system (Vectastain ABC Elite Kit, Vector L aboratories, Burlingame, CA), and with immunocomplex visualization via chromogen 3,3 diaminobenzidine. Sections were counterstained with hematoxylin. All slides were scanned using an Aperio ScanScope CS (40 magnification; Aperio Technologies Inc., Vista, CA), and images of representative areas Aperio Technologies Inc.). Double Labeling Immunofluorescence Analysis of Mouse Brain Tissue Paraffin embedded tissue sections were dep araffinized and hydrated through a series of graded ethanol solutions followed by 0.1M Tris, pH 7.6. The sections were blocked with 5% dry milk/0.1M Tris, pH 7.6 and were incubated simultaneously with combinations of primary antibodies diluted in 5% dry m ilk/0.1M Tris, pH 7.6. After extensive washing, sections were incubated with secondary antibodies conjugated to Alexa 594 or Alexa 488 (Invitrogen, Eugene, OR). Sections were post fixed with formalin, incubated with S udan Black, and stained with 5 g/mL 4 , 6 diamindino2 phenylindole (DAPI). The sections were coverslipped with Fluoromount G (SouthernBiotech, Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera to capture images. Immunoblotting analysi s Mouse brains were lysed in 2% SDS/50 mM Tris pH 7.5 by sonication and heated to 1000C for 10 minutes. Protein concentration was quantified using the bicinchoninic acid (BCA) assay and bovine serum albumin as a

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74 standard (Pierce Bi otechnology, Rockford, I L). 15 g of total protein was resolved by SDS PAGE on 13% polyacrylamide gels, followed by electrophoretic transfer onto nitrocellulose membranes. Membranes were blocked in Tris buffered saline (TBS) with 5% dry milk, and incubated with primary antibodies which were followed by either goat anti mouse conjugated horseradish peroxidase (HRP) (Amersham Biosciences, Piscataway, NJ) or goat anti rabbit HRP (Cell Signaling Technology, Danvers, MA). Protein bands were detected using chemiluminescent reagent (NEN Boston, MA) and a FluorChem E and M Imager (Proteinsimple, San Jose, California). MALDI TOF mass spectrometry of full Recombinant human full 0.1% TFA (trifluoroacetic aci d) solution. 1 L diluted sample was mixed with 1L cyano 4 hydroxycinnamic acid) solution (acetonitrile: methanol = sample mixture was loaded to ACCA pretreated MSP 96 target (Bruker Daltonics Inc., Billerica, MA, USA). The sa mples were analyzed with a Bruker Microflex (Bruker Daltonics Inc., Billerica, MA, USA) mass spectrometer in linear positive model. Spectra were calibrated with Bruker protein calibrate standard. Results To further investigate induction of S inclusion formation following brain injection of exogenous S and to generate a higher throughput experimental model, we injected fibr ils comprised of 21 3 1). We use aminotruncated 21140 S, as fibrils comprised of this protein can seed S similarly to the fulllength protein in cultured cells (Luk et al. 2009; Sacino et al. 2013b; Waxman & Giasson, 2010; Waxman &

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75 Giasson, 2011a) and it provides the ability to definitively assess aggregation of the endogenous S by detection with aminoterminal specific S antibodies. The presence of the exogenous S (25 g injected) could be readily detected in the needle track 2 days post injection in nT g mouse brains using an antibody to human S (Figure 3 1). By 4 days post injection, exogenous S was not detectable, consistent with the findings recently reported by MasudaSuzukake and colleagues who also showed exogenous human S injected into the br ain was detectable only within the first 7 days post injection. We did not detect local or distal induction of intracellular pathology at 4, 8, and 16 days post injection of 25 g fibrillar S. Analysis of nTg mouse brains neonatally injected with 2 g of exogenous fibrillar 21140 S and aged up to 8 months did not reveal the presence of any S pathology (Table 3 1). Injection of 25 g of exogenous fibrillar 21 140 S resulted in rare pathology in only one mouse at 2 months post injection, but at 8 months post injection, 4 out of 13 nTg mice injected with this dose of S showed sparse S neuronal inclusion pathology primarily localized to cortical neurons (Table 3 1 and Figure 3 2). In n Tg cohorts injected with either 2 g or 25 g nonamyloidogenic 71 82 S (Giasson et al. 2001b; Luk et al. 2009; Sacino et al. 2013b; Waxman et al. 2009) we did not observe any pathology in nTg mice (see Table 3 1). In these st udies 7182 S was used as a control for conformational templating mechanisms, as we and others have extensively studied this protein and showed that it is deficient in the ability to form or directly affect (induce or inhibit) the formation of S amyloid fibrils in vitro or in culture models (Giasson et al. 2001b; Luk et al. 2009; Sacino et al. 2013b; Waxman et al. 2009; Zibaee et al. 2007) After prolonged incubations at high concentrations, 71 82 S can form oligomers as observed by

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76 negative staining electron micr oscopy, but these are not amyloidogenic in nature. The 7182 S used for the current studies was not preincubated and is in the soluble form as previously described. We have also recently shown that this same preparation of 7182 S cannot directly seed the formation of S inclusions in primary neuronal (Sacino et al. 2013b) In contrast the same preparation of fibrillar S can seed inclusion formation very efficiently in those cultures. inclusion pathology formation in vivo we performed neonatal brain injection of fibrillar 21140 S in M20 Tg mice, which overexpress wild type human S. In adult M20 Tg mice there is ~5fold over expression of human S in the brain, but these mice do not develop S pathology during their lifespan in the absenc e of additional manipulations (Emmer et al. 2011; Giasson et al. 2002) These mice als o overexpress transgenic human S during development that can be observed as early as P0 (Figure 3 3), and as previous ly reported, the expression of S increases during mouse brain development (Hsu et al. 1998) Thus, they make an ideal model to explore paradigms for i nduction of S pathology. The neonatal brain injection of 2 g fibrillar 21 140 S did not induce the formation of intraneuronal pathology at times up to 4 months, but by 8 months sparse cortical pathology could be observed (Table 3 2). Similar challenge to 7182 S did not result in the formation of pathology. Increasing the treat ment to 25 g fibrillar 21 140 S resulted in sparse brain S pathology as early as 1 month and 4 months, but it was extensively distributed throughout the neuroaxis by 8 months (Table 3 2, Figures 3 4 and 3 5) at a higher de nsity than in nTg mice showing S pathology at 8 months post injection of 25 g of fibrillar 21 140 S. Interestingly, S inclusions were rarely observed

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77 in nigral dopaminergic neurons (Figure 3 6). Similar to the S aggregates in symptomatic M83 Tg mice, which spontaneously develop agedependent pathology (Emmer et al. 2011; Giasson et al. 2002) the inclusions in M20 Tg mice induced by the brain injection of fibrillar 21140 S were comprised of endogenous ly expressed S as they were reactive with aminoterminal specific antibodies Syn506 and SNL 4 (Figures 3 7 and 3 8). The inclusions were also reacti ve with p62 (sequestrosome; Figure 3 9), a robust marker of S inclusions (Kuusisto et al. 2003) Unexpectedly, similar brain injection of 7182 S also resulted in robust and widely distributed S brain pathology at 8 months in some of the injected M20 Tg mice (Table 3 2; Figures 3 6, 3 7, 3 8, 3 9 and 3 10). These S inclusions were also comprised of endogenous S (i.e. reactive with antibodies S yn506 and SNL 4), hyperphosphorylated at Ser129, and accumulated p62. For comparison, we show that some of the M20 Tg mice 8 months post injection with 25 g of 7182 S are devoid of S pathology ( Figure 3 1 3 ). Although none of the nTg or the M20 Tg mi ce were extensively analyzed for behavioral changes, the presence of S pathological inclusions was not associated with any overt behavioral abnormalities. To assess if there was an association between neuroinflammation (astrogliosis or microgliosis) an d induction of S aggregation, tissue sections from all injected ntg and M20 Tg mice were stained with antibodies to GFAP and IBA 1. As expected, control untreated M20 Tg mice at 8 months of age showed basal levels o f astrocytes and microglia (Figure 3 11, A and H) (Emmer et al. 2011; Giasson et al. 2002) Most injected nTg and M20 Tg mice at 1, 2 or 4 month post injection did not display increased astrogliosis or microgliosis as shown preventatively for M20 Tg mice 2

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78 months post injection with 25 g fibrillar S (Figure 3 11, B and I). At these ages some of the M20 mice with or without brain S pathology also revealed some modest increase in astrogliosis. In 8 month old nTg mice injected with fibrillar S and with modest S pathology, only minimal induction of astrocytes and microglia was observed (Figure 3 11, C, D, J and K). Conversely, in 8 month old M20 mice with significant S pathology induc ed by neonatal injection of 25 g fibrillar S, robust astrogliosis and modest microgliosis were observed (Figure 3 11, E, L). Fur thermore, in 8 month ol d M20 mice injected with 25 g 71 82 S, there was also robust astrogliosis (Figure 3 11, F, G, M, and N) regardless of whether S pathology had developed. These findings indicate that treatment with fibrillar or nonamyloidogenic S can induce a delayed activation of neuroinflammation that is significantly accentuated in M20 mice relative to nTg mice. To evaluate the purity, biophysical properties, and integrity of the recombinant 7182 S, we performed MS analysis (Fig ure 3 12). We conf irmed by K114 fluorometry that 7182 S protein was not amyloidogenic as previously described (Waxman et al. 2009) and that the addition of exogenous 71 82 S could not induce S inclusion formation in cultured cells (Sacino et al. 2013b) Discussion Our studies demonstrate that the brain injection of exogenous S can induce intraneuronal S pathology after prolonged in cubation t imes. Within days the injected S is rapidly cleared and the inclusion pathology that aris es from endogenously expressed S takes months to form. These findings are consistent with those of

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79 MasudaSuzukake and colleagues who showed t hat exogenous ly injected human S fibrils (10 g) into the brains of nTg mice can be detected for less than 1 week, but induction of S pathology is observed 3 months later (MasudaSuzukake et al. 2013) All prior studies of intraneuronal induction of S by cerebral challenge to exogenous fibrils have been interpreted as being indicative of a prionlike spread of S pathology (Luk et al. 2012a; Luk et al. 2012b; MasudaSuzukake et al. 2013) I ndeed the delayed induction of S pathology by exogenous S observed here and by MasudaSuzukake et al. (Masuda Suzukake et al. 2013) may be interpreted as stable S seeds that are present below detectable levels and over time induce pathology, which then may spread via a cycle of inclusion pathology giving rise to additional nucleation events that can be spread from cell to cell. Our findings that an injection of a non amyloidogenic form of S ( 7182) can induce similar delayed pathology indicate that it may be premature to conclude that the pathology induced is solely attributable to conformational dependent templating events. In both the MasudaSuz ukake et al. (MasudaSuzukake et al. 2013) and Luk et al. (Luk et al. 2012a) studies using nTg mice, soluble S was injected as controls and no induction of pathology was reported. As we find that at a higher dose of S the re is more robust induction of S pathology both in terms of extent of pathology and time to onset of pathology induction, and that inject it is possible that the lower doses (5 10 (Luk et al. 2012a; MasudaSuzukake et al. 2013) may account for the lack of pathology inductio n reported. Notably, a soluble S control injection was not reported in the study demonstrating pathology inducti on in adult M83 S Tg mice (Luk et al. 2012b) Here

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80 using neonatal S Tg mice, we observed that injection of soluble 71 82 S is capable of inducing S path ology similar to amyloidogenic S. Ind uction of robust intraneuronal S pathology by exogenous 7182 S challenge does not appear to be attributable to the neonatal injection paradigm as we have observed similar findings in adult mice (Sacino et al. in preparation). The finding that M20 S Tg mice are more prone to inclusion formation resulting from treatment w ith either exogenous fibrillar S or soluble 7182 S is likely due to a dosage effect of S expression, which could be akin to patients with duplication or triplication of the SNCA gene. Although the cohorts of mice used here are not large, they are comparable to those used by others to study the induction of brain pathology using injected exogenous S (Luk et a l. 2012a; Luk et al. 2012b; MasudaSuzukake et al. 2013) Larger cohorts of mice inje cted with various dosages of S are currently being aged to longer time points to further understand the me chanisms involved in exogenous S induction of brain pathology. Circumstantial evidence from post pathology transplanted neurons in the brains of some PD patients has been used to support the to cell by a prionlike mechanism (Braak et al. 2003a; Braak et al. 2003b; Kordower et al. 2008; Li et al. 2008; Mendez et al. 2008) However, many alternative explanations including chronic neuroinflammation, oxidative stress triggered by excitotoxicity, and loss of homeostasis from cellular stress, may lead to the failure of molecular chaperones and other (Brundin et al. 2008; Golde et al. 2013; Lema Tome et al. 2012; Sacino & Giasson, 2012)

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81 Our data that soluble nonpathology raises questions regarding the prionlike spread of pathology that has been reported, but it is premature to conclude that our studies definitively refute that mechanism. It is plausible that in the brain a noninto amyloidogenic seeds through additional modifications or interactions with lipids or protein chaperones. Thus, studies that track t will be necessary to evaluate these possibilities. Alternatively, these studies do strongly suggest that other mechanism(s) of extensive r response via toll like receptor pathways akin to lipopolysaccharide activation (Alvarez Erviti et al. 2011; Beraud et al. 2011; Codolo et al. 2013; Couch et al. 2011; Fellner et al. 2013; Kim et al. 2013; Klegeris et al. 2006; Klegeris et al. 2008; Lee et al. 2009; Reynolds et al. 2008; Roodveldt et al. 2008; Su et al. 2008; Tansey & Goldberg, 2010; Zhang et al. 2005) and single intracerebellar or intraperitoneal injections of lipopolysaccharide have been shown to result in the longneuronal inclusion formation (Gao et al. 2008; Gao et al. 2011) Importantly, S lacking residues 7182 has been shown to induce inflammation similar to full length S (Klegeris et al. 2008; Lee et al. 2009) In our current study, we have observed a delayed, longterm activation of neuroinflammation induced by brain treatment to both soluble and fibrillar and S that is accentuated in M20 Tg mice compared to nTg. These findings are consistent with cell culture studies that showed th at both soluble and aggregated S are potent activators of inflammati on (Alvarez Erviti et al. 2011; Beraud

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82 et al. 2011; Codolo et al. 2013; Couch et al. 2011; Fellner et al. 2013; Kim et al. 2013; Klegeris et al. 2006; Klegeris et al. 2008; Lee et al. 2009; Reynolds et al. 2008; Roodveldt et al. 2008; Su et al. 2008; Tansey & Goldberg, 2010; Zhang et al. 2005) It is possible t hat the exogenous treatment to S may trigger a slow positive feedback loop of inflammation and secretion followed by aggregation that may require a certain threshold of inflammation that builds overtime. Therefore, some of the M20 Tg mice with neuroinflammation, but without S inclusion s 8 months after treatment with 7182 S may not yet have reach the nec essary threshold. Exogenous 7182 S may not be as potent an inducer o f this process as is fibrillar S, because it may have a shorter half life than aggregated S, which could explai n why did was not as potent as fibrillar S, but this possibility will be investigated in future studies. However, the hypothesis that inflammation may play an i mportant role in the spread of S pathology induced by exogenous S is only one of several poss ible mechanisms that may act synergistically or independently to promote the spread of S pathology (Brundin et al. 2008; Golde et al. 2013; Lema Tome et al. 2012; Sacino & Giasson, 2012) Furthermore, there is abundant evidence that prionoid self protein a ggregates represent what are referred to immunologically as Danger Associated Molecular Patterns (DAMPs) and are capable of inducing robust immune responses (Rubartelli & Lotze, 2007) A number of studies show that when prionoids as sociated with CNS proteinopathies are applied exogenously to glial cells they can activate innate immunity through pattern recognition receptors (PRR) and induce a proinflammatory response (Czirr & Wyss Coray, 2012; Golde & Miller, 2009; Rubartelli & Lotze, 2007; Salminen et al. 2009) This innate immune response in turn could trigger inclusion pathology.

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83 Notably, these mechanisms are not mutually exclusive and may be mutually self reinforcing (Golde et al. 2013) Our impression from these studies is that amyloidogenic immunogenicity, or some combination of these factors. Our recent studies in c ultured cell provide strong evidence that amyloidogenic conformational templating of S can readily occur under certain conditions (Sacino et al. 2013b) but the situation in vivo is likely more complex and aggregate formation can involve several mechanisms. To this point, treatment with 7182 S did not induce S aggregation in c ultured cells, while fibrillar S was able to readily do so. A limitation of studies in culture cells is the duration of time (a few weeks) that the cells can be maintained experimentally, which may not be efficient to study mechanisms that are slower and more progressive. Collectively these data indic ate that exposure to exogenous S can induce intracellular aggregate formation by at least 2 mechanisms that are not mutually exclusions and could likely be synergistic. Further studies will be needed to determine the relative contribution of prionlike protein self templating versus other mechanisms in the induction and propagation of S pathology. Notably, the neonatal injection paradigm that we have developed can accelerate these mechanis tic studies by reducing the time needed to establish cohorts of mice necessary to conduct those studies. As nonamyloidogenic S can induce S pathology simi lar to fibrillar amyloidogenic S, it is possible that any form of brain injury that promotes release of normal cellular S could trigger intraneuronal S pathology.

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84 Extracellular S release could also occur during neurodegeneration when neurons die and this could be exacerbated if the protein is not cleared rapidly. More definitive elucidation of the m in vivo and synucleinopathies.

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85 Figure 3 1 Detection of injected human S in the needle track 2 days post neonatal injection. IHC staining with human S specific antibody LB509 2 days after neonatal injection of 25 g 21 140 human S fibrils in nTg mice. Staining shows the presence of human S in the brain injection tract adjacent to the lateral ventricle (black arrows). The tissue section was counterstained with hematoxylin. Scale bar = 250 m

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86 Figure 32 Schematic summary of the predominant cortical distribution of S pathology in nTg mice 8 months after brain neonatal injection of fibrillar 21140 S. nTg mice injected with 25 g 21140 fibrillar S. Map shows rostral caudal distribution of S inclusions via coronal sections. Equivalent density and distribution of S pathology was seen bilaterally. Pathology was detected with antibodies pSer129 and Syn506. As shown in representative images, small, rounded perinuclear S inclusion and neuritic profiles were found sparsely dis tributed in the cortex. The distribution of inclusions was very similar in mice with pathology. Scale bar = 50 m

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87 Figure 33 Inc reased postnatal expression of S in the brain of nTg and M20 Tg mice. Total protein mouse brain extracts from P0, P2, P4, P8, P16, P30 and adult (3 month) nTg and M20 human S Tg mice was resolved on 13% SDS polyacrylamide gels and analyz ed by immunoblotting with anti S antibody SNL 1, which detects both human and mouse S or anti human S antibody Syn211. Immunoblotting with an anti GAPDH antibody was performed as a loading control. The mobility of molecular mass markers in indicated on the left.

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88 Figure 34 Schematic representation of the distribution of S pathology at 8 months following brain neonatal injection of 21140 human S fibrils or 7182 human S in M20 S Tg mice M20 Tg mice injected with 25 g of fibrillar 21 140 human S A) or 7182 human S B). Maps show rostral caudal distribution of S inclusions via coronal sections. Equivalent density and distribution of S pathology was seen bilaterally. Pathology was detected with antibodies pSer129 and Syn506. A) P0 injection of 21140 human S fibrils results in the formation of S inclusions throughout the cortex, hippocampus, midbrain, brainstem, and spinal cord. B) P0 injection of 7182 human S also results in widespread S inclusions. The distribution of inclusions was very similar in mice with pathology.

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89 Figure 35 Induction of S pathology throughout the neuroaxis 8 months after neonatal brain injec tion of 25 g fibrillar 21 140 S in M20 human S Tg mice. Tissue sections were stained with pSer129. Dystrophic neurites were diffusely spread throughout the brain and spinal cord. The more rounded, Lewy body like pathology was seen predominantly in the olfactory bulb (OB), motor cortex (MC), amygdala (AMY), dentate gyrus of the hippocampus (DG), thalamus (TH), hypothalamus (HYP), substantia nigra (SN), ventral pons (VP), and both the ventral and dorsal horns of the spinal cord (VH SC and DHSC). Lewy neurite like pathology extending into the cellular processes was more predominantly seen in the piriform cortex (PC), entorhinal cortex (EC), striatum (ST), corpus callosum (CC), and CA1 of the hippocampus (CA1). Tissue sections were counterstained with hematoxylin. Scale bar = 50 m (OB), 100 m (MC), 50 m (PC), 50 m (EC), 100 m (ST), 200 m (CC), 50 m (AMY), 50 m (DG), 50 m (CA1), 50 m (TH), 50 m (HYP), 50 m (SN), 50 m (VP), 200 m (VH SC), and 200 m (DH SC).

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90

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91 Figure 36 The majority of S inclusions in the substantia nigra of M20 Tg S neonatally injected with exogenous S are not in TH positive neurons. M20 Tg mice 8 months after neonatal injection with 21140 human S fibrils A) or 7182 human S B). Doublelabeled immunofluorescence analysis for tyrosine hydroxylase (TH; red) labeling the dopaminergic neurons in the substantia nigra area, and pSer129 (green) labeling the hyperphosphorylated S inclusions show minimal colocalizatio n. The majority of S inclusions were not found in TH+ cells, except for a few neurons (white arrows). Cell nuclei were counter stained with DAPI. Scale bar = 100 m

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92 Figure 37 IHC showing similar S pathology induced by neonatal brain injection of f ibrillar human 21140 S and 7182 human S in M20 Tg mice com pared to symptomatic M83 human S A53T Tg mouse. Brainstem tissue sections from a 15 monthold symptomatic M83 mouse A) and 8 monthold M20 mice neonatally injected in the brain with 25 g 21140 fibrillar S B) or 25 g 7182 C) show similar staining of S inclusions as detected with pSer129 by IHC. S inclusions are also detected with Syn506 and p62 antibodies. Syn506 is a mouse monoclonal antibody that conformationally detects S inclu sions; and p62 is a rabbit polyclonal antibody, which nonspecifically recognizes intracellular protein aggregates. Scale bar = 100 m

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93 Figure 38 Labeling of S inclusions in M20 Tg mice 8 months after PO brain injection of exogenous S with both am ino terminal S antibody SNL4 and pSer129. Doublelabeled immunofluorescence of midbrain with SNL4 (red) and pSer129 (green) shows that pSer129+ hyperphosphorylated S inclusions are SNL4+ in a symptomatic 15 monthold M83 mouse A) and 8 monthold M20 mice neonatally injected in the brain with 25 g 21 140 fibrillar S B) or 25 g 71 82 C). Cell nuclei were counter stained with DAPI. Scale bar = 100 m

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94 Figure 39 Co localization of p62 with S inclusions in 8 monthold M20 Tg mice following neonatal brain injection of exogenous S. Doublelabeled immunofluorescence analysis in the of midbrain region for p62 (red) and pSer129 (green) showing that most pSer129+ hyperphosphorylated S inclusions are p62+ in a symptomatic 15 monthold M83 mouse A) and 8 month old M20 mice neonatally injected in the brain with 25 g 21 140 fibrillar S B) or 25 g 71 82 C). Cell nuclei were counter stained with DAPI. Scale bar = 100 m

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95 Figure 310 Induction of S pathology throughout the neuroaxis 8 months after neonatal brain injection of 25 g 7182 S in M20 human S Tg mice. Tissue sections were stained with pSer129. Round perikaryal inclusions and dystrophic neurites were diffusely spread throughout the brain and spinal cord. The more rounded, Lewy body like pathology was seen predominantly in the medial preoptic area (MPA) striatum (ST), thalamus (TH), hypothalamus (HYP), substantia nigra (SN), and ventral pons (VP). Lewy neuritelike pathology extending into the cellular processes was more predominantly seen in the entorhinal cortex (EC), amygdala (AMY), and CA3 region o f the hippocampus (CA3). Tissue sections were counterstained with hematoxylin. Scale bars = 100 m (MPA), 50 m (EC), 100 m (ST), 100 m (AMY), 200 m (CA3), 50 m (TH), 100 m (HYP), 50 m (SN), and 100 m (VP)

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96 Figure 3 11 Delayed induction of astroglios is and microgliosis in mice neonatally injected with soluble 7182 S or fibrillar S at 8 months post injection. Tissue sections were stained with GFAP antibody (A G), which detects astrocytes, and Iba1 antibody (H N), which detects microglia. Representative images were taken of the entorhinal cortex, where a high density of S pathology tends to form due to neonatal injection (see Figs. 2, 4, 5 and 10). An 8 month old control untreated M20 Tg mouse (A, H) and a M20 mouse injected with 25 g of 21140 fibrillar S at 2 months post injection (B, I) show basal levels of astrocytes and microglia. There was no significant increase from basal levels in the brains of a nTg mouse with pathology (C, J) relative to a similar nTg mouse without pathology (D, K) at 8 months post injection of 25 g of 21 140 fibrillar S. Robust astrocyte and microglia activation as observed 8 months after injection in M20 Tg mice with brain S pathology treated with 25 g of 21 140 fibrillar S (E, L). In addition, robust astrogliosis was also observed in M20 Tg mice 8 months after injection of 25 g of 71 82 S with (F, M), or without (G, N) brain S pathology. Tissue sections were counterstained with hematoxylin. Scale bar = 50 m

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97

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98 Figure 3 12 Mass spectrometric analysis of the 71 82 S used for neonatal brain injection. To verify that 7182 S was the correct protein and its integrity, we performed mass spectrometry and compared the molecular mass to full length S. Up panel, recombinant full

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99 F igure 313 Lack of induction of S pathology throughout the neuroaxis 8 months after neonatal brain injection of 25 g 7182 S in M20 human S Tg mice. Tissue sections wer e stained with pSer129. Brain regions that typically showed Lewy body/neuritelike p athology after injection of 25 g 71 82 S in M20 human S Tg mice, were blank in an unaffected mouse: the medial preoptic area (MPA), striatum (ST), thalamus (TH), hypoth alamus (HYP), substantia nigra (SN), ventral pons (VP), entorhinal cortex (EC), amygdala (AMY), and CA3 region of the hippocampus (CA3). Tissue sections were counterstained with hematoxylin. Scale bars = 100 m (MPA), 50 m (EC), 100 m (ST), 100 m (AMY), 200 m (CA3), 50 m (TH), 100 m (HYP), 50 m (SN), and 100 m (VP)

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100 Table 3 1. Summary of neonatal nonTg mice injected with S proteins Mouse Strain Inoculum Age at Harvest Number of Mice Pathological findings C57BL6/C3H Fib S (2 L of 1 mg/mL ) 1 month 9 No inclusions C57BL6/C3H Fib S (2 L of 1 mg/mL ) 2 months 3 No inclusions C57BL6/C3H Fib S (2 L of 1 mg/mL ) 4 months 6 No inclusions C57BL6/C3H Fib S (2 L of 1 mg/mL ) 8 months 4 No inclusions C57BL6/C3H Fib S (5 L of 5 mg/mL ) 1 month 9 No inclusions C57BL6/C3H Fib S (5 L of 5 mg/m L ) 2 months 7 1 of 7 mice show rare inclusions** C57BL6/C3H Fib S (5 L of 5 mg/m L ) 4 months 3 No inclusions C57BL6/C3H Fib S (5 L of 5 mg/mL ) 8 months 13 4 of 13 mice show rare cortical inclusions*** C57BL6/C3H 71 82 S (2 L of 1 mg/mL ) 1 month 4 No inclusions C57BL6/C3H 71 82 S (2 L of 1 mg/mL ) 2 months 3 No inclusions C57BL6/C3H 71 82 S (2 L of 1 mg/mL ) 4 months 2 No inclusions C57BL6/C3H 71 82 S (5 L of 5 mg/mL ) 1 month 9 No inclusions C57BL6/C3H 71 82 S (5 L of 5 mg/mL ) 2 months 8 No inclusions C57BL6/C3H 71 82 S (5 L of 5 mg/mL ) 8 months 6 No inclusions Non Tg mice were injected with 21140 human S fibrils or 71 82 human S at the different dosages indicated and analyzed for S pathology at 18 months post injection using pSer129 and Syn506 antibodies. ** Sparse inclusions were observed in the midbrain area of 1 mouse. *** See Figure 3 2 for a schematic neuroanatomical map showing the distribution of S pathology.

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101 Table 3 2. Summary of neonatal M20 human S Tg mice injected with S proteins*. Mouse Strain Inoculum Age at Harvest Number of Mice Pathological findings M20 (WT S) Fib S (2 L of 1 mg/mL ) 1 month 7 No inclusions M20 (WT S) Fib S (2 L of 1 mg/mL ) 2 months 5 No inclusions M20 (WT S) Fib S (2 L of 1 mg/mL ) 4 months 3 No inclusions M20 (WT S) Fib S (2 L of 1 mg/mL ) 8 months 4 4 of 4 mice show sparse cortical pathology M20 (WT S) Fib S (5 L of 5 mg/mL ) 1 month 4 3 of 4 mice show sparse cortical pathology M20 (WT S) Fib S (5 L of 5 mg/mL ) 2 months 5 4 of 5 mice show sparse cortical pathology M20 (WT S) Fib S (5 L of 5 mg/mL ) 4 months 3 2 of 3 mice show sparse cortical pathology M20 (WT S) Fib S (5 L of 5 mg/mL ) 8 months 12 12 of 12 mice show abundant pathology** M20 (WT S) 71 82 S (2 L of 1 mg/mL ) 4 months 3 No inclusions M20 (WT S) 71 82 S (2 L of 1 mg/mL ) 8 months 3 No inclusions M20 (WT S) 71 82 S (5 L of 5 mg/mL ) 1 month 7 No inclusions M20 (WT S) 71 82 S (5 L of 5 mg/m L ) 2 months 7 No inclusions M20 (WT S) 71 82 S (5 L of 5 mg/mL ) 8 months 6 2 out of 6 mice show abundant pathology** M20 Tg mice were injected with 21140 human S fibrils or 71 82 human S at the different dosages indicated and analyzed for S pathology at 18 months post injection using pSer129 and Syn506 antibodies. ** See Figure 3 4 for a schematic neuroanatomical map showing the distribution of S pathology.

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102 CHAPTER 4 AMYLOIDOGENIC SYNUCLEIN SEEDS DO NOT INVARIABLY INDUCE RAPID, WIDESPEAD PATHOLOGY Introduction Synucleinopathies, a spectrum of neurodegenerative disorders, most notably Parkinsons disease (PD), are characterized by the presence of intracellular synuclein ( S) inclusions. These inclusions are formed from the amyloidogenic aggregation of the nor mally soluble presynaptic protein S (Coo kson, 2005; Goedert, 1997; Waxman & Giasson, 2008a) Mature S inclusions are also labeled with other markers of intracellular protein aggregates including ubiquitin and p62 (Kuusisto et al. 2003; Lowe et al. 1988) A direct causal role for S in neurodegeneration is supported by missense mutations or increased copy number of the S gene ( SNCA ) in patients with familial PD (Appel Cresswell et al. 2013; Farrer et al. 1999; Kiely et al. 2013; Kruger et al. 2000; Lesage et al. 2013; Polymeropoulos et al. 1997; Proukakis et al. 2013; Singleton et al. 2003; Zarranz et al. 2004) Synucleinopathies are progr essive neurodegenerative disorders and several recent experimental and pathologic studies have been interpreted as supportive of a prion like spread mechanism where amyloidogenic S serves as a template to drive the conversion of soluble, natively unfolded S to a conformationally altered, aggregated form which transmits from cell to cell (Guo et al. 2013; Jucker & Walker, 2013; Kordower et al. 2008; Li et al. 2008; Li et al. 2010; Luk et al. 2012a; Luk et al. 2012b; Polymenidou & Cleveland, 2012; Sacino et al. 2013b; Volpicelli Daley et al. 2011) For ex ample, in post mortem studies of PD patients who had received therapeutic striatal transplants of fetal dopaminergic neurons, some of these neurons

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103 subsequently developed S pathology (Kordower et al. 2008; Li et al. 2008; Li et al. 2010) but as pointed out by Brundin and colleagues, many other mechanisms could explain this phenomenon (Brundin et al. 2008) Studies of stereotactic brain injections of amyloidogenic human S into nave M83 (A53T) S transgenic (Tg) mice (Luk et al. 2012b) as well as amyloidogenic murine S into nontransgenic (nTg)/native mice (Luk et al. 2012a) have been reported to induce S pathology at the injection site that also spreads following neuroanatomical pathways along white matter tracts to induce S pathology at distal sites. Such prionlike spread is also consistent with the proposed Braak staging of PD that to some degree follows neuroanatomical pathways (Braak et al. 2006a) Additionally, in cell culture studies, it was reported that the addition of preformed exogenous S amyloid seeds could induce intracellular S aggregates in nave primary mouse neuronal cultures (Dryanovski et al. 2013; Guo et al. 2013; VolpicelliDaley et al. 2011) Many of the previous studies reporting exogenous S seeding intracellular inclusion pathology, both in culture and in vivo have relied heavily on pSer129 immunoreactivity as the primary measure of pathology (Beach et al. 2010; Dryanovski et al. 2013; Guo et al. 2013; Luk et al. 2012a; Luk et al. 2012b; MasudaSuzukake et al. 2013; Mu et al. 2013; Volpicelli Daley et al. 2011) Recently, we have demonstrated that there is robust pS er129 S immunoreactivity, using antibody 81A, within neuritic processes in neuronal cultures from SNCA / mice (Sacino et al. 2013b) suggesting that staining with this antibody could have led to significant misinterpretation of data; herein, we experimentally identified the major nonS target of antibody 81A as the low molecular mass neurofilament subunit (NFL) specifically phosphorylated at

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104 Ser473. We show that native NFL staining and NFL proteinopathy can readily be misinterpreted as S pathology using pSer129/81A immunostaining. Therefore, we also tried to replicate the induction of S brain pathology via intracerebral injection of exogenous S fibrils and show that this challenge can induce significant S pathology in M83 Tg mice that express A53T human S, but in nTg mice and even other S Tg mice, induced S inclusions are primarily restricted to the injection site. These data indicate that there are significant barriers to widespread induction of pathology. Materials and Methods Ex The pRK172 cDNA with amino acid 7182 deleted ( 71 82), N terminal truncated wildtype 21and full length muri were previously described (Giasson et al. 2001b; Waxman & Giasson, 2010; Waxman & Giasson, 2011b; Waxman et al. 2009) E. coli BL21 (DE3) and purified to homogeneity as previously described (Giasson et al. 2001b; Greenbaum et al. 2005) . 21140 S, and A53T or E46K human S proteins were assembled into filaments by incubation at 37oC at 5 mg/mL in sterile phosphate buffered saline (PBS, Invitrogen) with continuous shaking at 1050 rpm (Thermomixer R, Eppendorf, Westbury, mbly was monitored as previously described with K114 fluorometry (Crystal et al. 2003; Waxman et al. 2009) S fibrils were diluted to a concentration of 12 mg/mL in sterile PBS and treated by mild water bath sonication for

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105 2 hours. These fibrils were tested for robust induction of intracellular amyloid inclusion format ion as previously described (Sacino et al. 2013b; Waxman & Giasson, 2010; Waxman & Giasson, 2011a) Mice husbandry and stereotactic injections. All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee or the Canadian Council on Animal Care and approved by the Faculty of Medicine Animal Care Committee at the University of Toronto, Canada. M47 and M83 Tg mice expressing human S with the E46K mutation or the A53T mutation, respectively, were previously described (Emmer et al. 2011; Giasson et al. 2002) SNCA / mice (Abeliovich et al. 2000) were obtained from The Jackson Laboratory (Bar Harbor, MA). NFHLacZ Tg mice that accumulate widespread brain NF i nclusions were previously described (Eyer & Peterson, 1994; Tu et al. 1997a) NFL null mice (Zhu et al. 1997) were a kind gift from Dr. JeanPierre Julien (Laval University, Canada) and maintained on a C57BL/6J background. nTg, SNCA / and M83 and M47 Tg mice at 2 months of age were bilaterally stereotaxically injected with 2 L of 1mg/mL S proteins or PBS in the hippocampus (coordinates from Bregma: A/P 1.7, L +/ 1.6, D/V 2.0). The inoculum was injected at a rate of 0.2 L per min (total volume of 2 L per hemisphere) with the needle in place for 15 min at each site. Mice n > 4 for all cohorts. Antibodies pSer129, also known as clone 81A, is a mouse monoclonal antibody (Waxman et al. 2008) SNL 4 and SNL1 are rabbit polyclonal antibodies raised against synthetic peptides corresponding to amino acids 212 and 104(Giasson et al. 2000b) Syn506 is an

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106 anti S mouse monoclonal antibody that recognizes the N terminus of S (Duda et al. 2002a; Waxman et al. 2008) PHF1 (generously provided by Dr. Peter Davies) is specific toward phosphorylation sites Ser396 and Ser404 in tau (Otvos et a l. 1994) AT100 (ThermoFisher) is specific toward tau phosphorylated at Ser212 and Thr214 (ZhengFischhofer et al. 1998) Anti p62 (SQSTM1; Proteintech; Chicago, IL) is a rabbit polyclonal antibody. Mouse anti actin (clone C4) monoclonal antibody reacts with all forms of vertebrate actin (Millipore, Billerica, MA). NR4 is a mouse monoclonal antibody specific for NFL (Sigma Aldrich). Chicken and rabbit polyclonal antibodies for NFL were generously provided by Dr. Gerry Shaw (Encor Biotechnology Inc.). Immunohistochemical analysis Mice were sacrificed with CO2 euthanization and perfused with PBS/heparin, followed by perf usion with 70% ethanol/150mM NaCl. The brain and spinal cord were then removed and fixed for at least 24 hours. Tissues were dehydrated and infiltrated with paraffin as previously described (Duda et al. 2000b) tions. Immunostaining of the sections was performed using previously described methods (Duda et al. 2000b) using avidinbiotin complex (ABC) system (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA) and immunocomplexes were visuali zed with the chromogen 3,3 diaminobenzidine. Sections were counterstained with hematoxylin. All slides were scanned using an Aperio ScanScope CS (40 magnification; Aperio Technologies Inc., Vista, CA) and images of representative areas of S pathology w ere taken using the ImageScopeTM software (40 magnification; Aperio Technologies Inc.). Double labeling immunofluorescence analysis of mouse brain tissue Paraffin embedded tissue sections were deparaffinized and hydrated through a series

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107 of graded ethanol solutions followed by 0.1M Tris, pH 7.6. The sections were blocked with 5% dry milk/0.1M Tris, pH 7.6 and were incubated simultaneously with combinations of primary antibodies diluted in 5% dry milk/0.1M Tris, pH 7.6. After extensive washing, sections w ere incubated with secondary antibodies conjugated to Alexa 594 or Alexa 488 (Invitrogen, Eugene, OR). Sections were post fixed with formalin, incubated with Sudan Black, and stained with 4',6diamidino2 phenylindole (DAPI)(Invitrogen, Eugene, OR). The sections were coverslipped with Fluoromount G (SouthernBiotech, Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera to capture images. Biochemical fractionation and immunoblotting analysis of mouse br ain tissue. Cortex and brainstem/spinal cord from 4 monthold nTg, SNCA / and NFL/ mice were biochemically fractionated as previously described (Giasson et al. 2002) Briefly, tissue samples were dissected, weighted, and homogenized in 3mL/g of highsalt (HS) buffer (50mM Tris, pH 7.5, 750mM NaCl, 20mM NaF, 5mM EDTA) with protease inhibitors and sedimented at 100,000 x g for 20 min. Pellets were reextracted in HS buffer, followed by subsequent extractions in HS/1% Triton X 100, HS/1M sucrose, RIPA buffe r (50mM Tris, pH 8.0, 150mM NaCl, 5mM EDTA, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS with the protease inhibitors), and 2%SDS/4M urea. SDS/urea fractions were then sonicated. 2% SDS was added to all the fractions, which were heated to 100C for 10 minutes, except for those fractions containing urea. For some experiments the RIPA homogenate was separated in half and the subsequent pellets were resuspended in 4M urea or 2% SDS by sonication. Protein concentrations

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108 were quantified using the bicinchoninic acid (BCA) assay and bovine serum albumin as a standard (Pierce Biotechnology, Rockford, IL). SDS PAGE and immunoblotting analysis Protein was resolved by SDS PAGE on 7% (w/v), 8% or 13% polyacrylamide gels, as indicated, followed by electrophoretic transfer onto nitrocellulose membranes. Some gels were directly stained with Coomassie R 250 or transferred by electrophoresis onto nitrocellulose membranes. For Western blotting, membranes were blocked in Tris buffered saline (TBS) with 5% dry mi lk, and incubated with primary antibodies in TBS/5% dry milk or TBS/5% BSA for pSer129/81A overnight. Following washes, membranes were incubated with a goat anti mouse antibody conjugated to horseradish peroxidase (HRP) or a goat anti rabbit antibody conjugated to HRP. Protein bands were detected using chemiluminescent reagent (NEN, Boston, MA) and a FluorChem E and M Imager (Proteinsimple, San Jose, California). In situ nitrocellulose protein dephosphorylation. For dephosphorylation of protein directly on the membrane, following transfer from SDS polyacrylamide gel, the membrane was blocked with 5% BSA in TBS overnight. The membrane was then rinsed in phosphatase buffer (50mM Tris, pH 9.2, 0.5mM MgCl2) and incubated in phosphatase buffer with or without 250U/mL bovine intestinal mucosa alkaline phosphatase (SigmaAldrich) overnight. The membranes were rinsed with TBS and blocked for 1 hour with 5% skimmed milk in TBS and processed for immunoblotting as described above. Immunoprecipitation. pSer129/81A antibody was preabsorbed with protein A/G agarose beads (Santa Cruz Biotechnology, Inc) and exchanged in CSK buffer

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109 (50mM Tris, 150mM NaCl, pH 7.4, 20mM NaF, 1mM EDTA, 1% Triton X 100). The SDS fraction from human cortical white matter was diluted in 20 volumes of CSK buffer with protease inhibitors and incubated with the pSer129/81A protein A/G agarose beads overnight at 4oC with rotation. After extensive washing with CSK buffer, the immunoprecipitated complex was eluted with SDS sample buffer and heating to 100oC for 5 minutes. Expression, purification, and phosphorylation of murine NFL from bacteria Recombinant NFL was expressed in Escherichia coli BL21 (DE3) using mouse NFL cDNA cloned into the pET 23d expression vector (Novagen, Inc. Madison, WI). The same construct with the Ser473Ala mutation was generated using the QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and specific oligonucleotides. Following transformation, bacteria were grown to an OD600 of 0.6 and the expression of the r ecombinant protein was induced with 1mM isopropyl d thiogalactopyranoside for 2 hours. NFL was purified using inclusion bodies procedure. Cells were pelleted, resuspended into lysis buffer (25% sucrose, 50mM Tris, pH 8.0, 1mM EDTA, 2mg/mL lysozyme, and a cocktail of protease inhibitors) and incubated on ice for 30 minutes. Ten mM MgCl2 to the homogenate, which was incubated on ice for another 30 minutes. Ten mL of detergent buffer (0.2M NaCl, 1% deoxycholic ac id, 1% IGEPAL CA630, 20mM Tris, pH 8.0, 2mM EDTA) per mL of lysis buffer were added and, after vigorous mixing, the insoluble material was sedimented at 5,000 g for 30 minutes. The supernatant was discarded and the pellet was repeatedly washed with 1mM E DTA/0.5% Triton X 100 to generate a highly compact pellet, which was solubilized in 8M urea, 10mM Tris, pH 8.0,

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110 1mM EDTA. Protein concentration was determined using Bradford assay (ThermoFisher) and bovine serum albumin as the standard. Recombinant NFL (1 mg/mL) was dialyzed overnight in 20mM Tris pH 7.5, 50mM KCl. Kinase reaction was performed by adding 10mM MgCl2, 200M ATP and 10000U casein kinase II/mL (control reaction had no kinase added). Reactions were incubated at 30oC for 1 hour. Sample buffer was added to the reactions and incubated at 100oC for 10 minutes. Mixed neuronal glial primary cultures Primary cultures were prepared from P0 C3HBL/6 mouse brains (Harlan Labs). Cerebral cortices were dissected from P0 mouse brains and dissociated in 2mg/ mL papain (Worthington) and 50 g/mL DNAase I (Sigma) in sterile Hanks Balanced Salt Solution (HBSS, Life Technologies) at 37C for 20 minutes. They were then washed three times in sterile HBSS to inactivate the papain and switched to 5% fetal bovine seru m (HyClone) in Neurobasal A growth media (Gibco), which includes 0.5mM Lglutamine (Gibco), 0.5mM GlutaMax (Life Technologies), 0.01% antibiotic antimycotic (Gibco), and 0.02% SM1 supplement (Stemcell). The tissue mixture was then triturated three times using a 5 mL pipette followed by a Pasteur pipette, and strained through a 70 m cell strainer. The cell mixture was then centrifuged at 200 g for 3 minutes, and resuspended in fresh Neurobasal A media. They were then plated onto poly D lysine coated c hamber slides (Life Technologies) or dishes at around 100,000200,000 cells/cm2. Cells were maintained in the Neurobasal A growth media without fetal bovine serum at 37C in a humidified 5% CO2 chamber.

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1 11 Results Pathological changes following intrahippocampal injection of preformed S fibrils in M83 Tg mice Recent studies have shown robust induction of widespread CNS S pathology using M83 Tg mice challenged with adult brain injection of human fibrillar (hfib) S (Luk et al. 2012b) M83 Tg mice intrinsically develop a severe motor phenotype associated with flamelike S inclusions starting at 7 months of age. Inclusion pathology occurs throughout the neuroaxis, but largely spares the hippocampus, striatum, and the cortex (Giasson et al. 2002) To limit confounds from intrinsic pa thology formation, intrahippocampal injection of 21140 hfib S was performed in young 2 monthold M83 Tg mice which were analyzed 2 months later, a time when no intrinsic pathology is observed. We injected N terminally truncated protein 21140 S, as we and others previously have shown that it can seed S inclusions in cultured cells as efficiently as full length S (Luk et al. 2009; Sacino et al. 2013b; Waxman & Giasson, 2010; Waxman & Giasson, 2011a) and it enables monitoring of the aggregation of endogenous S with Nterminal specific antibodies such as SNL4 and Syn506 (Duda et al. 2002a; Giasson et al. 2000b; Waxman et al. 2008) 21 140 hfib S intrahippocampal injection resulted in widespread formation of S inclusion formation that was found in all the regions where intrinsic S pathology would form. Abundant inclusions were also noted in areas where inclusion formation does not intrinsically occur in these mice including the hippocampus, where the protein was injected, and the striatum and cortex, though the striatal/cortical inclusions were less frequent (Figure s 4 1, 4 10, 411, 412). Intrahippocampal injection of PBS resulted in no S pathology formation, while injection of 7182 S, which has a deletion in the

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112 middle of the hydrophobic region of S that is required for amyloid formation and therefore lacks the ability to form or seed S amyloid in vitro and in cultured cells (Giasson et al. 2001b; Luk et al. 2009; Sacino et al. 2013b; Waxman et al. 2009) resulted in sparse altered pSer129 staining that rese mbles dystrophic synaptic terminals and perikaryal cell bodies at the site of injection ( Fig ure 4 13 ; data not shown). Similar to the S inclusions seen in symptomatic M83 Tg mice (Figure 4 1B), those seen post injection of 21140 hfib S are predominantly flame like, filling the somatodendritic compartment, and are robustly recognized by the S antibody Syn506 and by an antibody to p62, which is specific for inclusion formation (Kuusisto et al. 2003) (Fig ure 4 1 ; Fig ures 4 1 0, 4 11, 4 12). Using multiple S antibodies, we were unable to observe robust evidence of inclusion pathology within brain white matter tracts as previously reported (Luk et al. 2012b) The vast majority of the white matter staining by pSer129/81A antibody was not react ive with other S antibodies (Figure 4 2). This difference is significant as it was used to support the concept of axonal spread of inclusion pathology from the site of injection (Luk et al. 2012b) Only sparse S inclusions that could be labeled with other S antibodies were observed in the brain white matter tracts of h fib S injected M83 Tg mice. pSer129/81A readily labeled S inclusions as shown by double labeling; however, it did not stain areas abundant in normal S such as the neuropil areas of the cerebellum (Figure 4 2), consistent with previous findings that S i s only modestly phosphorylated at Ser129 under normal conditions but hyperphosphorylated in aggregates (Anderson et al. 2006; Fujiwara et al. 2002; Waxman & Giasson, 2008b) However, the intense pSer129/81A white matter tract staining was still present

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113 throughout the CNS in SNCA / mice as shown by immunofluorescence microscopy and IHC (Fig ure 4 2, 4 14). This finding is consistent with our recent studies that also revealed robust pSer129/81A staining of cultured neuronal processes from SNCA / mice (Sacino et al. 2013b) indicating that this staining is due to cross reactivity of the pSer129/8 1A antibody with a nonS target. Identification of the major axonal pSer129/81A crossreactive protein as NFL phosphorylated at Ser473 To identify the nonS pSer129 target, we first performed biochemical fractionations from CNS areas enriched in grey matter (cortex) and white matter (spinal cord and brain stem) from nTg and SNCA / mice. Western blot analysis of these fractions demonstrated that S is predominantly present in the HS fractions, and the presence of modestly pSer129 phosphorylated S in the HS soluble fraction of only nTg mice ( Figur e 4 15 ). This is consistent with previous characterizations of normal S protein demonstrating that it is largely soluble and only modestly phosphorylated at Ser129 (Anderson et al. 2006; Davidson et al. 1998; Fujiwara et al. 2002; Waxman & Giasson, 2008b; Weinreb et al. 1996) As previously reported, endogenous S is also more highly expressed in the cortex than the spinal cord (Giasso n et al. 2001a; Giasson et al. 2002) Importantly, this analysis demonstrates that pSer129/81A also strongly reacts with a 70 kDa non S target that is still present in SNCA / mouse brain and enriched in the white matter. Biochemically, this protein is enriched in the SDS urea fraction, but is also present in lesser amounts as a soluble form in the HS fraction ( Fig ure 4 15). This fin ding is also consistent with our previous characterization of this antibody where we showed that it can cross react with a detergent insoluble 70 kDa nonS target enriched in white matter brain tissue,

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114 like cerebellar white matter (Waxman & Giasson, 2008b) and we hypothesized that the reactivity with this protein was largely responsible for the intense pSer129/81A staining observed in neuronal processes still present in SNCA / mice (Sacino et al. 2013b) We found that the ~70 kDa pSer129/81A reactive protein was soluble in either 4M urea or 2% SDS and that it was similarly abundant in white matter from human cortex or cerebellum (Figure 4 3A). Compared to total protein Coomassie stained gel, we found that the pSer129/81A reactive 70 kDa band aligned with the low molecular mass neurofilament subunit (NFL) (Figure 4 3A). To ascertain that pSer129/81A could be reacting with NFL, we performed immunoprecipitation (IP) assays with pSer129/81A and showed by immunoblotting that pSer129/81A antib ody could immunoprecipitate NFL from both human and mouse white matter (Figure 4 3B). We then performed dephosphorylation studies to show that the reactivity of the pSer129/81A antibody with the 70 kDa protein band was completely dependent on phosphorylat ion (Fig ure 4 3C). To confirm that the 70 kDa protein recognized by pSer129/81A antibody was NFL, we analyzed extracts from NFL/ mice and showed that it was completely absent in tissue from NFL / mice (Fig ure 4 3D). To further confirm that pSer129/81A was reacting with phosphorylated NFL we purified recombinant NFL from bacteria and phosphorylated the protein with casein kinase II in vitro We found that the pSer129/81A antibody only reacted with recombinant NFL phos phorylated in vitro (Fig ure 4 3E). The major in vivo phosphorylation site in NFL is Ser473 (normally modified at ~73%), which corresponds to Ser472 in bovine NFL (Trimpin et al. 2004; Xu et al. 1990) Like the Ser129 phosphorylation site in S, this site in NFL is also an excellent substrate for casein kinase II (Ishii et al. 2007; Nakamura et al. 1999; Waxman & Giasson, 2008b) and

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115 analysis of the sequence revealed homology between each site: DEPPpSEGEAE in NFL versus AYEMPpSEEGYQ in S (the sequence used to make the pSer129/81A antibody) (Waxman & Giasson, 2008b) Furthermore, the amino acid residues surrounding Ser473 in NFL is conserved across all species (Geisler et al. 1985; Julien et al. 1987; Julien JP, 1986; Trimpin et al. 2004; Xu et al. 1990) Therefore, we generated recombinant NFL with the Ser473Ala mutation and showed that this mutation ablated the reactivity of pSer129/81A with NFL incubated with casein kinase II (Fig ure 4 3E). Collectively, these studies show that the major ~70 kDa protein recognized by pSer129/81A antibody is NFL phosphorylated at Ser473. Analysis of pSer129/81A detection of phosphorylated NFL in white matter t racts of nervous tissue. Since it has been previously suggested that S pathology spreads from the site of intracerebral injection of fibrillar S via white matter tracts based on pSer129/81A staining (Luk et al. 2012a; Luk et al. 2012b) we investigated the extent to which pSer129/81A detects phosphorylated NFL in the white matter tracts of nTg mice. In untreated nTg mice, IHC can readily detect NFL in the white matter tracts of the corpus callosum, hippocampus, striatum, brain stem, and cerebellum (Fig ure 4 4). IHC staining with pSer129/81A shows a similar neuroanatomical staining pattern of white matter tracts as NFL antibody staining. Conversely, IHC with anti S antibody Syn506 does not stain white matter tracts since S is normally a presynaptic protein (George et al. 1995; Iwai et al. 1995a; Jakes et al. 1994) (Figure 4 4). Immunofluor escent analysis also demonstrated that pSer129/81A immunoreactivity colocalizes with phosphorylated NFL in the majority of white matter tracts and also in a subset of cell bodies ( Figure 4 16 ). For comparison, we stained nervous tissue from

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116 NFL / mice and show that the loss of the NFL staining pattern is accompanied by a similar loss of white matter pSer129/81A staining, but the pSer129/81A and NFL staining of white matter tracts are unaltered in SNCA / mice ( Fig ure 4 16). This data indicates that pSer129/81A can readily detect phosphorylated NFL in white matter tracts and some cell bodies. To further investigate if pSer129/81A could react with inclusions comprised of neurofilaments (NFs), we then stained the nervous tissue from NFH LacZ Tg mice, wh ich develop intracellular NF inclusions throughout the brain and brain stem (Eyer & Peterson, 1994; Tu et al. 1997b) We found that pSer129/81A strongly detects the Lewy body like perikaryal NF inclusions and these inclusions do not contain S, as they are not detected by the S antibo dy SNL 4 (Fig ure 4 5). The NF inclusions in NFHLacZ Tg mice were also not stained with more than 10 other S antibodies. Expanding upon our findings in murine nervous tissue, we next tested the specificity of pSer129/81A staining in human nervous tissue and in murine primary mixed neuronal cultures. In nervous tissue from a control human subject, we found that pSer129/81A also stains phosphorylated NFL in the white matter tracts in the absence of accumulated endogenous S (Fig ure 4 6). Furthermore, we also show that similar to staining in mouse CNS tissue, anti pSer129/81A can robustly cross react with the nonS target in white matter tracts in human nervous system tissue that can be easy misinterpreted as S pathology ( Fig ure 4 17). We and others have used primary neuronal cultures as in vitro modeling systems for S pathology (Luk et al. 2009; Sacino et al. 2013b) W e have previously shown in mixed neuronal cultures from untreated nTg and SNCA / mice that pSer129/81A

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117 antibody demonstrated robust cross reactivity with an unknown nonS target in the cell bodies and processes (Sacino et al. 2013b) We show that in these cultures the pSer129/81A antibody can completely colocalize with the NFL profile indicating that the maturity of the pSer129 staining in native neuronal cultures is due to reactivity with Ser473 phosphorylated S (Fig ure 4 7). Analyses of intrahippocampal injection of preformed S fibrils in nTg mice To further study the pathological changes resulting from cerebral challenge of exogenous preformed S fibrils, we performed similar intrahippocampal injection of 21140 hfib S in nTg mice. This treatment resulted in the accumulation of neuronal pSer129 immunostaining localized to the site of injection with very limited pathology in the cortex (Figure 4 8). Intrahippocampal injections also resulted in the formation of some S inclusions that could be detected with Syn506 and p62 antibodies (Figure 4 8). Injection of 71 82 S resulted in pSer129/81A accumulations that resemble dilated synaptic ter minals as shown in Figure 413, but these were not detectable with other S antibodies. pSer129, Syn506, and p62 immunoreactive perikaryal and neuritic inc lusions in the hippocampus of nTg mice challenged with 21140 hfib S persisted for up to 4 months, although with decreasing abundance (Fig ure 4 8). During this time period there was no evidence of spread beyond the initial sites of pathology. Altered pSer 129/81A staining was not detected in nTg mice challenged with 7182 S beyond 1 month. The limited level of S pathology induced after intrahippocampal injection of 21140 hfib S caused us to examine whether there is a cross species barrier inhibiting in clusion formation. We performed intrahippocampal injection of full length mouse

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118 fibrillar (mfib) S. At 2 months post injection of mfib S, there were S inclusions readily detected with pSer129, Syn506, and p62 antibodies at the site of injection, and t he amount of pathology was more robust than for 21140 hfib S (Fig ure 4 8); however, there was no difference in the distribution pattern of pathology. The more robust induction of local pathology by mfib S could simply be due to the fact that mouse S i ntrinsically have the A53T substitution that promotes S aggregation and seeding (Conway et al. 1998; Giasson et al. 1999; Rochet et al. 2000) Therefore, our findings are consistent with those recently reporting that mfib S can induce the formation of some S inclusions in nTg mice (Luk et al. 2012b; MasudaSuzukake et al. 2013) ; however, S pathology in our studies was highly localized to the site of injection. Analyses of intrahippocampal injection of preformed S fibrils in M47 Tg mi ce. Since we observed rapid and widespread induction of S pathology in M83 Tg mice, but not in nTg mice, we next examined induction of S inclusion formation following stereotactic brain injection of fib S in a Tg mouse model expressing human S with the E46K missense mutation (line M47). These mice typically develop Lewy body like S inclusions with a distribution similar to M83 Tg mice but not before 15 months of age (Emmer et al. 2011) At 4 months post hippocampal injection of hfib S S inclusions were predominantly found at the site of injection in the hippocampus, with modest amounts in the cortex, midbrain, and brainstem (Figure 4 9A). The rounded, perinuclear and neuritic inclusions similar to those seen in symptomatic M47 Tg mi ce were also detectable by Syn506 and p62 (Fig ure 4 9B). Because some p62labeled

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119 inclusions were not reactive with S antibodies (Figure 4 9C; lower panel) and these mice can develop tau inclusions later in age (Emmer et al. 2011) we examined whether tau pathology was induced. We found that hippocampal injection of hfib S resulted in the formation of endogenous tau aggregation readily detectable with phosphotau antibodies PHF 1 and AT100 (Otvos et al. 1994; ZhengFischhofer et al. 1998) A significant percentage of tau aggregates were also reactive for p62 ( Fig ure 4 18). We further investigated whether S fibrils with pathogenic mutants such as A53T or E46K could be more potent at inducing S pathology in M47 Tg mice. The hippocampal injection of fibrils comprised of E46K S in M47 Tg mice also recapitulates the condition where the same protein is injected as is expressed, alleviating concerns of crossspecies differences. The location and formatio n of S pathology in M47 Tg mice injected with A53T or E46K S fibs was similar to the mice injected with the 21140 hfib S ( Fig ure s 4 19, 420 ). Discussion Our studies demonstrate that direct intracerebral injection of fib S can induce S inclusion pathology in both S Tg and nTg mice. These studies confirm this primary observation reported in several recent studies (Luk et al. 2012a; Luk et al. 2012b; MasudaSuzukake et al. 2013) However, a number of observations in our current study challenge the interpretation from these previous studies that direct intracerebral injection of S results primarily in progressive prionlike spread of the pathology. pSer129 immunostaining has been used as a major method to assess the presence of, and to infer the spread of, S pathology in these recent mouse studies and in human studies (Beach et al. 2010; Luk et al. 2012a; Luk et al. 2012b; MasudaSuzukake et

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120 al. 2013; Mu et al. 2013) Although anti pSer129 reactivity is a sensitive marker of S pathology because S is normally only marginally phosphorylated while it is hyperphosphorylated in inclusions (Anderson et al. 2006; Fujiwara et al. 2002; Waxman & Giasson, 2008b) we show that pSer129/81A immunostaining is not always a reliable marker of S pathology as it can detec t phosphorylated NFL. This cross reactivity is especially concerning since it was used to document induction of S inclusion formation in cultured cells and spread of S pathology in mouse brain injected with exogenous S fibrils (Dryanovski et al. 2013; Guo et al. 2013; Luk et al. 2012a; Luk et al. 2012b; Volpicelli Daley et al. 2011) Staining of NFL is likely responsible for most of the white matter tract staining in human and m ouse tissue, and the staining of neuronal processes in cultured neurons shown here and in our previous publication (Sacino et al. 2013b) since S is normally a presynaptic protein (George et al. 1995; Iwai et al. 1995a; Jakes et al. 1994) Similarly, we show that pSer129/81A immunoreactivity in some human sections can appear to look like Lewy pathology, and it can readily label the Lewy like NF inclusions in NFHLacZ Tg mice. Although other pSer129 antibodies are available commercially and some have been used to document peripheral S inclusion pathology (Beach et al. 2010; Mu et al. 2013) it is likely that some preparations of rabbit polyclonal pSer129 can also react with NFL phosphorylated at Ser473 due to the homology between both epitopes, but each preparation of pSer129 S antibodies will need to be specifically tested. We have only specifically shown crossreactivity with the pSer129/81A antibody, which is the most commonly used antibody for seminal reports (Dryanovski et al. 2013; Guo et al. 2013; Luk et al. 2012a; Luk et al. 2012b; Volpicelli Daley et al. 2011) We do not know if pSer129 antibody 1175 that has

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121 been used in one study to report delayed induction of S pathology f ollowing cerebral injection of S fibrils cross reacts with phosphorylated NFL (Masuda Suzukake et al. 201 3) but this antibody was not biochemically characterized for specificity, and MasudaSuzukake and colleagues did not document S inclusion formation with any other S antibodies (Masuda Suzukake et al. 2013) Phosphorylation of NF subunits has been extensively studied and many antibodies specific for phosphorylated NFH and NFM are commercially available. The NFM and NFH subunits become hyperphosphorylated when they are transported from the cell body to the axons, but they can also become hyperphosphorylated in the perikarya under many stress conditions (Giasson & Mushynski, 1996; Julien & Mushynski, 1998) The mechanisms that can modulate the phosphorylation of Ser473 in NFL in vivo have not been extensively studied. We are not aware of any commercial antibody for NFL phosphorylated at Ser473, although one has been generated by another group (Nakamura et al. 1999) and it is unknown if stress conditions can affect this phosphorylation. Nevertheless, we did not observe any major changes in the distribution of the phosphoNFH or NFM staining pattern in S fib injected mice. Until antibodies that do not cross react and are specific for S and NFL phosphoepitopes are available, it will remain unclear how much of the alterations in the pSer129 staining is solely due to S. When we tracked S inclusion induction with multiple markers, we found that there is limited induction outside of the site of injection in nTg and M47 Tg mice. The low level of induction of S inclusion formation in fibrillar S injected nTg mice reported here is consistent with the recent report by a second study from the Lee group

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122 indicating that co ntrary to their previous report (Luk et al. 2012b) they could not observe the induction of brain S inclusions following similar treatment in mice that do not overexpress S (Guo et a l. 2013) Furthermore in nTg mice our data show that the S inclusion pathology decreases over time rather than increases. The issue of whether hyperphosphorylation of Ser129 in S can influence aggregation has not been definitively answered (Fujiwara et al. 2002; Paleologou et al. 2008; Sato et al. 2013; Waxman et al. 2008) Therefore, it is unclear whether an initial induction of pSer129 immunoreactivity in nTg mice may be involved in subsequent formation of mature inclusions. It is also unclear if the initial local formation of S aggregation following injection of preformed fibrils is due to local seeding of S pathology resulting from uptake of S seeds from surgical neuronal damage or reported biological activities (Ahn et al. 2006; Desplats et al. 2009; Emmanoui lidou et al. 2010; Lee et al. 2008a) Although it could be argued that the time frame (4 months) for our studies was not sufficiently long to result in prion like spread of S pathology in nTg and M47 Tg mice, it is longer than some of the time points that were reportedly demonstrated to result in the spread of S pathology in nTg mice (Luk et al. 2012a) At this time, we cannot exclude that similar to our recent studies and those of MasudaSuzukake and colleagues that injection of exogenous S may lead to longer term and delayed induction of S inclusion pathology (MasudaSuzukake et al. 2013; Sacino et al. 2013a) However, we would point out that these findings do not directly address the issue of prionlike mechanism since i) initial diffusion of the seed has not been tracked in these prior studies and ii) brain injection of an amyloiddeficient S protein ( 7182

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123 S) could also induce delayed (for several months) S aggregation following exposure and after the initial inoculum could no longer be det ected (Sacino et al. 2013a) Consistent with previous reports, in M83 Tg mice more robust and widespread formation of matur e S inclusions stained with multiple markers was observed within a shorter time frame following injection of the same preparation of hfib S as used in the nTg and M47 Tg studies. Inclusion formation was more widely distributed in hippocampal injected M83 Tg mice than typically observed when they naturally become symptomatic and show inclusion pathology (Giasson et al. 2002) In these mice, S pathology was observed around the injection site, but also in all the areas of the neuroaxis where intrinsically develop S inclusion pathology normally develops (Giasson et al. 2002) We could detect only sparse authentic S inclusions in white matter tracts with multiple markers in contrast to previous studies (Luk et al. 2012b) Instead, we find that most of white matter tract pSer129/81A immunostaining is attributable to the phosphorylated NFL. A key concept in prion like transmission is the notion that different conformations of the seeds will likely induce different pathologies and that the interaction between seed and host protein may create both complete and subtler kinetic barriers to templating (Aguzzi et al. 2007; Coll inge & Clarke, 2007; Eisenberg & Jucker, 2012) Indeed, it is thought that subtle conformational differences in the seed underlie the strainlike phenomena reported for prionlike templating of many amyloidogenic proteins. Consistent with this notion, our recent cell culture studies show that different mutant S fibrils can exert a dominant effect on inclusion morphology (Sacino et al. 2013b) Even more recent culture and in vivo studies reporting that

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124 certain S preparations can seed tau whereas others do not, support the concept that S may exist in different strains (Guo et al. 2013) Although it is conceptually attractive that different S conformations (strains) can induce different pathologies, much more work needs to be done to replicate and extend these initial findings and whether they have relevance to human disease. Given that most of the S used for these seeding studies is recombinant protein that is purified and aggregated using varying protocols, strain like phenomena may be very challenging to reproduce across laboratories. As in (Atwood et al. 2003; Canevari et al. 2004) issues with reproducibility will likely be best settled by rapid exchange of S preparations between laboratories as well as rigorous quality control regarding the purity of the recombinant proteins. We also note that there appears to be sig nificant intrinsic susceptibility differences to pathology induction from injected fibrillar S that is inherent to the different models used. M83 Tg mice, which intrinsically develop S pathology more rapidly than M47 Tg mice (Emmer et al. 2011) appear to be more primed for synchronous induction of S pathology. In addition, in the M47 Tg mice, more extensive tau inclusions pathology is induced (Emmer et al. 2011) It is not clear why the M83 Tg mice are more susceptibl e to induction and spread of S pathology by exogenous S fibrils compared to M47 Tg mice, but it is possible that the threshold for this process is different between these mice, which would be consistent with the latter onset in native M47 Tg mice. Our c urrent data along with a recent study from our group showing that soluble 7182 S, which is nonamyloidogenic in vitro and in cell culture studies, can induce

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125 delayed S pathology in vivo following neonatal brain injection (Sacino et al. 2013b) raise several questions regarding the interpretation of data suggesting that similar intracerebral injection studies demonstrate spreading of S pathology via prionlike mechanisms. Indeed, our data suggest that there may be multiple barriers that can limit spread of pathology. First, both our data and that of others, rather than being indicative of spread may be just as easily attributable to initial diffusion of the injected S which then induces pathology at sites distal to the site of injection. One would predict that there would be a decreasing gradient of injected protein away from the injection site possibly along normal anatomical connections; thus, progressive development of S pathology might appear as spreading when in fact it represents nothing more than the initial dispersion gradient of the injected material. Second, given that there appears to be an intrinsic susceptibility between the various models to inclusion formation, it is possible that specific neuronal populations may be selectively vulnerable to form S inclusions in these Tg S mouse models, and th at exogenous S results in a stress signal that simply accelerated the development of the intrinsic phenotype. Indeed, it has previously been shown that lipopolysaccharide can trigger S pathology in the M83 Tg mouse line (Gao et al. 2008; Gao et al. 2011) As there is growing evidence that exogenous amyloidogenic proteins like S, especially when a ggregated, act as DAMPs (Danger Associated Molecular Patterns) to activate innate immunity ( Golde et al. 2013; Rubartelli & Lotze, 2007) the studies conducted to date cannot exclude induction of innate immune signaling pathways or other injury responses as trigger s that indirectly induce S pathology.

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126 As we have hypothesized in a recent review the spread of S and other pathologies is likely to be complex and not mediated by a single mechanism (Golde et al. 2013) Prion like seeding, induction of a toxic environment, a nd intrinsic disruptions of proteostasis may all synergistically contribute to the induction and spread of S inclusion pathology as well as the proteinopathies that underlie other neurodegenerative CNS diseases (Golde et al. 2013) Further studies will be needed to m ore conclusively determine the relative contribution of these various mechanisms to induction and propagation of S pathology. Resolving the mechanism(s) of disease progression is important for biological, experimental, medical, and social reasons, but it also has direct therapeutic implications.

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127 Figure 41 Induction of S pathology at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice. A) Schematic map showing rostral caudal distribution of S pathology in M83 Tg mice followi ng hippocampal injection of 21140 hfib S. In these mice there was robust induction of S pathology at the site of injection, but also throughout the CNS. Red dots indicate locations of intrinsic S pathology observed when M83 Tg mice become symptomatic with aging (Giasson et al. 2002) and blue dots indicate locations of additional S pathology uniquely induced by hippocampus injection with 21140 hfib S. Therefore, in M83 Tg mice injected with 211 40 hfib S, S neuronal inclusions were observed in the location indicated by both red and blue dots. Similar density and distribution of S pathology was seen bilaterally. This distribution of S inclusions was assessed with both pSer129/81A and Syn506 a ntibodies. B) IHC of brainstem tissue sections from a symptomatic 15 monthold M83 Tg mouse and a M83 Tg mouse at 2 months post intrahippocampal injection of 21140 hfib S showing pSer129/81A+ inclusions that fill the cell bodies and extend out into the processes along with the presence of dystrophic neurites. These S inclusions are also readily detected by staining with Syn506 and p62 antibodies. Tissue sections were counterstained with hematoxylin. C) Double immunofluorescence analysis of the hippoc ampal region for pSer129 (green) and SNL4 (A; upper panels) or p62 (B; lower panels) shows that all pSer129/81A+ perikaryal inclusions are also SNL 4+ and that the majority of pSer129/81A+ inclusions are also p62+. Cell nuclei were counter stained with D API. Scale bar = 100 m and 25 m (inset).

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129 Figure 42 Non specific staining by anti pSer129/81A antibody in the white matter tracts of M83 Tg mice 2 months post intrahippocampal injection of 21140 hfib S. Double immunofluorescence analysis of the cerebellar region in M83 Tg mice for pSer129 and anti S antibody SNL4 shows that while there is colocalization of pSer129 with SNL4 indicating the presence of S inclusions (A; arrows) there is also detection of a nonS target by pSer129 in the white matter tracts (A; arrowheads). Neuropil in the molecular layer (ML) and granular layer (GL) of the cerebellum, while enriched with S, rarely formed S inclusions. This same detection of a nonS target in the white matter tracts of the cerebellum was seen in SNCA / mice (B; arrowheads), in which the absence of S is seen by the absence of SNL4 detection (B). Cell nuclei were counter stained with DAPI. Scale bar = 250 m.

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131 Figure 43 The ~70 kDa protein recognized by pSer129/81A antibody is phosphoserine 473 in NFL. A) Immunoblotting with pSer129/81A and anti NFL antibody NR4 or Coomassie R250 stained 8% polylacrylamide gels of 2% SDS or 4M urea fractions from the cortical (CX) or cerebel lar (CB ) white matter of human brain. B) pSer129/81A antibody immunoprecipitates NFL from human cortical white matter or mouse spinal cord/brain stem. Immunoprecipitation was performed as described in Material and Methods. Input, unbound, and immunoprecipi ated fractions were analyzed by immunoblotting with pSer129/81A and anti NFL antibody NR4. C) In situ alkaline phosphate treatment showing that the pSer129/81A reactive ~70 kDa protein is a phosphoprotein in both human and mouse brain. Nitrocellulose membranes with SDS fractions from human cortical white matter or murine SDS/urea spinal cord and brain stem were incubated without ( ) or with (+) alkaline phosphatase (AP) as described in Material and Methods and immunoblotted with pSer129 or anti NFL NR4 ant ibodies. D) Immunoblotting of SDS/urea fractions from nTg/wildtype (WT) and NFL/ mice with pSer129/81A or anti NFL NR4 antibodies showing that th e ~70 kDa protein band is NFL. E) In vitro casein kinase II phosphorylation of recombinant wildtype (WT) and Ser473Ala NFL followed by immunoblotting with either anti NFL antibody NR4 or pSer129/81A antibody demonstrating that pSer129/81A antibody recognized NFL phosphorylated at Ser473. GFAP, glial fibrillary acid protein; NFL, low molecular mass neurofilament subunit; NFM, mid sized molecular mass NF subunit; NFH, heavy molecular mass NF subunit.

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133 Figure 44 Immunocytochemical pSer129/81A staining of the white matter tracts in nTg/native mice is absent in NFL/ mice. A) White matter tracts of the corp us callosum and internal capsule in nTg mice are detected by staining for NFL. Areas within those tracts are also detected with pSer129/81A, but not by S marker Syn506, indicating that pSer129/81A is detecting a nonS target in the tracts. In NFL / mi ce, there is no staining for NFL or pSer129/81A in these white matter tracts. B) Similarly, white matter tracts in the cerebellum of nTg mice, which are detected by NFL, also stain with pSer129/81A and not Syn506. These are also unstained for NFL and pSer129/81A in NFL/ mice. Tissue sections were counterstained with hematoxylin. Scale bar = 150 m and 50 m (inset).

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135 Figure 45 NF inclusions in NFHLacZ Tg mice are stained by pSer129/81A. A) IHC of hippocampal (HC), brain stem (BS), and cerebellar (CB) tissue sections from a NFHLacZ Tg mouse show pSer129/81A+ Lewy body like perikaryal inclusions which are also detected by NFL. Tissue sections were counterstained with hematoxylin. B) Double immunofluorescence analysis of the brain stem region for anti S antibody SNL4 (top panel) or pSer129/81A (bottom panel) with NFL shows that the NF inclusions detected by NFL do not contain S but are nonetheless detected by pSer129/81A (arrows). Cell nuclei were counterstained with DAPI. Scale bar = 100 m.

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137 Figure 46 pSer129/81A detects NFL in white matter tracts from human nervous tissue sections. A) IHC of human CNS tissue shows that in the white matter tracts of the pons in a control subject, pSer129/81A stains in a similar pattern t o that seen with NFL. Tissue sections were counterstained with hematoxylin. B) Double immunofluorescence analysis of the pons in human CNS tissue for anti S antibody SNL4 (green; top panel) and pSer129/81A (green; bottom panel) with NFL (red) shows that pSer129/81A colocalizes with NFL staining in the white matter tracts (arrows). Cell nuclei were counterstained with DAPI. Scale bar = 100 m.

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138 Figure 47 pSer129/81A detects phosphorylated NFL in mixed primary neuronal cultures. Double immunofluoresc ence analysis of untreated mixed primary neuronal cultures at 7 days in vitro for NFL (red; A, B) and pSer129/81A (green; A, B) shows that pSer129/81A staining colocalizes almost completely with NFL in both the processes and cell bodi es (arrowheads; A). C) Confocal Z slice analysis midway through a neuronal cell body shows that there is complete colocalization of pSer129/81A staining with NFL. Cell nuclei were counterstained with DAPI. Scale bar = 50 m (A); 25 m and 12.5 m (B; insets).

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140 Figure 48 Induction of S inclusions in nTg mice after intrahippocampal injection of 21140 hfib S or full length mfib S. A) Schematic map showing rostral caudal distribution of S in nTg mice at 4 months post injection. Similar density and distribution of S pathology was seen bilaterally. Inclusions were mainly localized around the site of injection (hippocampus) with sparse pathology seen in the cortex for both human and mouse derived S amyloid ogenic fibrils. B) Representative images of IHC showing decreas ing pSer129/81A pathology at 1 month, 2 months, and 4 months post injection of hfib S. Some inclusions reactive with Syn506, a mouse monoclonal antibody that conformationally detects S aggregates, and p62, a rabbit polyclonal antibody that nonspecificall y recognizes protein aggregates, were also observed. Similar staining also shows S inclusion formation resulting from the hippocampal injection of mfib S at 2 months post injection. Some of these S inclusions are also detected by Syn506 and p62. Tissue sections were counterstained with hematoxylin. Scale bar = 50 m.

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142 Figure 49 Characterization and distribution of S inclusion pathology at 4 months post intrahippocampal injection of 21140 hfib S in M47 Tg mice. A) Diagram shows rostral/caudal distribution of S inclusion pathology in M47 Tg mice. Tissue sections were stained with antibodies pSer129/81A and Syn506 to detect S inclusion pathology. Intrahippocampal injections resulted in the induction of S pathology at the site of injection with some additional pathology in the midbrain and brainstem. Similar density and distribution of S pathology was seen bilaterally. B) Brain tissue sections with representative regions of the hippocampus in i njected M47 Tg mice show rounded, perinuclear inclusions and neuritic pathology stained with pSer129/81A. Inclusions were also recognized by Syn506, a mouse monoclonal antibody that conformationally recognizes S inclusions, and p62, a rabbit polyclonal a ntibody that nonspecifically recognizes protein aggregates. C) Double immunofluorescence analysis of the hippocampal region stained for pSer129/81A (green) and SNL4 (red; upper panels) or p62 (red; lower panels) shows that most hyperphosphorylated S inc lusions are SNL 4+ and that a large portion are also p62+ in M47 Tg mice injected with 21140 hfib S. There is also significant p62 immunoreactivity that does not co localize with pSer129 indicating the formation of more than one type of protein aggregat e. Arrows show aggregates that are double labeled. Tissue sections were counterstained with hematoxylin (B) and DAPI (C). Scale bar = 50 m (B), and 100 m and 25 m (C; insets).

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144 Figure 410 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice using antibody pSer129/81A. Representative images depicting the distribution of S aggregates in brain/spinal cord tissue sections of M83 Tg mice following hippocampal injection of 21140 hfib S stained with antibody pSer129/81A. Perinuclear Lewy body like pathology and Lewy neuritelike pathology extending into the processes was seen in the motor cortex (MC), somatosensory cortex (SSC), hippocampus (dentate gyrus, DG; CA1), amygdala (AMY), hypothalamus (HYP), thalamus (TH), substantia nigra (SN), superior colliculus (SC), ventral pons (VP), ventral horn of the cervical spinal cord (VH SC), and dorsal horn of the cervical spinal cord (DH SC). Tissue sections were counterstained with hematoxylin. Scale bar = 100 m (MC), 100 m (SSC), 50 m (DG), 50 m (CA1), 50 m (AMY), 50 m (HYP), 50 m (TH), 50 m (SN), 50 m (SC), 50 m (VP), 200 m (VH SC), and 200 m (DH SC).

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146 Figure 411 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice using antibody Syn506. Representative images depicting the distribution of S aggregates in brain/spinal cord tissue sections of M83 Tg mice following hippocampal injection of 21140 hfib S stained with antibody Syn506. Perinuclear Lewy body like pathology and Lewy neuritelike pathology extending into the processes was seen in the motor cortex (MC), somatosensory cortex (SSC), hippocampus (d entate gyrus, DG; CA1), amygdala (AMY), hypothalamus (HYP), thalamus (TH), substantia nigra (SN), superior colliculus (SC), ventral pons (VP), ventral horn of the cervical spinal cord (VH SC), and dorsal horn of the cervical spinal cord (DH SC). Tissue se ctions were counterstained with hematoxylin. Scale bar = 100 m (MC), 100 m (SSC), 50 m (DG), 50 m (CA1), 50 m (AMY), 50 m (HYP), 50 m (TH), 50 m (SN), 50 m (SC), 50 m (VP), 200 m (VH SC), and 200 m (DH SC).

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148 Figure 412 Distribution of S pathology throughout the neuroaxis at 2 months post intrahippocampal injection of 21140 hfib S in M83 Tg mice using an antibody to p62. Representative images depicting the distribution of S aggregates in brain/spinal cord tissue sections of M83 Tg mi ce following hippocampal injection of 21140 hfib S stained with p62. Perinuclear Lewy body like pathology and Lewy neuritelike pathology extending into the processes was seen in the motor cortex (MC), somatosensory cortex (SSC), hippocampus (dentate gy rus, DG; CA1), amygdala (AMY), hypothalamus (HYP), thalamus (TH), substantia nigra (SN), superior colliculus (SC), ventral pons (VP), ventral horn of the cervical spinal cord (VH SC), and dorsal horn of the cervical spinal cord (DH SC). Tissue sections we re counterstained with hematoxylin. Scale bar = 100 m (MC), 100 m (SSC), 50 m (DG), 50 m (CA1), 50 m (AMY), 50 m (HYP), 50 m (TH), 50 m (SN), 50 m (SC), 50 m (VP), 200 m (VH SC), and 200 m (DH SC).

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149 Figure 413 Immunohistochemical analysis of pSer129/81A staining in M83 Tg mice 2 months post intrahippocampal injection with PBS and 71 82 S. Brain tissue sections were stained with pSer129/81A antibody and representative images are shown. Changes in pSer129 st aining were not detected in M83 Tg mice injected with PBS (A). Injection of 7182 S (B) resulted in the sparse formation of small pSer129 reactive accumulations in the hippocampus that appear to be dystrophic synaptic terminals near the site of injection. Tissue sections were counterstained with hematoxylin. Scale bar = 100 m and 25 m (insets).

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150 Figure 414 Non S staining of white matter tracts by pSer129/81A antibody in nTg and SNCA / mice. IHC of brain tissue sections from 4 monthold nTg (A, B) and SNCA / (C, D) mice stained with pSer129 shows cross reactivity with a nonS target in the white matter tracts of the corpus callosum (red box; A, C), white matter tracts within the striatum region (blue box; A, C), and in the cerebellum (green box; B, D). Tissue sections were counterstained with hematoxylin. Scale bars = 50 m.

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151 Figure 415 Western blot analysis demonstrating the detection of a major ~70 kDa major nonS target reactive with pSer129/81A in mouse brain. Samples from the cortex and brain stem/spinal cord, which are enriched in white matter, of untreated nTg and SNCA / mice were sequentially extracted, as described in Materials & Methods. HS and SDS/urea(U) fractions were analyzed by Western blotting using anti S antibody SNL1 and pSer129. (A) Detection of endogenous S with antibody SNL1 in the HS fraction from the cortex and brainstem/spinal cord of nTg mouse ( 17 kDa; arrow). Expectedly, this protein band is not present from extracts of SNCA / mice. (B, C) Detection of S weakly phosphorylated at pSer129 in the HS cortical fraction of nTg mouse (arrow). pSer129 also detects a 70 kDa (asterisk) nonS target in both HS and SDS/U fractions from nTg and SCNA / mice. (D) Western blot for actin is shown as a loading control. In A, B, and D equal amounts (30 g) of total protein from each fraction were resolved on 13% SDS polyacrylamide gels, and in C equal amounts (30 g) of total protein from each fraction were resolved on 7% SDS polyacrylamide gel to better resolve the major ~70 kDa nonS protein reacting with pSer129/81A antibody. The positions of molecular mass markers are indicated on the left.

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153 Figu re 4 16 pSer129/81A detects phosphorylated NFL in the white matter tracts and cell bodies of nTg mice. Double immunofluorescence analysis of the cerebellar region in (A) nTg, (B) SNCA / and (C) NFL / mice with anti NFL (red) or pSer129/81A (green). In nTg mice there is c o localization of pSer129/81A with NFL in some cell bodies (arrows) and in the white matter tracts (arrowheads). In SCNA / mice, there is still robust staining of white matter tracts for NFL and pSer129/81A antibodies, but some processes are only labeled with NFL antibody similarly to nTg mice. In NFL/ mice, there is no staining for NFL or pSer129 in the white matter tracts, further indicating that it is specifically detecting phosphorylated NFL. Cell nuclei were counterstained with DAPI. Scale bar = 2 50 m.

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154 Figure 417 pSer129 antibody can intensely detect white matter tracts that is not due to S in human nervous tissue sections. IHC (A) and immunofluorescence (B D) analyses of fixed CNS tissue from a control subject or a dementia with LBs (DLB) patient stained with pSer129 and SNL 4. White matter tracts in the pons of the control subject and DLB patient were detected with pSer129 by both IHC (A) and IF (B) staining; however, this reactivity was not detectable when staining for S with SNL 4 (C). pSer129 staining in the gray matter area of the cingulate cortex of a DLB patient depicting cortical LBs (indicated by arrows) detected by both IHC (A) and immunofluorescence analysis (B) that was also detectable with SNL4 (C, D). Cell nuclei were counter stained with hematoxylin (A) and DAPI (B D). Scale bar = 100 m and 25 m (insets).

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155 Figure 41 8 Tau aggregate formation 4 months post intrahippocampal injection of 21140 hfib S in M47 Tg mice. Double immunofluorescence analysis of the hippocampal region with PHF 1 (green) or AT 100 (green), which recognize phosphorylated tau epitopes, and p62 (r ed) shows the presence of tau inclusions in M47 Tg mice 4 months post injection of 21140 hfib S. Arrows show aggregates that are double labeled. Cell nuclei were counter stained with DAPI. Scale bar = 100 m and 25 m (insets).

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156 Figure 419 Schematic summary of S pathology distribution at 4 months post intrahippocampal injection of hfib 21140, E46K, and A53T S in M47 Tg mice. Diagrams show rostral/caudal distribution of S pathology in M47 Tg mice. Tissue sections were stained with pSer129 and Syn506 to detect S pathology.

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157 Figure 420 Immunohistochemistry showing similar S pathology induced by intrahippocampal injection of 21140 WT, E46K, or A53T hfib S in M47 T g mice at 4 months post injection. Brain tissue sections with representative regions of the hippocampus in M47 Tg mice injected with 21140 (A), E46K (B), or A53T (C) hfib S show rounded, perinuclear inclusions and neuritic pathology regardless of the type of inoculum detected with pSer129/81A antibody. Inclusions were also recognized by Syn506, a mouse monoclonal antibody that conformationally recognizes S inclusions, and p62, a rabbit polyclonal antibody that nonspecifically recognizes protein aggregates. Tissue sections were counterstained with hematoxylin. Scale bar = 50 m.

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158 CHAPTER 5 PERIPHERAL INJECTION OF RECO M BINANT SYNUCLEIN INDUCES CNS SYNUCLEIN PATHOLOGY AND A RAPIDONSET, SYNCHRONIZED MOTOR PHENOTYPE Introduction Synucleinopathies are a group of diseases unified by the presence of amyloidogenic synuclein ( S) inclusions that can occur in neurons and glial cells of the central nervous system (Cookson, 2005; Gasser, 2009; Goedert, 2001a; Waxman & Giasson, 2008a) In Parkinsons disease (PD), a causative role for S has been established via the discovery of mutations in the S gene S N CA resulting in autosomal dominant P D (Farrer e t al. 2004; Kiely et al. 2013; Kruger et al. 1998; Polymeropoulos et al. 1997; Proukakis et al. 2013; Singleton et al. 2003; Waxman & Giasson, 2008a; Zarranz et al. 2004) Although S inclusions (e.g., Lewy bodies) are the hallmark pathology of PD, how they contribute to disease pathogenesis remains controversial (Cookson, 2005; Dawson et al. 2002; Goedert, 2001a; Waxman & Giasson, 2008a) Post mortem studies have suggested that S inclusion pathology may spread following neuroanatomical tracts (Braak et al. 2006a; Braak et al. 2003a; Braak et al. 2003b) and between cells (Kordower et al. 2008; Li et al. 2008; Li et al. 2010) S pathology has also been found in the peripheral nervous system (PNS), for example, in the enteric plexus and the pelvic plexus (Braak et al. 2006b; Wakabayashi et al. 2010) ; and it has been suggested that S pathology might start in the nerves of the PNS and spread to the CNS (Braak et al. 2006a) Experimentally, it has been reported that intracerebral injections of preformed amyloidogenic S fibrils in non transgenic (Tg) and S transgenic (Tg) mice can induce the formation of intracellular S inclusions that

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159 appear to progress from the site of injection (Luk et al. 2012a; Luk et al. 2012b) Collectively, these studies have been used to support the notion that S inclusion pathology may propagate via a prionlike conformational self templating mechanism (Jucker & Walker, 2013; Polymenidou & Cleveland, 2012) A caveat with the direct intracerebral injection of S is that the surgical procedure directly alters brai n homeostasis making it difficult to assess the relative contribution of the experimental trauma in the induction of S pathology, especially since incidents such as traumatic brain injury can induce the formation of S pathology (Uryu et al. 2003) Here we report that the intramuscular (IM) injection of amyloidogenic S in M83 Tg mice expressing human A53T S can result in the rapid and synchronized development of hind limb motor weakness and robust widespread CNS S pathology. Materials and Methods Mice husbandry All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. M83 Tg mice expressing human mouse prion protein promoter were previously described (Emmer et al. 2011; Giasson et al. 2002) All animals were maintained on ad libitum food and water with a 12 h light/dark cycle. Intramuscular inje ction into M83 Tg or M20 Tg mice Bilateral injection of S proteins or lipopolysaccharide (LPS) SigmaAldrich (St. Louis, MO) control was performed by inserting the needle about 0.5 to 1 cm deep into the biceps femoris or gastrocnemius muscle (as summ ari zed in Tables 51 and 52 ). Mice at 2 months of age

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160 were anesthetized with isoflurane (15%) inhalation. Injections were made using a 10 L Hamilton syringe with a 25gauge needle. Different syringes were used for each type of inoculum to prevent any contamination. Post injection, mice were placed on a heating pad for recovery before being returned to their home cage. Recombinant N terminal truncated 21m ino acid 21), 7182 deletion ( 71 purified to homogeneity as previously described (Giasson et al. 2001b; Greenbaum et al. 2005; Waxman et al. 2009) 21 140 human S was shown to induce S inclusion formation similar to full length protein in cultured cells (Luk et al. 2009; Waxman & Giasson, 2010) but provided the added advantage of being able to determine that inclusions are comprised of the endogenously expressed using aminoterminal S antibodies (Giasson et al. 2000b; Waxman et al. 2008) 7182 S has a deletion in the middle of the hydrophobic region of S that is required for amyloid formation and therefore lacks the ability to form or seed S amy loid in vitro and in vivo (Giasson et al. 2001b; Luk et al. 2009; Sacino et al. 2013b; Waxman et al. 2009) into filaments by incubation at 37oC at 5 mg/mL in sterile phosphate buffered saline (PBS, Invitrogen) with continuous shaking at 1050 rpm (Thermomixer R, Eppendorf, K114 fluorometry (Crystal et al. 2003; Waxm an et al. 2009) sterile PBS and treated by mild water bath sonication for 2 hours at room temperature. These fibrils were tested for induction of int racellular amyloid inclusion formation as

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161 previously described (Waxman & Giasson, 2010) The 71 studies was no t pre incubated and is in the soluble form as previously described (Giasson et al. 2001b; Waxman et al. 2009) We have also recently shown that this same preparation of 71prima inclusion formation very efficiently in those cultures (Sacino et al. 2013b) Immunohistochemical (IHC) analysis Mice were sacrificed with CO2 euthanization and perfused with PBS/heparin followed by 70% ethanol/150mM NaCl. The brain and s pinal cord were then removed and fixed for at least 48 hours followed by immunostained using previously described methods and immunocomplex visualization via chromogen 3,3 diaminobenzidine (Giasson et al. 2002) Sections also were counterstained with hematoxylin. All slides were scanned using an Aperio ScanScope CS (40 magnification; Aperio Technologies Inc., Vista, CA), and images of representative areas were taken using the ImageScopeTM software (40 magnification; Aperio Technologies Inc.). Quantification of immunoreactivity for the levels of astrogliosis and microgliosis was accomplised using ImageScopeTm software to measure the density of staining per area. Thr ee independent regions were quantified per animal and two animals were used per condition. The data is expressed as averaged immunoreactivity/area normalized to the control SEM. Stained tissue sections from untreated young (3M to 4M) M83 Tg mice without S inclusion pathology were used as the baseline control.

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162 Double Labeling Immunofluorescence Analysis of Mouse Brain Tissue Paraffin embedded tissue sections were deparaffinized and hydrated through a series of graded ethanol solutions followed by 0.1M Tris, pH 7.6. The sections were blocked with 5% dry milk/0.1M Tris, pH 7.6 and were incubated simultaneously with combinations of primary antibodies diluted in 5% dry milk/0.1M Tris, pH 7.6. After extensive washing, sections were incubated with secondary antibodies conjugated to Alexa 594 or Alexa 488 (Invitrogen, Eugene, OR). Sections were post fixed with formalin, incubated with S udan Black, and stained with 5 g/mL 4, 6 diamindino2 phenylindole (DAPI). The sections were cover slipped with Fluoromount G (SouthernBiotech, Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera to capture images. Antibodies phosphorylated at Ser129 (Waxman & Giasson, 2008b) SNL 4 is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 2(Giasson et al. 2000b) Syn506 is an anti N that preferentially (Waxman et al. 2008) Anti p62 (SQSTM1; Proteintech; Chicago, IL), anti glial fibrillary acidic protein (GFAP) (Promega; Madison, WI), and anti ionized calcium binding adaptor molecule 1 (Iba1) (DA KO; Glostrio, Denmark) are rabbit polyclonal antibodies. Results Native M83 Tg mice show signs of motor impairment from 816 months of age with an arched back as the initial disease presentation which progresses to quadriparesis and a moribund state requi ring euthanasia within 2 weeks (Giasson et al. 2002) The presentation of this phenotype is associated with the formation of S

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163 inclusion pathology throughout most of the neuroaxis, but this phenotype never occurs spontaneously before 7 months of age and most often occurs around 12 months of age (Emmer et al. 2011; Giasson et al. 2002) Therefore, we preformed IM injections with various S preparations into the hind limb muscles of 2 monthold M83 Tg mice and monitored them for induction of disease (Figure 5 1 and Table 5 1). 10 g of recombinant full length mouse fibrillar S (mfib), 21 140 fibrillar human S (hfib), human 7182 S, or LPS (25 g) were injected. Both mfib and hf ib are amyloidogenic, and can act as seeds for conformational templating in vitro in culture, and in vivo Moreover, the truncated 21140 S hfib protein was used to enable assessment of aggregation by endogenous S through detection with aminoterminal specific S antibodies (Luk et al. 2009; Sacino et al. 2013b; Waxman & Giasson, 2010; Waxman & Giasson, 2011b) Non amyloidogenic human 7182 S has a deletion in the middle of the hydrophobic region of S required for amyloid formation and therefore lacks the ability to form or seed S amyloid in vitro and in cultured cells (Giasson et al. 2001b; Luk et al. 2009; Sacino et al. 2013b; Waxman et al. 2009) IM injection of human 71 82 S was used to test the hypothesis that induction of S pathology occurs solely through amyloidogenic conformational templating. Lipopolysaccharide (LPS) was used as a control for potential inflammatory effects induced by S (Codolo et al. 2013; Fellner et al. 2013; Kim et al. 2013) 40 90 days post IM injection with hfib or mfib S, M83 Tg mice developed a unilateral foot drop that progressed rapidly to a bilateral foot drop followed by full hind limb paralysis within a week of onset (Figure 5 1 ). Mice reached a moribund state at a median of 53 and 77 days post injection for mfib and hfib S, respectively. Bilateral injection of hfib S in the biceps femoris muscles and

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164 unilateral injection in the gastrocnemius muscle resulted in the same phenotype. Though M83 Tg mice injected with mfib S showed a more rapid disease onset (Figure 5 1; Table 5 1), there was no detectable difference in disease progression. Mice injected with fib S developed S inclusion pathology that was nearly indistinguishable both in terms of morphology and anatomical distribution from that seen in aged (> 8 m onth old) untreated M83 Tg mice (Figure 51 ) (Giasson et al. 2002) In contrast to IM injection of fibrillar S, no S inclusion pathology or any overt behavioral changes was observed in mice injected with LPS (Figure 5 1; Table 5 1). S inclusion pathology in mice injected with preformed fibrillar S was robust in the spinal cord, brain stem and midbrain structures, but sparse in the cortex (Figures 5 2; 5 5 ). There was a high density of S pathology in the midbrain, but inclusions were rarely observed in dop aminergic neurons (Figure 56 ). The S inclusions were composed of endogenous S, as shown by reactivity with other S markers, Syn506 and SNL4, and they were are also strongly reactive for p62, a nonspecific marker of intracellular protein aggregates (Figure s 5 1, 5 7 ) (Kuusisto et al. 2003) S inclusions were not found in the sciatic nerve or musc le injection site ( Figur e 58 ). These data indicate that a peripheral IM hfib or mfib injection dramatically accelerates onset of disease in the M83 Tg model with complete penetrance in this study. In contrast to the results with amyloidogenic forms of S, the IM injection of the nonamyloidogenic 7182 resulted in delayed onset of disease and incomplete penetrance of the accelerated pathology. 6 of 11 71 82 S IM injected M83 Tg mice became moribund with 2 of these 6 developing a motor phenotype at 120 dpi indistinguishable from that observed in the fibS injected mice. These 2 mice and 3

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165 others sacrificed at 139 dpi without motor signs showed S pathology similar to untreated, aged M83 Tg mice (Figures 5 1, 5 2, 5 7 ; Table 5 1). In summary, IM injection of 71 82 S into 11 M83 Tg mice resulted in 2 mice with both the motor phenotype and S pathology, while 4 became moribund with no S pathology and another 3 mice developed S pathology in the absence of any phenotype. To assess whether the development of S pathology in the brain and spinal cord was associated with a neuroinflammatory state, tissue sections from all injected mice were stained for the astrocyte marker, GFAP, and the microglia m arker, Iba1 (Figure 5 3). In untreated, aged M83 Tg mice with CNS S inclusion pathology, there was concurrent astrogliosis in the areas with S pathology; however, there was no significant increase in microgliosis (Figure 5 3). Interestingly, M83 Tg mi ce IM injected with LPS or 7182 S that did not develop S pathology even up to 4 months post injection, did not show elevated levels of astrogliosis or microgliosis (Figure 5 3). On the other hand, M83 Tg mice injected with 71 82 S or fib S (no diff erences seen between mfib or hfib, bilateral or unilateral injections) that developed S pathology, also showed massive astrogliosis in the areas with S inclusion pathology as seen in aged M83 Tg mice that develop a motor phenotype, as well as elevated mi crogliosis in the areas with S pathology (Figure 5 3). Collectively, this data indicates that IM injections in M83 Tg mice leading to the development of S pathology are associated with increased microglosis and astrogliosis. IM S injection studies wer e also conducted in a cohort of M20 Tg mice, which overexpress human wildtype S, but never develop a phenotype or S inclusion

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166 pathology (Emmer et al. 2011; Giasson et al. 2002) In contrast to the M83 Tg mice, this cohort of IM injected M20 Tg mice did not develop any overt motor phenotype. However, at 78 months after IM injection of hfib S, induction of S inclusion pathology was observed in the CNS, albeit less robustly than in M83 Tg mice (Figure 5 4; Table 5 2). S pa thology in hfib S IM injected M20 Tg mice was predominantly found in neuronal processes and some cell bodies in the periaqueductal gray area of the midbrain and gray matter at all levels of the spinal cord. A few mice also displayed S inclusions in the white matter tracts of the spinal cord. Discussion Our findings show that S pathology in the CNS can be efficiently induced with peripheral injections of amyloidogenic S and less efficiently with non amyloidogenic S in M83 Tg mice. In both cases S path ology is associated with a rapidly progressive motor phenotype subtly distinct from that in untreated, aged M83 Tg mice due to the initial presentation of foot drop, and with a heightened neuroinflammatory response. Previous experimental studies have provided evidence for in vivo prionlike spread of S pathology (Luk et al. 2012a; Luk et al. 201 2b) ; however, several findings here and in other recent studies from our group suggest that additional mechanisms for induction of pathology should be considered (Golde et al. 2013; Kim et al. 2013; Sacino et al. 2013a) Indeed, i) there was no evidence of S aggregation in the sciatic nerve, ii) the distribution of S pathology in M83 Tg mice IM injected with various forms of S was identical to that seen in untreated, aged M83 Tg mice, iii) IM injection of a nonamyloi dogenic form of S induced CNS S inclusion pathology albeit less efficiently

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167 than fibrillar S and iv) there was significant increases of both astrogliosis and microgliosis associated with the regions where S pathology was induced. These studies also highlight the fact that as we have noted in other studies (Sacino et al. 2013a) that there appears to be significant differences in susceptibility of different models to induction of pathology, with the M83 Tg mice being apparently susceptible to rapid onset of a neurodegenerative phenotype. Although the studies in wild type M20 Tg mice are best viewed as works in progress, as it is possible that longer incubation times will result in more robust pathology and a complete neurodegenerative cascade, in the current studies it is clear that they are less susceptible to pathology induction by IM injection of fibrillar S. Studies are currently underway to more specifically i) identify mechanisms responsible and ii) the factors (e.g., age, transgene expression, strainlike properties of the injected S) that may influence susceptibility to disease induction. Such studies will be needed to clarify whether the spread of S pathology is occurring in a manner similar t o that seen in prion disease or involves additional mechanisms (Golde et al. 2013) Although our finding that injection of a nonamyloidogenic form of S induces S pathology, albeit less efficiently than fibrillar S, certainly suggest that mechanism besides prionl ike templating may contribute to induction of disease, we think this data should be viewed cautiously. Indeed, it is possible that additional factors, analogous to the Protein X in prion disease (Telling et al. 1995) absent in in vitro systems could facilitate the conversion of the non amyloidognic soluble S to an amyloidogenic form in vivo Notably, the synchronous and rapid induction of a motor phenotype and S pathology by a peripheral injection of S will likely prove invaluable in future studies

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168 exploring mechanisms of pathology induction and S toxicity. The M83 Tg mice are one of the most widely used models of S pathology; however, the ~8month time window over which disease can present imposes a major limitation on identifying disease modifiers and testing novel therapies Our finding that diseaseonset can be shortened, predicted, and synchronized through a simple manipulation provides a valuable model to accelerate studies designed to fully understand the mechanisms underlying induction of the inclusion pathology and mot or phenotype, but also to enable much more rapid and cost effective pre clinica l testing of novel PD therapies

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169 Figure 51 S IM injection reduces survival in M83 Tg mice. A) KaplanMeier survival plot shows a decreased time to moribund state for M83 Tg mice injected with amyloidogenic S versus 7182 S and LPS, and noninjected agematched M83 Tg mice. Median times to moribund state were: within 88 dpi (median 62 dpi) for mice IM injected bilaterally with amyloidogenic S (n=21); within 80 dpi (medi an 56 dpi) for those injected unilaterally with amyloidogenic S (n=9); and within 120 dpi for phenotypic mice injected with 7182 S [n=6; red arrowhead indicates time to moribund state for the 2 mice with foot drop/paralysis]. Mice IM injected with LPS (n=5) and noninjected M83 Tg mice (n=56) remained disease free (p<0.0001; 2, 123.7; DF, 5). All mice IM injected bilaterally with mfib S died within 57 dpi (n=7; median 53 dpi) and with hfib S within 88 dpi (n=14; median 77 dpi)(p<0.01; 2, 10.62; DF, 1). B) Double immunefluorescence analyses of an untreated, aged (> 11 months old) M83 Tg mouse, a M83 Tg mouse that is 2 months post injection with 10 g hfib S, and a M83 Tg mouse that is 4 months post injection with 10 g 7182 S. pSer129+ S inclusions (green) colocalize with SNL 4+ marker for endogenous S (red top panels) and with a nonspecific intracellular inclusion marker, p62 (redbottom panels). Tissue sections were counter stained with DAPI (blue). Scale bar = 100 m and 25 m (inset).

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171 Figure 52 S pathology in M83 Tg mice IM injected with S is similar to that seen in untreated, aged M83 Tg mice. Distribution of S inclusion pathology is shown as CNS schematics with inclusions depicted as red dots. Representative IHC images of S inclusion pathology fr om various regions are shown. A ) S inclusion pathology distribution in untreated, aged (> 11 months old) M83 Tg m ice. S pathology is abundant in the spinal cord, brain stem, and midbrain areas, rare in the cortex and absent the hippocampus (Giasson et al. 2002) M83 Tg mice IM injected with 10 g hfib S B) or 10 g 7182 S C ) s how similar bilateral pathology Sc ale bar=50 m.

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173 Figure 53 Accelerated onset of astrogliosis and induction of microgliosis in M83 Tg mice IM injected with 7182 S and hfib S compared to untreated, aged M83 Tg mice. A) For each condition, quantitative analyses of GFAP immunoreactivity, a marker of the astrogliosis, and Iba 1 immunoreactivity, a marker of microgliosis, per area was performed and normalized relative to the staining signal in untreated M83 Tg mice without S inclusion pathology. M83 Tg mice IM injected with either 7182 S or hfib S and that develop robust S CNS inclusion pathology demonstrated a 68 fold increase in the abundance of GFAP staining signal sim ilar to that seen in untreated, aged M83 Tg mice that presented with S inclusion pathology (associated with aging and motor impairment) (*, p<0.001). Mice IM injected with S protein and that developed CNS S inclusion pathology also showed an ~ 5 fold i ncrease in Iba 1 staining signal, which was not seen in motor impairment untreated, aged M83 Tg mice (**, p<0.001). No quantitative increase in GFAP or Iba1 staining was observe in M83 Tg mice IM injected with LPS or 7182 S and that did not develop CNS S inclusion pathology. B) Representative images of ventral pons or cervical spinal cord tissue sections stained by IHC for GFAP or Iba1. In untreated, aged M83 Tg mice with robust S pathology in the pons and spinal cord there is increased astrogliosis in the pons and gray matter of the spinal cord with limited microgliosis. M83 Tg mice IM injected with 10 g LPS or 10 g 7182 S that has no S inclusion pathology at 4 months post injection, showed baseline levels of astrocytes and microglia in the pons and spinal cord. M83 Tg mice IM injected with 10 g 7182 S or 10 g fib S that developed S pathology demonstrated elevated astrogliosis and increased microgliosis in both these affected areas. Scale bar=100 m.

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175 Figure 54 Induction of S pathology at 7 months post IM injection of 10 g hfib S in M20 Tg mice. Schematic summary of rostral caudal S pathology distribution in M20 Tg mice 7 months after bilateral IM injection with hfib S. A moderate density of S pathology was seen throughout the spinal cord and midbrain with no pathology in the striatum, hippocampus, or cortex. The hyperphosphorylated S inclusion s were also detected by Syn506, an anti N terminal S antibody that conformationally detects S inclusions and p62, an antibody that nonspecifically recognizes intracellular protein aggregates. Equivalent density and distribution of S pathology was observed bilaterally. Scale bar=50 m

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176 Figure 55 Induction of S pathology throughout the CNS 2 months after IM injection of 10 g hfib S in M83 Tg mice. Tissue sections were stained with pSer129/81A antibody. S inclusions filling neuronal cell bodies and processes were found throughout the cervical spinal cord [VH SC (ventral hornspinal cord) and DH SC (dorsal hornspinal cord)]; brain stem [VP (ventral pons), SuVe (superior vestibular nuclei), SC (superior colliculus), and PAG (periaqueductal gray area)]; substantia nigra (SN); striatum (ST); thalamus (TH); and hypothalamus (HYP). The majority of animals have no or scant pathology in the cortex (MC, motor cortex) and none have pathology in the hippocampus (CA1 region). Scale bar=50 m.

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177 Figure 56 S inclusions in the substantia nigra of M83 Tg mice are not found in dopaminergic neurons. Midbrain tissue sections from a 15 monthold untreated, aged M83 Tg mouse that developed motor impairments and a M83 Tg mouse that is 2 month after IM injection with hfib S. Double immunofluorescent labeling of dopaminergic neurons of the substantia nigr a with tyrosine hydroxylase (TH; red; A, B, D, F ) and of hyperphosphorylated S i nclusions with pSer129 (green; B, C, E, F) shows that S inclusions are predominantly found dorsal to the substantia nigra in the midbrain. Overlay images shown in C and F Sc ale bar=100 m.

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178 Figure 57 S pathology post IM injection of 10 g hfib S or 10 g 7182 S shows similar IHC staining profile compared to mature inclusions seen in untreated, aged M83 Tg mice that developed motor impairments. Brainstem tissue sections from a 15month old, untreated M83 Tg mouse that developed motor impairments (A ), a M83 Tg mouse that is 2 months post IM injection with hfib S (B ), and a M83 Tg mouse that is 4 months post IM injection with 7182 S (C ). Sections were stained with 3 separate markers for mature S pathology: pSer129, an antibody detecting S inclusions that ar e hyperphosphorylated at Ser129; Syn506, an antibody that specially recognized the N terminus of S; and p62, an antibody that nonspecifically recognizes intracellular protein aggregates. Each marker was able to detect S pathology with similar staining densities in both untreated, aged symptomatic M83 Tg mouse and IM injected M83 Tg mice. Scale bar=50 m.

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179 Figure 58 S inclusions are not present in the sciatic nerve or skeletal muscle post IM injection of 10 g hfib S in M83 Tg mice. A ) Motor imp aired hfib S injected M83 Tg mice with robust CNS S pathology did not exhibit any pathology in the sciatic nerve at the time of demise and revealed similar staining as untreated nontransgenic and SNCA / controls. There was, however, background staining with pSer129 that was not detected with Syn506 or p62, which we have recently determined to be due to cross reactivity to a nonS axonal, cytoskeleton protein that is present in SNCA / mice. B ) No S inclusion pathology was observed in the skeletal hin d limb muscles post injection of hfib S at the time when the mice were harvested. Scale bar=100 m.

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180 Ta Table 51 Summary of M83 Tg mice injected with S proteins and LPS control* Strain Inoculum Site of injection Numbe r of mice Median survival (dpi) Phenotype Pathology M83 (A53T bilateral (5 L of 2mg/mL) biceps femoris muscle 14 77 12 of 14 with foot drop/paralysis ** 12 of 14 with robust pathology** M83 (A53T bilateral (5 L of 2mg/mL) biceps femoris muscle 7 53 7 of 7 with foot drop/paralysis 7 of 7 with pathology M83 (A53T unilateral (5 L of 2mg/mL) gastrocne mus muscle 9 56 8 of 9 with foot drop/paralysis *** 8 of 9 with pathology** M83 (A53T bilateral (5 L of 2mg/mL) biceps femoris muscle 11 120 2 of 11 with foot drop/paralysis 5 of 11 with pathology M83 (A53T bilateral LPS (5 L of 5mg/mL) biceps femoris muscle 5 Undefine d 0 of 5 with foot drop/paralysis 0 of 5 with pathology M83 Tg mice received injections at 2 months of age with human or mouse fibor LPS and were analyzed at time of death or at 4 months post IM injection. See Figure 5 2 for ** Two mice died suddenly without an observed phenotype and were not available to analyze for *** One mouse died suddenly without an observed phenotype and was not available to analyze Two of these mice had a similar foot drop phenotype and extensive hind limb paralysis as were sacrificed upon veterinary recommendation. 5 2c. Three Additional

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181 Table 52 *. Strain Inoculum Site of Injection Number of mice bilateral hfibL of 2mg/mL) biceps femoris muscle 7 *All mice received injections at 23 months of age and did not present a motor or overt neurological phenotype.

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182 CHAPTER 6 DISCUSSION Summary of Results Presented in this Dissertation My graduate studies have led to the development of three novel, practical, and highly efficient models of S inclusion induction to identify therapeutic targets. Studies using these models have provided 5 major contributions to the field of S proteinopathy: i) wild type and mutant S can exist as biologi cally distinct strains that differ in their induction properties; ii) S does not have to misfold into an amyloidogenic form in order to induce S pathology; iii) there are biological barriers to the spread of S pathology, indicating the potential for additional nonmutually exclusive mechanisms(s) of induction; iv) pSer129/ S immunoreactivity, a major readout that was used to document the spread of S pathology in seminal animal studies and the presence of peripheral S inclusion pathology in human subjects, also detects NFL, a major cytoskeletal component of neurons ; and v) a single IM injection of S can induce a rapid degenerative phenotype with transmission of S pathology to the brain. Finally, by not focusing on the genetic component of a part icular disease as has been classically done with PD and other disorders, we have developed models that can be used in the study of over 20 diseaseassociated proteinopathies. Introduction Prion diseases or transmissible spongiform encephalopathies (TSEs) are currently classified in a group called protein misfolding disorders (PMDs), which also includes S and other hallmark neurodegenerative proteins such as Abeta and tau. The 20 diseaser elated amyloidogenic PMD proteins share a general mechanism of misfolding and aggregation in association with neurotoxicity; however the best

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183 characteri zed are the TSEs [ reviewed in (Costanzo & Zurzolo, 2013) ] The five properties of prions, which define their causative role in disease are: efficient self propagation; strainlike properties of the PrPSc protein, which allow a single protein to encode multiple disease phenotypes; resistance to biological clearance mechanisms; bioavailability, transport, and spread of the PrPSc protein; and the ability of th e PrPSc protein to induce toxicity and transmit phenotypic changes to its host [as listed in (Soto, 2012) ] The question of whether another PMD protein, such as S, behaves in a prionoid manner can be answered by providing results that S also has these defining characteristics (Table 61) Efficient Propagation and Strainlike Properties Once the scrapie PrPSc protein had been isolated, the inoculation of host subjects in order to study disease transmission yielded a major finding that although the amino acid sequence of the PrPSc inoculum matched that of the host PrPC, there could be differences in the patterns of neuropathology induced and in the incubation periods to producing disease (Collinge & Clarke, 2007; Fraser, 1973; Pattison IH, 1961; Prusiner, 1998; Telling, 2004) At first these differences were interpreted as disease specific nucleic acids, which transmitted disease along with the PrPSc p rotein. It is now believed that these differences denote prion strains, and strain specificity is linked to conformational differences in the misfolding of a single PrPSc protein. The prion strain hypothesis has been supported by several studies indicat ing that an individual strain has its own distinct biochemical properties in addition to differential infectivity patterns [ (Bessen et al. 1995; Korth et al. 2003; Telling et al. 1996) reviewed in (Tanaka et al. 2004a) ] Similarly, differences have been seen between wildtype S and some of its

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184 missense mutants (A53T, E46K, A30P) in terms of amyloid conformation, incubations periods, and morphology of S pathology induced. Although slightly dissimilar from prion disease in that the primary amino acid sequence is changed at one position, these studies provide evidence that S may have strai n like properties. The initial studies performed on the synthetic S fibrils were structural and kinetic studies of the amyloidogenic fibrils derived from both wildtype and mutant S. formation of elongated filaments from the previously monomeric protein (Conway et al. 1998; Crowther et al. 1998; El Agnaf et al. 1998a; Giasson et al. 1999; Han et al. 1995; Hashimoto et al. 1998; Iwai et al. 1995b; Narhi et al. 1999; Wood et al. 1999; Yoshimoto et a l. 1995) By electron microscopy it has been shown that wildfibrils were predominantly straight filaments, with a subset of twisted fil aments (Giasson et al. 1999) differences: type, to be more bundled compared to wildas t wo thick helical protofilaments (Giasson et al. 1999; Greenbaum et al. 2005; Ono et al. 2011) A more recent study has shown via NMR that these ultrastructural differences in the wildin secondary structure by the mutants, which influences the overall conformation of even the misfo (Lemkau et al. 2013) distinct amyloidogenic filamentous structures, another commonality is their ability to

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185 self propagate (also known as seed or nucleate), or to act as a conformational helical self protein into the pathogenic sheet form. In prion disease, different strains of PrPSc can serve as distinct templ ates in order to propagate their specific disease phenotype; however, these strains inefficiently crossseed each other and have not been known to catalyze the misfolding of other aggregateprone proteins [ (Prusiner, 1998; Telling, 2004) reviewed in (Collinge & Clarke, 2007) ] In vitro type and mutant forms (either by de novo formation or exogenous addition) can serve as the initial nucleus to self Bousset et al. has further shown that slight alterations in the chemical compositions of amyloid (referred to as fibrils and ribbons) (Bousset et al. 2013) The second is that the seeding process follows first order kinetics, where the rate of amyloid formation is the mutants or s seed wildof amyloid formation; however, these studies providing data on cross seeding did not convert the ultrastructure of wildconversion without influencing the structure of the wild(Bousset et al. 2013; Conway et al. 1998; Crowther et al. 1998; El Agnaf et al. 1998a; El Agnaf et al. 1998b; Giasson et al. 1999; Greenbaum et al. 2005; Hashimoto et al. 1998; Narhi et al. 1999; Wood et al. 1999) seed

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186 other PMDs, such as Abeta (Han et al. 1995; Yoshimoto et al. 1995) Therefore, it was shown by in vitro its prionoid properties of conformational templating to propagate aggregation and of strainlike differences in biochemical and biophysical properties. propagation, and mutati onspecific strain like differences in incubation periods and induced pathology. Several groups have shown that under certain experimental conditions t culture models can be taken up, and intracellularly catalyze the (Bousset et al. 2013; Desplats et al. 2 009; Luk et al. 2009; Nonaka et al. 2010; Sacino et al. 2013b; Volpicelli Daley et al. 2011; Waxman & Giasson, 2010) Strain ulture have been reported on by 2 separate groups (Bousset et al. 2013; Sacino et al. 2013b) I defined wild type rences in incubation periods mirror those discussed previously with the in vitro measurements of nucleation rates among the wildin vivo in mouse m odels on the same genetic background and PrP promoter, but overexpressing human wildtype, A53T, or (Emmer et al. 2011; Giasson et al. 2002) Bousset et al. reported unique strains of wildultrastructural differences on electron microscopy, and showed that in c ulture, these

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187 strains differ in incubation periods and toxicity induced (Bousset et al. 2013) ; however, there were no structural differences in the inclusions they seeded. Similarly, Guo et al. has recently reported the development of amyloidogenic wildseed intr acellular tau inclusions (Guo et al. 2013) Overall, although all three studies do provide evidence for prionoid in cellbased systems, they have not shown data to make the distinction between the exogenous fibrils acting as an actual conformational template versus acting as a general catalyst to promote amyloidogenic conversion in an aggregateprone environment. Resistance of Biological Clearance As mentioned previously, one of the most dangerous and worrisome aspects about the infectivity and transmission of prion disease is its resistance to biological agents and extreme conditions (Chatigny & Prusiner, 1980; Greenlee, 1982; Latarjet et al. 1970; Walker et al. 1983) Therapeutic studies in prion disease have largely focused on promoti ng the intracellular clearance of PrPSc, for example by use of lysosomotropic factors such as quinolone, or by stimulation of macrophages and dendritic cells, which can degrade prions; however, although some chemotherapies slow the course of prion disease, they are unable to completely halt or cure disease (Gilch et al. 2007; Marzo et al. 2013; Sim & Caughey, 2009) fortunately is not completely able to evade degradation (Crews et al. 2010; Kuusisto et al. 2001; McLean et al. 2002; Shin et al. 2005) ; however, as disease progresses, intracellular degradation machinery is overwhelmed and this results in a net (Ebrahimi Fakhari et al. 2012)

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188 Studies have shown that the missense mutations (A53T and A tightly to cell mediated autophagy receptors on lysosomal membranes and this in turn inhibits their degradation, allowing for increased intracellular levels of the protein (Alvarez Erviti et al. 2010; Cuervo et al. 2004; Ghavami et al. 2014) Combined with the increased affinity of the mutant forms to misfold into amyloid, this may contribute to the increased propensity of the mutations to form intracellular aggregates. Mutations in Parkin (an E3 ubiquitin ligase) and ubiquitin carboxyl te rminal hydrolase L1 (UCH L1) that lead to Parkinsonism, cause impairments in the proteasome system and (Andersson et al. 2011; Cartier et al. 2012; Ghavami et al. 2014) Similarly, mutations in Pink1/PARK7 and Parkin disrupt mitochondrial function, which leads to impairment of autophagy, ggregates (Gautier et al. 2013; Ghavami et al. 2014) Furthermore, there have been conflicting studies on t he ability of cellular pathway, which typically clears misfolded proteins and cellular debris in bulk, as being (Watanabe et al. 2012) ; and also impairs the process (Dehay et al. 2010; Tanik et al. 2013) The latter appears and autophagy markers such as ubiquitin and p62/SQSTM1, along with proteasome ons (Crews et al. 2010; Kuusisto et al. 2001) Even in vitro

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189 certain proteases, such as protease K (Miake et al. 2002) It is then thought that evading extracellular protease cleavage in order to transmit pathology (Ebrahimi Fakhari et al. 2012; Ross & Poirier, 2005) Therefore, this resistance to degradation of the bulk aggregate prionoid agent to transmit disease. Recent therapeutic studies have focused on (Park & Kim, 2013) as well as using immunotherapy to he (Bae et al. 2012) Bioavailability, Transport, and Spreading Studies from natural pri on disease to animal and cellular models have shown that infection and transmissibility of the PrPSc protein depend on 4 factors: the site of entry, the strain of the prion, the dose, and overcoming the species barrier (Kovacs & Budka, 2008) The main obstacle to transmissibility of prion disease is the species barrier. The PrPSc protein is only highly infectious if the sequence of the invading PrPSc is identical to the host PrPC (Moore et al. 2005; Prusiner et al. 1990; Scott et al. 1989; Scott et al. 1997) This limits cross species known transfer of PrPSc from animals to humans, except for the human variant CJD infection by beef contaminated with bovine spongiform encephalopathy (BSE) (Barria et al. 2014; Hueston, 2013) As mentioned previously, even within species there are PrPSc strain barriers to infection [ reviewed in (Kretzschmar & Tatzelt, 2013) ] Regardless, prions are still readily bioavailable in high enough doses for infection through environmental contagions (Beekes & McBride, 2007; Gough & Maddis on, 2010; Mathiason et al. 2006; Safar et al. 2008; Tamguney et al.

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190 2012) and in humans, through iatrogenic contamination or cannibalistic practices [ reviewed in (Brown & Mastrianni, 2010; Imran & Mahmood, 2011) ] Animal models have shown that prion infection can occur through multiple direct injection sites (muscle, peritoneum, bloo d, and brain) Site of entry for natural forms of prion disease include: the alimentary tract including the oral cavity, lymphatic system, peripheral nervous system, blood, and muscle [ reviewed in (Beekes & McBride, 2007; Kovacs & Budka, 2008) ] Interestingly, cell cultures models have not been as effective in modeling disease. There is an inherent resistance to infection by PrPSc; however, stable cell lines propagating PrPSc have been established without showing any toxicity. Although membrane receptors for PrPC have been established, it still remains elusiv e how PrPSc is taken up and released by the cell. Intercellular transmission of PrPSc has only been shown to occur via direct cell to cell contact. Treatment with media or lysate from infected cells is not able to passage infection onto nave cultures [ reviewed in (Grassmann et al. 2013; Krauss & Vorberg, 2013; Miesbauer et al. 2010) ] In summary, although it is known that PrPSc is infectious and capable of being transmitted from peripheral tissues to the CNS to induce toxicity and cause disease, little is known about the mechanism(s) at the molecular level. nt as with prion disease, multiple studies have shown a correlation between environmental factors and eripheral tissues (Barbeau et al. 1987; Dick et al. 2007; Elbaz et al. 2009; Giasson & Lee, 2000; Gorell et al. 1998; Koller et al. 1990; Lai et al. 2002; Liou et al. 1997; Ltic et al. 2004;

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191 Norris et al. 2007; Rajput et al. 1987; Seidler et al. 1996; Semchuk et al. 1992; Uversky et al. 2001b; Veldman et al. 1998) As mentioned previously, results from post mortem histological studies of PD pa (Braak et al. 2003a; Braak et al. 2003b) Additionally, colonic biopsies from patients with early stage PD in nerve fibers of the colonic submucosa (Shannon et al. 2012) In vivo al injection (Luk et al. 2012a; Luk et al. 2012b; MasudaSuzuka ke et al. 2013; Mougenot et al. 2012; Recasens et al. 2013; Sacino et al. 2013a; Watts et al. 2013) or peripheral ad ministration (Lee et al. 2011; MasudaSuzukake et al. 2013; Rey et al. 2013) Parkinsonism patients. While the intracerebral studies are useful for looking at bioavailability, due to the general nature of the studies, they cannot produce conclusive evidence about transmission of S fibrils or brain homogenate are directly into the brain; therefore, although it can be concluded pathology to different brain regions via white matter tracts (Luk et al. 2012b) used the antibody, pSer129/ 81A, as the sole marker. I recently discovered that pSer129/81A crossreacts with NFL, a major component of neuronal processes, which comprise the white m atter tracts 3) The M83 transgenic mouse models overexpressing human A53T thology post injection (Luk

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192 et al. 2012b; Mougenot et al. 2012; Watts et al. 2013) ; however, in each of the site is identical to that seen in untreated, aged M83 mice (Giasson et al. 2002) or whether cellular stress induced pathology formation in a primed, vulnerable population of neurons. 4) Of the studies completed, 5 have involved injections in nTg mice (Luk et al. 2012a; MasudaSuzukake et al. 2013; Recasens et al. 2013; Sacino et al. 2013a) site was reported (Luk et al. 2012a; MasudaSuzukake et al. 2013; Recasens et al. 2013) ; however, I recently reported that the data cannot be reproduced (Guo et al. 2013; Sacino et al. 2013a) Also, those studies used a pSer129 antibody as their marker for reactivity. 5) It has never been shown that the brain homogenates or synthetic fibril preparations (which are made in bacteria) used as inoculums in the intracerebral injection studies do not contain another cytotoxic substance, which induces a neuroinflammatory state that could promote aggregate formation. Models of peripheral administration of brain homogenate or synthetic fibrils have yielded varying results. I ntranasal administration of synthetic fibrils in nTg mice did not administration (MasudaSuzukake et al. 2013) ; however, Rey et al. (Rey et al. 2013) Injection of brain homogenate from a DLB patient into the gastric walls of M83 t

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193 months post injection, but with no evidence of retrograde transmission to the CNS (Lee et al. 2011) The only robust CNS has been reported in intramuscular injections in M83 and particularly M20 (overexpressing human wildnsgenic mice. While the M83 transgenic mice have a pattern of pathology sim ilar to that of untreated, aged M83 transgenic mice, as described above, M20 transgenic mice generally do not develop pathology (Giasson et al. 2002) these mice may be due to peripheral transmission. One premise for the nearby cells and serve as a conformational template for the conversion and disease, cell culture studies have shown that both monomeric and aggregated forms of sms for each process te the plasma membrane of cells (Danzer et al. 2007; Danzer et al. 2011; Dunning et al. 2012; Hansen et al. 2011; Jang et al. 2010; Lee et al. 2005; Lee et al. 2008a; Lee et al. 2008b; Luk et al. 2009; Nonaka et al. 2010; Paillusson et al. 2013; Volpicelli Daley et al. 2011; Waxman & Giasson, 2010) Regardless, cell culture studies have shown that there is neuronto neuron as well as neuronto astrocyte transfer of aggregates (Desplats et al. 2009; Lee et al. 2010a; Lee et al. 2010b; Sacino et al. 2013b; VolpicelliDaley et al. 2011; Waxman & Giasson, 2010) I have also shown that, unlike PrPSc

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194 pathology in nave cultures (Sacino et al. 2013b) Ultimately, both in vivo and cell pathology may at least contrib synucleinopathies; however, the specific mechanisms remain to be determined. Toxicity Induction and Transmission of Phenotypic Changes The pathogenic component of prion disease is not solely the transmission of the PrPSc, but the associated neurotoxicity causing cell death, which ultimately leads to brain dysfunction and a disease state. The mechanism by which PrPSc confers toxicity to cells is still unknown; however, three separate mechanisms for relaying toxicity have been proposed: inherent toxicity of the PrPSc protein itself, altered or loss of function of the PrPC protein, and microglia activation (Aguzzi & Falsig, 2012; Brown & Kretzschmar, 1997; Giese & Kretzschmar, 2001; Kretzschmar et al. 1997) It has been found both in cell culture and animal models that in the absence of the PrPC protein, PrPSc is rendered innocuous. Furthermore, PrPC expression control s the progression of prion disease and overexpression of mutant forms of PrPC result in cellular degeneration. Detection and uptake of PrPSc by microglia lead to its activation causing the recruitment of more cells (gliosis) and the release of cytokines a nd reactive oxygen species, which are toxic to neurons. The end result is the induction of apoptosis leading to neuronal loss and spongiform degeneration (reviewed in (Aguzzi & Falsig, 2012; Giese & Kretzschmar, 2001; Kretzschmar et al. 1997) ) Regardless of who the major player in the prion degeneration cascade is, infection of a host by the PrPSc protein results in a neurotoxic state and a transmittable neurodegenerative phenotype in anim al models, and animal and human disease consisting of a decline in neurological functioning

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195 (motor and/or cognitive) (reviewed in (Brown & Mastrianni, 2010; Head & Ironside, 2012; Imran & Mahmood, 2011) ) Although it has been reported that LBs/LNs may be neuroprotective (Olanow et al. 2004; Tanaka et al. 2004b) S pathology has been associated with cellular toxicity and a range of subsequent neurologic phenotypes seen with the synucleinopathies (Dickson, 2012; Halliday et al. 2011; Puschmann et al. 2012) Studies have shown cellular upregulation of wild type S as a defense against stress (Alves Da Costa et al. 2002; Colapinto et al. 2006; da Costa et al. 2000; Hashimoto et al. 2002; Lee et al. 2001; Seo et al. 2002) ; however, it has also been shown that the overexpression of wild type and mutant S, as well as oligomeric and fibrillar forms increase cellular toxicity via [as partly listed in (Waxman & Giasson, 2008a) ]: prote asomal inhibition, altered signal transduction pathways, mitochondrial alterations, increased production and release of free radicals, intracellular S inclusions, and clustering of dopamine transporters. As a result of this increased toxicity, different cellular phenotypes have resulted, including: neuronal electrophysiological dysfunction and cell death (VolpicelliDaley et al. 2011) ; increased caspase3 activation leading to cell death (Desplats et al. 2009) ; and up regulation of various cy tokines and chemokines in astrocytes, such as ICAM and IL 6 (Klegeris et al. 2006; Lee et al. 2010a) As seen in cell culture studies, induced toxicity and phenotypes have been reported in transgenic mouse models overexpressing human wildtype and mutant S, and S i njection mouse models. Apart from the formation of S inclusion pathology, evidence of toxicity in transgenic mouse models includes: Wallerian degeneration of ventral root axons and muscle denervation; entrapment of mitochondria; impaired

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196 axonal transpor t leading to axonal swelling; neuron loss; accumulation of intracellular phosphorylated neurofilaments; microglia activation; degeneration of axonal terminals, and decreased hippocampal neurogenesis [ reviewed in (Waxman & Giasson, 2008a) ] Injection of fibrillar and soluble S, apart from the induction of S pathology, results in a heightened neuroinflammatory response, including massive astrogliosis and microgliosis (Lee et al. 2011; Luk et al. 2012b ; Recasens et al. 2013; Sacino et al. 2013a; Watts et al. 2013) There have also been reports of loss of tyrosine hydroxylase positive striatal fibers and dopaminergic neurons post intracerebral injection of PD brain extract and S fibrils into nTg mice (Luk et al. 2012a; Recasens et al. 2013) Additionally, in both transgenic and injection mouse models, over t neurodegenerative phenotypic changes have been observed coinciding with the induction of S pathology, cellular toxicity, and a heightened neuroinflammatory response (Emmer et al. 2011; Freichel et al. 2007; Giasson et al. 2002; Luk et al. 2012a; Masliah et al. 2001; Nuber et al. 2008; Recasens et al. 2013; Watts et al. 2013) For example, I have shown that post intramuscular injection of both amyloidogenic and nonamyloidogenic S, M83 Tg mice develop a unilateral foot drop, which progresses to bilateral hind limb paralysis within a week of ons et. In human cases of Parkinsonism, it currently is not possible to correlate the onset of disease with induction of S pathology. Additionally there are cases of Parkinsonism in which no S pathology is present (Gaig et al. 2008; Gaig et al. 2007) However, apart from S pathology, there are other indicators of neurotoxicity in patients. Parkinsonism results in cell death, particularly seen in the dopaminergic neurons of the SNpc in PD pat ients. This cell death is accompanied by a massive

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197 neuroinflammatory response in the form of gliosis and microvascular proliferation, which also allows more opportunity for peripheral toxins and cytokine releasing cells to interact with neuronal cells (Barci a et al. 2004; Dunning et al. 2013; Fahn & Sulzer, 2004) Regardless, in the majority of Parkinsonism cases, the hallmark pathology is LBs/LNs associated with cellular toxicity and brain dysfunction, which can result in a myriad of neurodegenerative phenotypes, similar to the pathophysiol ogical course seen with prion disease (Dickson, 2012; Halliday et al. 2011; Puschmann et al. 2012) Alternative inclusion pathology induc tion The in vivo that there may be other processes occurring in tandem with a potential prionoid spread (Brundin et al. 2008) and that the current models proposed may be a simplified version of what is really occurring. Since most of the models are completed via intracerebral solely because of the amyloidogenic i noculum or also in part due to the procedure itself. Studies have shown that head trauma can result in the accumulation of certain (Uryu et al. 2007; Uryu et al. 2003) Also there is a separate field of study on chronic traumatic encephalopathy, in which patients who have suffered from head injurie other proteinopathies without any known specific predispositions (McKee et al. 2013) Recently, I have shown that intracerebral injections into mice has resulted in the intracellular accumulation of NFL and peripherin at the injection site, which is not seen in untreated or peripherally injected animals Therefore, this data indicates that direct

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198 bra in injection of S may be causing a disruption of cellular proteostasis resulting in the simultaneous accumulation and aggregation of multiple prote The major premise of prion disease is that an endogenous, soluble protein is able to misfold and at that point becomes a toxic agent of disease. The soluble form of the protein is unlikely to be a diseasetransmitting agent. I performed a n intracerebral 7182), that is unable to fibrillize (Giasson et al. 2001b; Waxman et al. 2009) and showed that at 8 months post injection into M20 transgenic mice overexpressing wild (Sacino et al. 2013a) I have als o shown that peripheral intramuscular injecti on of the same solubl e protein around 4 months post injection. In additional studies of intracerebral injection or peripheral adminis soluble controls either were not provided (Luk et al. 2012b) or reported to not induce (Luk et al. 2012a) The refore, this form of S that is soluble, natively unfolded, and incapable of seeding S pathology in culture, can seed pathology and transmit phe notypic changes in vivo albeit less efficiently. It could be argued that the truncation mutant is filling the role of the Protein X hypothesis, in that a protein not intrinsically capable of misfolding, is able to help catalyze the misfolding of another more prone protein (Telling e t al. 1995) Another possibility is that the deletion mutant i s able to undergo a conformational change in vivo which allows it to template the formation of S inclusion pathology.

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199 Although this finding does not discredit the ability of S to behave like a prion in the induction of pathology, it does show for the f irst time that the amyloidogenic form of the protein may behave in a similar manner. Future studies will have to piece apart the versus a general disruption of proteostasis causing accumulation of this aggregate prone protein. Is There Really a Prionoid S pread of S P athology? Misfolding of soluble proteins occurs as normal cell processes and has been seen as an evolutionary tool to adapt to environmental changes; however, the inability to refold properly, along with the subsequent accumulation and resulting toxic effects, is what makes PMDs detrimental. Post mortem studies on Parkinsonism patients along e arguably the most useful tools for studying its prionoid pro perties. From these studies, I have sheet, amyloidogenic form. This amyloidogenic form can serve as a nucleus to self periods to onset of disease and in the characteristics of the pathology induced, which is point of resistance to biological clearance, which allows for the formation and studies have

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200 phenotypes. Although studies have provided results indicating the prionoid behavior of additional mechanisms contributing to the induction and spread of S pathology must be addressed neurodegenerative phenotype in vivo albeit less efficiently and at a slower rate. 2) We still a re not in vivo Yes, it has been shown in one cell culture study ; however, in the in vivo studies, there are confounds (as discussed in the sections above) and even in the human graft studies, it inclusions vulnerable grafted neurons. 3) Separate from the idea of transmissibility at the cellular level, there is no evidence of infectivity at an organismal level. In other synucleinopathy. ehaving like a prion is not simple, and there are still pivotal questions that need to be answered, including discerning the trigger in naturally occurring disease that leads to permanent misfolding and intracellular accumulation of mechanism; however, this has not been convincingly demonstrated at this point. The fact that there are differences in vulnerability among different cellular populations for induction of pathology, and at the organismal level for development of disease, indicates that there are probably mult iple biological mechanisms intertwined to modulate

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201 induction and progression of disease, then this might enable the development of synuclei nopathies, but also for PMDs in general.

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202 Table 61 Su mmary of the prionoid characteristics of S Prion properties Prionoid characteristic Efficient propagation Both wild nucleus for templating the conver s amyloidogenic form following first order kinetics. Generation of different strains Wild type and mutant conformations, incubation periods, and inclusion morphologies in vitro in cell culture, and in vivo Resistance of biological Clearance the autophagic/lysosomal system and the proteasomal system, and by cellular proteases. Furthermore, the A53T and A30P mutants are able t o bind cell mediated autophagy receptors on lysosomal membranes to inhibit their degradation. Bioavailability multiple studies have reported that there is a correlation between certain environmental factors, such as heavy metal or pathology. Transport and spreading peripheral nerves of PD p atients, indicating neuroanatom ical spread to the brain intrastriatal fetal dopaminergic grafts in PD patients In animal models, intracererbral and intramuscular injection of athology. Cell culture studies have also shown that there is passaging of pathology, and neuron to neuron and neuron to astrocyte transmission Toxicity induction Overexpression of wild or fibrillar forms increase cellular toxicity via: proteasomal inhibition, altered signal transduction pathways, mitochondrial alterations, increased production and rel e ase of free radicals, transporters. This leads to neuronal loss and the stimulation of a neuroinflammatory response. Transmission of phenotypic changes The only study sho wing transmission of a phenotypic change mice which develop bilateral hind limb paralysis post intramuscular injection of both amyloidogenic and non

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231 BIOGRAPHICAL SKETCH Amanda Nicole Sacino was born in Fort Lauderdale, Florida in 1987 to parents Liberty and Mark Sacino, and is number one of two s iblings. She graduated from Cardinal Gibbons High School in 2005 and proceeded to Boston University in Boston, Massachusetts, where she earned a dual Bachelor of Science degree in Biochemistry and Molecular Biology, and Neurochemistry in 2009. She then entered the MD PhD program at the University of Florida College of Medicine. She completed the College of Medicines interdisciplinary program (IDP) in biomedical science and graduated in May 2014 with her PhD in Neuroscience.