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Understanding the Protective Mechanisms of Two Engineered Anti-Amyloid-Beta Agents in Drosophila

Permanent Link: http://ufdc.ufl.edu/UFE0045618/00001

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Title: Understanding the Protective Mechanisms of Two Engineered Anti-Amyloid-Beta Agents in Drosophila
Physical Description: 1 online resource (45 p.)
Language: english
Creator: Khare, Swati
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: alzheimers -- amyloid -- hsp70 -- scfv
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alzheimers disease (AD) is a progressive neurodegenerative disease with no current cure,affecting one in eight Americans above the age of 65 years. Symptomatic treatments with cholinesterase inhibitors, N-methyl-D-aspartate antagonists,anti-psychotic and anti-depressant drugs offer beneficial effects but do not address the cause of the disease. There is clearly a need for the development of effective treatments for AD. Disease modifying treatments aiming to target the root cause of AD are handicapped due to our incomplete knowledge of the mechanisms of AD pathogenesis. Through my Masters thesis, I analyzed the biochemical aspects of two anti-amyloid beta strategies in Drosophila to better understand the mechanisms underlying its toxicity. One of the strategies involves screening of single chain variable fragments (scFvs) against amyloid-beta 1-42 and the other involves the usage of a novel engineered secretable heat shock protein 70 (secHsp70) factor. Rescue data of the Drosophila AD eye phenotype has indicated robustness of these approaches thus being the motivation for thisstudy. I compared the distribution of the secHsp70 with cytosolic Hsp70 via immunostaining and found that it differs due to its ability to be secreted in the extracellular space. I used western blots and sandwich enzyme-linked immunosorbent assays (sELISAs) to study the distribution of amyloid beta 42 in detergent soluble or insoluble fractions and found a difference in its quantities across fractions. Preliminary results from these studies are a promising starting point to understanding the anti-amyloid effects of both engineered factors.
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.
Statement of Responsibility: by Swati Khare.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Ormerod, Brandi K.
Local: Co-adviser: Fernandez-Funez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045618:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045618/00001

Material Information

Title: Understanding the Protective Mechanisms of Two Engineered Anti-Amyloid-Beta Agents in Drosophila
Physical Description: 1 online resource (45 p.)
Language: english
Creator: Khare, Swati
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: alzheimers -- amyloid -- hsp70 -- scfv
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alzheimers disease (AD) is a progressive neurodegenerative disease with no current cure,affecting one in eight Americans above the age of 65 years. Symptomatic treatments with cholinesterase inhibitors, N-methyl-D-aspartate antagonists,anti-psychotic and anti-depressant drugs offer beneficial effects but do not address the cause of the disease. There is clearly a need for the development of effective treatments for AD. Disease modifying treatments aiming to target the root cause of AD are handicapped due to our incomplete knowledge of the mechanisms of AD pathogenesis. Through my Masters thesis, I analyzed the biochemical aspects of two anti-amyloid beta strategies in Drosophila to better understand the mechanisms underlying its toxicity. One of the strategies involves screening of single chain variable fragments (scFvs) against amyloid-beta 1-42 and the other involves the usage of a novel engineered secretable heat shock protein 70 (secHsp70) factor. Rescue data of the Drosophila AD eye phenotype has indicated robustness of these approaches thus being the motivation for thisstudy. I compared the distribution of the secHsp70 with cytosolic Hsp70 via immunostaining and found that it differs due to its ability to be secreted in the extracellular space. I used western blots and sandwich enzyme-linked immunosorbent assays (sELISAs) to study the distribution of amyloid beta 42 in detergent soluble or insoluble fractions and found a difference in its quantities across fractions. Preliminary results from these studies are a promising starting point to understanding the anti-amyloid effects of both engineered factors.
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.
Statement of Responsibility: by Swati Khare.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Ormerod, Brandi K.
Local: Co-adviser: Fernandez-Funez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045618:00001


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1 UNDERSTANDING THE PROTECTIVE MECHANISMS OF TWO ENGINEERED ANTI AMYLOID AGENTS IN DROSOPHILA By SWATI KHARE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Swati Khare

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3 To everyone who has inspired me in one way or the other

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4 ACKNOWLEDGMENTS First and foremost, I would like to thank my mentors Drs. Pedro Fernandez Funez and Diego Rincon Limas for their constant support guidance and I extend my gratitude to my committee chair, Dr. Brandi Ormerod for her valu able advice and support at various occasions. I would like to thank Dr. Keselowsky for discussions and his inspiring and interesting class on biomaterial immunomodulation. I cann ot thank the members of the lab enough for sharing their expertise wit h me and for being kind and patient. The postdocs Jonatan (he would have earned a million dollars if he had charged me one dollar per question), Yan for guiding and teaching me biochemical assays, Kurt for his perspective, expertise and the soundtrac k music and Alfonso for his advice with designing experiments, writing, statistics and the party music. Graduate students Krishanu, Amrutha and Jose have been great friends and excellent lab mates, I thank them for that. I thank Brittney Otero, the extr emely talented undergraduate volunteer who has helped me in numerous ways Last but not the least, I want to thank my family (Mamma, Papa, Ankit and Nithya) who have unfailingly believed in me and have been the best support system I could have asked for. H eidi, Vikram, Mini and Hridis have contributed to the richness of my life and have been excellent friends. I am grateful to them for their kindness

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATION S ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 ................................ ................................ ............................... 11 Amyloid Cascade Hypothesis ................................ ................................ ................. 11 Treatments for AD ................................ ................................ ................................ .. 12 Immunotherapeutic Applications in AD ................................ ................................ ... 13 Active and Passive Immunotherapy ................................ ................................ 13 Single ............................ 14 ................................ ................................ ............... 15 Heat Shock Protein 70 and its Role in Neurodegenerative Diseases ............... 15 Hsp70 and AD ................................ ................................ ................................ .. 15 Drosophila Models for AD ................................ ................................ ....................... 16 Mechanistic Studies of Anti Amyloid Strategies ................................ ...................... 16 ScFvs in a Drosophila AD Model ................................ ................................ ...... 16 Secretable Hsp70 in a Drosophila AD Model ................................ ................... 17 2 MATERIALS AND METHODS ................................ ................................ ................ 18 Dros ophila Genetics ................................ ................................ ............................... 18 Generation of ScFv Constructs and Transgenic Flies ................................ ...... 18 Generation of Secretable Hsp70 (secHsp70) Constructs and Transgenic Flies ................................ ................................ ................................ .............. 18 Stocks ................................ ................................ ................................ ............... 18 Drosophila Eye images ................................ ................................ ........................... 19 Immunostaining ................................ ................................ ................................ ...... 19 Protocol ................................ ................................ ................................ ............ 19 Antibodies ................................ ................................ ................................ ......... 19 Western Blots ................................ ................................ ................................ ......... 20 Preparation of Homogenates ................................ ................................ ............ 20 Immunoblotting Protocol ................................ ................................ ................... 20 Antibodies ................................ ................................ ................................ ......... 21 ................................ ................................ ........... 21 ................................ ................................ ................. 21 Quantification of SDS ................................ ....... 22

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6 Statistical Analysis ................................ ................................ ................................ .. 23 3 RESULTS ................................ ................................ ................................ ............... 24 ................................ ................................ ................................ ..... 24 Specific ScFvs Rescues the Ey e Phenotype Induced by ................................ ................................ ................................ .............. 24 Co ................................ ................................ .... 25 ................................ ................................ .......... 26 Effect of ScFvs on SDS ................................ ............................. 27 ................................ ................................ ............................... 28 Distribution of SecHsp70 ................................ ................................ .................. 28 Expression of SecHsp70 Rescues the Eye ........ 29 Co ................................ ............................ 30 ................................ ................. 32 Analysis of TBS SDS and FA .. 33 .............. 34 4 DISCUSSION ................................ ................................ ................................ ......... 36 LIST OF REFERENCES ................................ ................................ ............................... 40 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 45

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7 LIST OF FIGURES Figure page 3 1 Expression of ScFvs. ........................... 24 3 2 Co ................................ ..................... 25 3 3 .............................. 26 3 4 Quantification of SDS ................................ ............... 28 3 5 Distribution of SecHsp70. ................................ ................................ ................... 29 3 6 ..................... 30 3 7 Co localization of SecHsp70 and ................................ ............................. 31 3 8 SecHsp70 or Hsp70 or ER Chaperone BiP. ................................ ....................... 32 3 9 SecHsp70 or Hsp70 or ER Chaperone BiP. ................................ ....................... 33 3 10 Western Blots of TBS, 1% SDS and FA Soluble Fractions. ................................ 34 3 11 Quantification of SDS ................................ ................ 35

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8 LIST OF ABBREVIATIONS AD APP Amyloid precursor protein Amyloid beta BSA Bovine serum albumin cDNA complementary DNA FA Formic acid Gmr Glass multiple reporter HRP Horse radish peroxidase Hsp70 Heat shock protein 70 IgG Immunoglobulin G NA Numerical Aperture PBS Phosphate buffered saline PBST Phosphate buffered saline + Triton X PI Protease Inhibitor PSEN1 Presenilin 1 PSEN2 Presenilin 2 scFv Single chain variable fragment SDS Sodium dodecyl sulfate secHsp70 Secretable Hsp70 TBS Tris buffered saline UAS Upstreaming activating sequence sELISA sandwich enzyme linked immunosorbent assay

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science UNDERSTANDING THE PROTECTIVE MECHANISMS OF TWO ENGINEERED ANTI AMYLOID AGE NTS IN DROSOPHILA By Swati Khare May 2013 Chair: Brandi Ormerod Co chair: Pedro Fernandez Funez Major: Biomedical Engineering with no current cure affecting one in eight Americans above the age of 65 years. Symptomatic treatments with cholinesterase inhibitors, N methyl D aspartate antagonists, anti psychotic and ant i depressant drugs offer beneficial effects but do not address the cause of the dise ase There is clearly a need for the development of effective treatments for AD. Disease modifying treatments aiming to target the root cause of AD are handicapped d ue to our incomplete knowledge of the mechanisms of AD pathogenesis. esis, I analyzed the biochemical aspects of two anti amyloid beta strategies in Drosophila to better understand the mechanisms underlying its toxicity One of the strategies involves s creening of single chain variable fr agments (scFvs) against amyloid 1 42 and the other involves the usage of a novel eng ineered secretable heat shock p rotein 70 (secHsp70) factor Rescue dat a of the Drosophila AD eye phenotype has indicated r obustness of the se approaches thus being the motivation for this study. I compared t he distribution of the secHsp70 with cytosolic Hsp70 via immunostaining and found that it differs due to its ability to be secreted in the

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10 extracellular space I used western blots and sandwich enzyme linked immunosorbent assays (sELISAs) to study the distribution of amyloid beta 42 in detergent soluble or i nsoluble fractions and found a difference in i ts quantities across fractions. Preliminary results from these studies are a promising starting point to un derstand ing the anti amyloid eff ects of both engineered factors.

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11 CHAPTER 1 INTRODUCTION isease (AD) is a lethal incurable neurodegenerative d ementia accounting for about 60% of the total dementia cases worldwide AD is increasingly gaining importance as a medical and social problem with 5.4 million Americans suffering from it as of 2012 and healthcare costs of about 200 billion USD in the US. Of the total number of people with AD, 4% fall under the age group of 65, with the rest of the AD patients being over 65 years of age ( The Alzheimer's Association report 2012) The progression of AD is classified into five stages preclinical AD, mild cognitive imp airment, and mild, moderate and severe dementia. AD symptoms include progressive memory loss, cognitive dysfunction affecting judgment and decision making, language deficits, dis orientation and social symptoms such as withdrawal from activities, depression, apathy, delusions and anxiety. Many of these symptoms warrant patient care and in 2011 it was estimated that 15 million friends and relatives of AD patients in the US provided unpaid care for 17.4 billion hours va lued at more than 210 billion USD ( The Alzheimer's Association report 2012) Amyloid C ascade H ypothesis The causes of AD are not well understood. Our current understanding of the etiology is based on the genetic and pathological hallmarks of AD, which include the a ccumulation of senile (neuritic) plaque s and neurofibrillary tangles These hallmarks are not restricted to AD patients and can also be found in cognitively normal older adults. H owever, the heightened density of this pathology in AD helps discriminate it from normal aging (Price et al., 2009) The identification of amyloid beta ( ) in senile

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12 plaques (Glenner and Wong, 1984) was th e first clue for the amyloid cascade hypothesis. This favored hypothesis proposes that the a ccumulation of the peptide is the initial pathological event leading to AD (Hardy and Selkoe, 2002) The identification of m utations on Amyloid precursor p rotein ( APP ) and P resenilin s ( PSEN1 and PSEN2 ) linked to early onset familial AD cases (Goate et al., 1991; Sherrington et al., 1995) contributed to the formulation of the a myloid cascade hypothesi s The p athogenic mechanisms of AD have not been clearly elucidated but APP metabolism is suggested to be the first step in the disease cascade. and secretase s via the amyloidogenic pathway to form heterogeneous including aggregation prone, neurotoxic which is attributed as a major factor in AD pathology (Hardy and Selkoe, 2002; Iijima et al., 2004; Arimon et al., 2005) Neurotoxi city of has not been conclusively explained but recent studies point towards soluble oligomeric forms as causative agents (Shankar et al., 2008; Ono et al., 2009; Brouillette et al., 2012) Treatments for AD S ymptomatic treatments currently available for AD include t hree cholinesterase inhibitors (donepezil, galantamine and rivastigmine ) for m ild AD (Birks, 2006) These are acet ylcholinesterase blockers, and thus inhibit the breakdown of acetylcholine, an important neurotransmitter associated with memory. The N methyl D aspartate antagonist (m emantine) prescribed for moderate to severe AD acts by preventing neuronal damage caused by excitotoxicity (McShane et al., 2006) Antidepressant and a ntipsychotic drugs are used to control AD behavioral symptoms (Ballard et al., 2009) These drugs exhibit beneficial effects against cognitive decline but do not alter disease

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13 progression T hus there is a dire need for the development of disease modifying therapies against AD Novel disease modifyin g therapies involving a tau pathology and mitochondrial dysfunction are currently undergoing efficacy and safety testing in clinical trials The anti amyloid strategies include : (i ) drugs that production by inhibiting/modulating and secretase (Tomita, 2009; Tang and Ghosh, 2011; D'Onofrio et al., 2012) as well as secretase activators (Marcade et al., 2008) ; (ii ) drugs that (Adlard et al., 2008; Lannfelt et al., 2008) ; and (iii ) strategies that which include active and passive immunotherapy. Many of these strategies have fail ed in clinical trials due to adverse side effects or low efficacy (Gauthier et al., 2009; Imbimbo and Giardina, 2011) while some others are in early clinical trial phases (Mangialasche et al., 2010) Immunotherapeutic A pplications in AD Active and Passive I mmunotherapy Immunothe rapy has gained popularity as a potential treatment option for AD over the past decade E xciting pre clinical data (Schenk et al., 1999; Bard et al., 2000) le d to phase II clinical trials f or active immunizatio n with pre aggregated (AN1792 QS21) The discovery of 6% instances of meningoencephalitis in patients raised safety concerns with this strategy. These adverse effects have been explained by T cell autoimmune response activation. However, the efficacy levels looked promising with slower cognitive decline and decreased cerebrospinal fluid levels of tau (Gilman et al., 2005) Passive immunotherapy is considered a safer, more controllable alternative Passively administered antibodies against reduced the amyloid load in an AD

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14 mouse model (Bard et al., 2000) The mechanism of action of these antibodies was initially hypothesized to be via fragment c rystallizable (Fc) receptor mediated phagocytosis but further studies indicate d that non Fc mediated clearance mechanisms were also involved (Bacskai et al., 2002) The use of full antibodies poses new concerns like reduced blood brain barrier penetration and adverse side effects like cerebral hemorrhage (Pfeifer et al., 2002) Recent failures of passive immunotherapy trials using humanized anti monoclonal antibodies Bapineuzumab and Solanezumab (Aisen and Vellas, 2013) have increase d the interest in engi neering anti antibodies to increase their efficacy and safety (Galimberti et al., 2013) Single Chain Variable F ragments isease Recent s tudies indicate that it may not be necessary to use complete anti antibodies for their specific action and that using fragments of effective antibodie s can potentially reduce side effects (Tamura et al., 2005) Single chain variable fragments ( scFvs ) comprise the variable heavy and light chain s of an antibody connected through a peptide linker (Ahmad et al., 2012) scFv s retain the antigen binding capacity of the original, complete antibody that they are derived from In vitro s tudies showed the efficacy of engineered scFvs in their binding action (Zameer et al., 2006) followed by in vivo studies with promising results of alleviating AD pathologies by different scFvs (Marn Argany et al., 2011) Recombinant anti scFvs (scFv 9 against f ibrillar 1 42 scFv 35 42 and scFv 40.1 against 1 40 ) injected i n brains of CRND8 mi ce displaying AD like phenotype s led to reduce d and reduced plaque formation (Levites et al., 2006) Despite the se evaluations of AD pathologies, neurodegeneration was not assessed because APP mouse models display little or no neuronal cell death Studies of these scFvs on

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15 neurotoxicity will provide us with important clues regarding efficacy of these amyloid binding agents. Engineered Agents a Heat Shock P rotein 70 and its Role in Neurodegenerative D iseases The Heat shock protein 70 ( Hsp70 ) belongs to a family of molecular chaperones that play an important role in protein folding during protein synthesis (Georgopoulos and Welch, 1993) Hsp70 is mostly found intracellularly and is induced by a variety of stressful conditions like hyperthermia, oxidative stress, etc. Previous research in a Drosophila model for polyglutamine disease indicated a beneficial neuroprotective role of Hsp70 (Warrick et al., 1999) Similar beneficial roles of Hsp70 have been noted in Drosophila and mouse models of Parki (Auluck et al., 2002; Klucken et al., 2004) Hsp70 and AD Elevated levels of Hsp70 expression have been observed in AD brains (Muchowski and Wacker, 2005) towards the involvement of Hsp70 in the mediated toxicity (Magran et al., 2004) In vitro studies suggest that Hsp70 suppress es possibly through its interaction with the toxic oligomeric species but not fibrils. It has therefore assembly (Evans et al., 2006) In vivo studies with AD phenotypic mice expressi ng mutant APP ( APPswe ) and overexpressing intracellular Hsp70 showed reduced AD like cogn itive phenotypes (Hoshino et al., 2011) The exact mechanism of action of this observed improvement in cognition and the effects on neurodegeneration are not well elucidated and t he role interactions and compartmentalization of in AD remain a mystery Hsp70

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16 seems to be a promisin g candidate for engineering molecular therapeutic approaches against AD. Drosophila M odel s for AD Drosophila is a time and cost efficient animal model for studying di sease pathogenesis and effects of therapeutics (Rincon Limas et al., 2012) S everal independent models are available for studying AD in Drosophila (Finelli et al., 2004; Iijima et al., 2004; Crowther et al., 2005; Iijima and Iijima Ando, 2008; Casas Tinto et al., 2011) Drosophila AD models of fer several advantages like fast generation time and high progeny allow ing us to screen rapidly for potential therapeutics. Genet ic manipulations are easy and quick in the flies increasing the po ssib ilit y of exploring combinat orial effects of genes/therapeutics F or studying AD, most mouse models mimic pathogenic mutations in APP which restricting the potential to study effects of the ind ividual toxic/non toxic species. Drosophila AD flies exhibit pathological characteristics of AD like extensive neuronal cell loss (Iijima and Iijima Ando, 2008) Further, the bipartite Gal4/upstream activat ing sequence (UAS) in Drosophila allows for superior spatial control of gene expression (Elliott and Brand, 2008) Mechanistic S tudies of Anti Amyloid S trategies Two potential AD therapeutic strategies described below are c urrently under study in our lab S cFvs in a Drosophila AD M odel Two scFvs (213 and Ab9) against were previously characterized in mice (Levites et al., 2006) and introduced in Drosophila in an attempt to set up a rapid screen ing system for the effectiveness of recombinant scFvs and to study neurotoxicity

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17 effects. Co expression of both scFvs ( 213 and Ab9 ) which was not previously tested in the mouse model was also explored in the fly study S ecretable Hsp70 in a Drosophila AD M odel mostly intracellular, a secretable form of H sp70 was engineered and introduced in the Drosophila model. A signal peptide attached to Hsp70 would allow the protein to be transported outside the cells and thereby be available for possible interaction with the During the course of my reliminary studies with the Drosophila AD model led to observations of rescue of the eye phenotype in both cases This convinced me to pur sue biochem ical studies to decipher the underlying mechanism (s) of actio n with a hope to further our understanding about and the potential application in future AD therapies

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18 CHAPTER 2 MATERIALS AND METHODS Drosophila G enetics Generation of ScFv C onstructs and Transgenic F lies Two anti scFvs ( 1 42 and 35 42 ) were selected for analysis of effects on in Drosophila cDNA s of the scFvs ( a gift from Dr. Todd Golde, University of Florida) were inserted in to the pUAST vector for micro injection in yellow white flies at Rainbow Transgenics (CA) followed by selection of transgenic flies expressing a copy of scFv Ab9 and scFv 213 individually and co expressing scFv Ab9 + 213 (Krishanu Mathur, Biomedical Engineering, University of Florida). Generation of Secretable Hsp70 (secHsp70) C onstructs and Transgenic F lies The SignalP 4.0 program (Petersen et al., 2011) was used to select the p repoinsulin signal peptide for attachment with human Hsp70 (HSPA1L, a gift from N. Bonini) The cDNA was cloned in the Drosophila expression vector pUAS T and the resulting pUAS T secHsp70 plasmid was microinjected into Drosophila yellow white embryos at Rainbow Transgenics (CA) Ten independent lines were established from which the strongest expressors of secHsp70 were selected (Dr. Diego Rincon Limas and S hei la Emani, University of Florida) Stocks UAS stocks carrying the cDNAs for the reporter gene La cZ, cytoplasmic Hsp70 (Warrick et al., 1999) and the ER chaperone BiP were obtained from the Bloomington Drosophila Stock Center (flystocks.bio.indiana.edu). The mushroom body specific driver (OK107 Gal4) and eye specific driver (gmr Gal4) were also obtained from this stock center Transgenic flies expressing two copies of (Casas Tinto et al., 2011) under

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19 control of relevant driver s were crossed with the scFv and Hsp70 stock flies described above Gmr Gal4 crosses were made at 28.5C and day 1 flies were stored at 20C for eye imaging. OK107 Gal4 crosses were maintained at 27C and the hea ds of day 1 and day 20 flies were separated and frozen at 80C for biochemical analyses All flies and larvae were grown in vials containing jazz mix Drosophila food (Fisher Scientific). Drosophila E ye imag es Fresh eye images of one day old female flies were taken with a Leica Microsystems MSV266 microscope Z stacks of the whole eye were taken and merged to form a montage using the Leica Application Suite version 4.1. Immunostaining P rotocol E ye imaginal discs of larva e and brains of adult flies we re dissected in cold 1X p hosphate b uffered s aline (PBS) followed by 30 min fixation in 4% formaldehyde. The tissue was washed thrice for 10 min each in 1X PBS with 0.1% TritonX 100 ( PBS T) follo wed by blocking in PBST containi ng 0.3% b ovine serum albumin ( BSA) for 30 min. Overnight incubation with primary antibody three 10 min washes with PBST and 30 min blocking with PBST BSA ensued. Secondary antibody treatment followed for 2 hr after which the tissue was mounted on slides with Vectashield ( Vector Labor atories, CA). Fluorescent images were acquired using a Carl Zeiss ApoTome microscope with 2 0X (NA 0.8) and 63X (NA 1.4) objectives T hese images were analyzed with the AxioVision software C orresponding figures were prepared in Adobe Photoshop. Antibodies amyloid 1 7 24 Covance) was used to detect and SPA 812 (1: 250 inducible Hsp70, Enzo Life Sciences) was used to detect Hsp70 c Myc chicken

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20 antibody ( Invitrogen ) was used to detect the myc tagged scFvs. Cy3 (1:600, Jackson Imm unoResearch Laboratories) an d Alexa 488 (1:200, Invitrogen ) were used as secondary antibodies. Western Blots Preparation of H omogenates Ten to fifteen f rozen fly heads were homogenized in a mix ture of 28.5 l of Pierce RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP 40 1% sodium deoxycholate, 0.1% sodium dodecyl s ulfate ( S DS ) ) and 1.5 l of 20X Protease inhibitor ( PI complete Mini, EDTA free Protease Inhibitor cocktail, Roche Diagnostics GmbH) using a Kontes motorized pestle The sample s were centrifuged at 13,000 rpm f or 1 min and the supernatants were collected in separate tube s 10 l of l oading buffer (NuPAGE LDS Sample buffer 4X) was then added to each sample followed by heating the mixture at 95C for 5 min Immunoblotting P rotocol The samples were then separated for 80 min on 12% NuPAGE Bis Tris precast gels in the XCell SureLock Mini Cell Electrophoresis System filled w ith NuPAGE MES Running Buffer. The separated proteins were transferred to supported nitrocellulose membranes of 0 .2 m ( Bio Rad ) using the Trans Blot Cell transfer system ( Bio Rad ) for half an hour at 800mA, utilizing the transfer buffer (25 mM Tris, 192 mM Glyc ine, 20% w/v methanol, pH 8.3) For detection, the membrane was boiled for 5 min i n 1X PBS and then b locked for 30 min in 5% non fat dry milk in Tris buffered saline with 0.1% Tween 20 (TBST). The blocked membrane was exposed to the primary antibody overnight at 4C. Then, the membrane was washed thrice for 10 min each in TBST Secondary antibody trea tm ent followed for 2 h after which the membrane was washed

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2 1 thrice for 10 min each with TBST. The immunoblots were then exposed to SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) for 5 min and then developed using the Konica Medical film processor SRX 101A system to detect the protein of interest. Antibodies Mouse antibody amyloid 1 16, Covance) was used for detection of at around 4 kD a. M ouse anti tubulin antibody ( 1:5 000 000, Sigma Aldrich) was used to detect th e loading marker tubulin at about 55 kD a in all samples. Rabbit SPA 812 (1:1000, inducible Hsp70, Enzo Life Sciences) was used to detect Hsp70 at 70 kD a. Goat anti mouse HRP or g oat anti rabbit HRP IgG ( 1:2000, Jackson ImmunoResearch ) secondary antibodies were used against the primary antibodies Total Quantification A ssays Ten fly heads of the relevant genotypes were homogenized and subjected to western blotting as d escribed in above The i ntensity of the bands was determined using ImageJ ( http://rsbweb.nih.gov/ij/ ) for triplicate runs. The intensity of bands was normalized against the loading control tubulin. Means and standard deviations (SD) were calculated and used to plot column graphs using Microsoft Excel. Separation of F ractions To separate into detergent soluble and insoluble fractions t he first fraction was prepared by homogenizing 13 fly heads (8 females + 5 males) of the relevant genotypes in 28.5 l 1X Tris buffered saline (TBS) + 1.5 l 20X PI using a Kontes motorized pestle followed by sonication for 2 min in the Branson 1510 sonicator The homogenized samples were then centrifuged at 21 1 00 x g 4C in a Thermo Scientific Sorvall Legend Micro 21R centrifuge for 20 min The supernatant was collected as

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22 and the pellet s were washed with 1X TBS followed by centrifugation in the aforementioned conditions. The supernatant from this step was discarded. 28.5 l of 1% SDS in water + 1.5 l 20X PI was then added to the pellets and the samples were vortexed fo r 2 min using the Labnet Vortex mixer. The samples were centrifuged as per previously mentioned conditions and the supernata nt was collected A wash with 1% SDS and centrifugation for 20 min followed with the supernatant from this step being discarded. 10 l of LDS sample buffer 4X was added to the fractions 1 and 2 followed by heating the samples at 95C. These samples were stored at 20C until further use. To the pellet remaining after the SDS wash, 20 l 70% formic a cid (FA) was added foll owed by vortexing and sonicating for 2 min each. The centrifugation step was repeated and the supernatant was collecte d. The samp les were neutralized by adding 180 l of 2M Tris HCl pH 9.1. For protein pr ecipitation, 800 l of ice cold methanol was added to each sample followed by overnight incubation at 20 C. The protein suspensions were centrifuged at 21100x g at 4C for 30 min The supernatant was discarded without disturbing the pellet The pellet was re suspended in 25 l of RIPA buffer with addition of LDS buffer and then heated. For qualitative analysis, a ll three fractions were loaded in 12% Bis tris gels for immunoblotting Quantification of SDS S oluble by S andwich ELISA The SDS soluble fract ions were quantitatively analyzed using sandwich ELISA (Dr. Yona Levites, Kim et al., 2008) The primary monoclonal antibody A b 2.1.3 (human 42 specific ) was used to capture and detection was done by HRP conjugated mAb Ab9 Five biological re plicates were analyzed and averaged.

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23 Statistical Analysis Western blot d ata exported from ImageJ was analyzed by calculating means and standard deviations using Micros oft Excel. GraphPad Prism was used to carry out o ne way ANOVA statistical analysis to determine differences in total levels for separate time points Two way ANOVA technique was used to analyze quantitative data obtained from s ELISA to compare between genotypes and different time points.

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24 CHAPTER 3 RESULTS S cFvs and Expression of S pecific ScFvs R escues the Eye Phenotype I nduced by The scFvs Ab9 and 213 showed a decrease in the AD pathology in mouse models (Levites et al., 2006) T o establish a rapid in vivo screening syst em for such scFvs and to study neurotoxicity we utilized a Drosophila AD model expressing two copies of (Casas Tinto et al., 2011) The Drosophila eye provides a quick screen for e ffects on neurotoxicity as a strong AD eye phenotype develops in young flies. W e co expressed the scFvs and specifically in the eyes of flies using the gmr Gal driver. The non control flies show ed well organized eyes with neatly arranged ommatid ia (Control, Figure 3 1). The flies expressing had small disorganized eyes with necrotic spots and depigmentation ( /LacZ Figure 3 1) The expression of scFvs 213, Ab9 and Ab9 + 213 show ed a partial rescue of this phenotype (Figure 3 1, K. Mathur ) We found a reduction in the number of necrotic spots better organization of the eye and lesser depigmentation in all three cases T he flies expressing the combination scFv Ab9 + 213 showed the maximum rescue followed by those expressing scFv s Ab9 and 213 Figure 3 1. Rescue of the Eye Phenotype by Expression of S cFvs. A reduction in the number of necrotic spots and better organization of the ommatidia can be seen in the flies co expressing scFVs (213, Ab9 and Ab9+213) and

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25 Co localization of S cFv and The rescue of the AD eye phenotype was the first clue that the scFvs were correctly expressed folded, and secreted in Drosophila Since the scFvs are A specific binding agents we decided to study their localization profiles in our Drosophila AD model that expresses extracellular We expressed scFv Ab9 + 213 and in the fly brain using the OK107 Gal4 driver and immunostained for scFvs (c myc, green) and ( 4G8, red) A cluster of interneurons in the antennal lobe (Whole brain, Figure 3 2) showed the overlap of both fluorescent signals along the secretory pathway T hus the scFvs co localized with supporting the idea that the scFvs suppress neurotoxicity by binding to (Figure 3 2 Dr. Pedro Fernandez Funez) Figure 3 2. Co localization of S cFv Ab9+213 and A cluster of interneurons in the brain specifically driven to co express (4G8, red) and scFv Ab9+213 (myc, green) show co localization of the proteins.

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26 Effect of S cFvs on T otal Previous studies in AD mouse models suggest that one possible mechanism of rescue of the AD phenotype by A specific scFv s is by reducing the levels of (Levites et al., 2006) T o determine if the rescue of the phenotype in AD flies expressing scFvs was due to a reduction in the A prote in level, I quantified total by western blot (Figure 3 3) The negative control of non transgenic flies did not show cross 1 16 ) whereas flies expressing produced a specific band at 4kD which is the expected band size of One way ANOVA of t he levels of total in flies expressing the scFvs show ed no significant d ifference s against the controls in triplicate western blots (Figure 3 3 ) when normalized to the internal loading cont rol (tubulin) Hence the mechanism of action of the scFv rescue in the Drosophila AD model was not due to the reduction in total levels Figure 3 3 Quantification of T otal eads. A) Immunoblot for and tubulin B) Mean of band intensity normalized against tubulin as loading control. The bars indicate SD from triplicate blots.

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27 Effect of S cFvs on SDS S oluble Another possible mechanism of action of the scFvs may induce a change in the levels o f pathogenic species of Earlier reports suggested that highly aggregated fibrillar A is the most pathogenic species in AD (Stphan et al., 2001) but recent studies point toward s the role of soluble oligomers in disease pathogenesis (Brouillette et al., 2012) W e investigated the distribution of in the detergent soluble and insoluble fractions to understand how the scFv s rescue neurotoxicity I extracted the protein in TBS, SDS and FA fractions as described in the Materials and methods section Q uantification of the SDS soluble in AD flies expressing the scFvs by sELISA revealed no significant difference ( using one way ANOVA) in the soluble levels in day 1 flies. However, there was a significant increase in the levels of soluble in AD flies expressing scFv 213 and the Ab9+213 combination when compared to LacZ controls at day 20 (Figure 3 4A). I did not find a significant difference in the soluble levels between the different time points of each genotype using GraphPad Prism two way ANOVA (Figure 3 4B). The TBS and FA fractions are yet to be analyzed by s ELISA a nd we are hoping to get further clues about the rescue mechanism through tho se results. Further studies comparing the effect of age on neurodegeneration will also be required to help explain the differences in soluble with age.

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28 Figure 3 4 Quantification of SDS S oluble by sELISA. A) scFv, day 1 flies reveal no significant differences in quantity of SDS soluble Day 20 flies show significant difference when comparing LacZ with 213 and *p<0.05 versus con trol, **p<0.01 versus control. B) No significant difference was noticed in the levels of SDS soluble between different time points for each genotype. Error bars indicate SD. S ecHsp70 and Distribution of S ecHsp70 Our lab created a novel, engineer ed secHsp70 construct to replicate the extracellular compartmentalization of Preliminary s tudies carried out by Dr. Rin c on Limas determined that secHsp70 was indeed being secreted out of cultured S2 cells in vitro (data not shown) To confirm the secretable nature of secHsp70 in vivo, we compared its distribution against the cytosolic Hsp70 in larvae. We specifically expressed Hsp70 and secHsp70 in the posterior part of larval eye discs using the gmr Gal4 driver Following i mmunostaining analysis we noticed that th e distribution of Hsp70 (Hsp70, Figure 3 5) was restricted to the gmr expression pattern in the posterior part of the eye disc (gmr Gal4, Figure 3 5) In contrast, secHsp70 appeared to diffuse into other parts of the eye disc and the ad joining antennal disc ( secHsp70, Figure 3 5 ). This result confirm ed that the secHsp70 differ s from the cytosolic form of Hsp70 in its distribution and is secreted to the extracellular space, thereby facilitating this diffusion.

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29 Figure 3 5. Distribution of S ecHsp70. The larval eye discs are stained with 6E10 against in red and SPA 812 against Hsp70 in green. The staining against the gmr Gal4 driver. SecHsp70 diffuses th rough the eye disc into the antennal disc and shows a distribution different from Hsp70 which is localized in the eye disc. Expression of S ecHsp70 Rescues the Eye Phenotype I nduced by On the basis of earlier reports indicative of a possible neuroprot ective role of Hsp70 in AD, we tested the effect of overexpression of secHsp70 against the eye phenotype of the Drosophila AD model using the gmr Gal4 driver. Preliminary studies at 27C revealed a potent rescue of the eye phenotype by secHsp70 (data not s hown) We chose to push the system with increased expression levels by rearing the flies co expressing secHsp70 or Hsp70 and at a higher temperature ( 28.5C ) to see if the rescue could still be maintain ed I imaged the eyes of young flies and noticed that the control flies expressing showed higher disorganization and reduction in size along with necrotic spots compared to the flies grown at 27C To our delight, even at a higher temperature, the eyes of secHsp70 f lies ( /secHsp70, Figure 3 6 ) showed significant rescue of the eye phenotype with better organization of cells,

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30 minimal necrotic spots and eye shape and size comparable to the LacZ control not expressing (Control, Figure 3 6 ) /Hsp70 flies s howed less er rescue compared with the /secHsp70 flies with more necrotic spots a nd disorganization of ommatidia, but were still better than the control flies ( /Hsp70, Figure 3 6 ). These results prove that secHsp70 has a powerful neuroprotective role against overexpression of in our Drosophila model. Figure 3 6. Rescue of the Eye Phenotype by Expression of S ecHsp70. A reduction in the number of necrotic spots and better organization of the ommatidia can be seen in the flies co expressing secHsp70 and A relatively lesser rescue is shown by flies co expressing Hsp70 and with some necrotic spots and more disorganization compared to the secHsp70 flies. Co localization of S ecHsp70 and Promising results of the AD eye phenotype rescue led us to speculate that the localization of secHsp70 may play a role in the mechanism of action as both and secHsp70 are found in extracellular compartments in our model. We expressed Hsp70 or secHsp70 in the AD fly brain using the OK107 Gal4 driver and immunostained for Hsp70 or secHsp70 (SPA 812 green) and ( 4G8, red). The pattern of localization of Hsp7 0 ( Hsp70, Figure 3 7) seemed predominantly cytosolic as expected and

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31 was distinct from that of secHsp70. We observed co localization of secHsp70 and as indicated by the overlap of fluorescent signals along the secretory pathway in a cluster of l ocal interneurons in the antennal lobe ( secHsp70, Figure 3 7) It was thus clear that the secHsp70 co localized with in brain neurons a key prerequisite to demonstrate that secHsp7 0 protects by directly binding to (Figure 3 7 Dr. Pedro Fernandez Funez). Figure 3 7. Co localization of S ecHsp70 and A cluster of interneurons in the whole brain specifically driven to co express (4G8, red) and secHsp70 (SPA 812, green) show co localization of the proteins.

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32 SecHsp70 D oes Not A ffect T otal L evels To test the hypothesis that secHsp70 may offer protection against the AD phenotype by reduc ing total I quantified the amount of total in fly heads co expressing and secHsp70 in the fly brain via the OK107 Gal4 system using triplicate immunoblots When compared with the controls, the levels of total were not significantly different in the secHsp70 or Hsp70 flies in day 1 (Figure 3 8 ) and day 20 flies (Figure 3 9 ) Thus, reduction in total is proba bly not the mechanism of action of alleviating neurotoxicity by secHsp70. Figure 3 8. Quantification of T otal in D ay 1 F lies C o expressing and S ecHsp70 or Hsp70 or ER C haperone BiP. A) Immunoblot for and tubulin B) Mean of band intensity normalized against tubulin as loading control. The bars indicate SD from triplicate blots.

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33 Figure 3 9. Quantification of T otal in Day 20 Flies C o expressing and SecHsp70 or Hsp70 or ER C haperone BiP A) Immunoblot for and tub ulin B) Mean of band intensity normalized against tubulin as loading control. The bars indicate SD from triplicate blots. A nalysis of TBS SDS and FA Soluble F ractions of b y Western B lots The roles of different soluble/insoluble species of are not well understood in the context of AD. To test how the different fractions of are affected in the secHsp70 rescue setup, I separated the protein from LacZ, secHsp70 and Hsp70 flies into three fractions: TBS soluble, SDS soluble and FA soluble Immunoblotting these fractions with 6E10 ( 1 16) revealed an increase in the amount of with age of flies in all fractions (D1 versus D20, Figure 3 10) The TBS fraction showed a decrease in in the secHsp70 and Hsp70 day 1 flies (TBS fraction, Figure 3 10). I did not notice a difference in the levels of SDS soluble fractions within genotypes for day 1 and day 20 flies (SDS fraction, Figure 3 10). Moreover, I noticed an increase in the amount of FA soluble protein in the da y 20 secHsp70 flies when compared to the control ( secHsp70, FA fraction, Figure 3 10) There was also an increase in the Hsp70 FA soluble fraction when compared to the control and this increase is lesser than the one observed in secHsp 70 flies ( Hsp70,

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34 FA fraction, Figure 3 10) This data indicates that there may be a difference in the quantity of soluble and insoluble between genotypes and this may contribute to the mechanistic action of secHsp70 in the rescue of AD phenotype Figure 3 10. Western Blots of TBS, 1% SDS and FA Soluble F ractions. All three fractions show an increase in quantity of with age of flies. In fraction 3 (FA soluble), there seems to be an increase in compared to the control. Analysis of TBS, SDS and FA Soluble F ractions of by sELISA We utilized sELISAs to quantify in the different fractions since sELISAs are quantitatively more sensitive than western blots. I prepared the same three fractio ns as described earlier for western blots and Dr. Yona Levites performed the sELISA to determine levels of SDS soluble I analyzed the data with GraphPad Prism (two way ANOVA) and did not observe a significant difference in the levels of SDS soluble against controls between genotypes (Figure 3 1 1 A) and between time points (Figure 3 11B). The TBS and FA soluble fractions a re yet to be analyzed by sELISA

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35 and I believe completion of these experiments will provide vital clues to the mechanistic ac tion of secHsp70 in the Drosophila AD model Figure 3 11 Quantification of SDS S oluble by sELISA. A) secHsp70 and Hsp70 day 1 and day 20 flies reveal no significant differences in quantity of SDS soluble compared to each other and the control B) No significant difference was noticed in the levels of SDS soluble between different time points for each genotype. Error bars indicate SD.

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36 CHAPTER 4 DISCUSSION With one person developing AD every 68 seconds in America, and no disease modifying therapies currently available in the clinic, the emphasis is high on developing new robust treatments to lessen the impact of AD. A major hurdle in the development of therapeutic strategies is our inadequate knowledge of AD pathog enesis. APP transgenic mouse models are extensively used for studying AD, but these models display limitations, the major one being the lack of prominent neurodegeneration. Our Drosophila AD system offers an advantage here, by allowing us to rapidly screen for the effects of therapeutics on induced neurotoxicity. It may be argued that over expressing derived from APP may play a role in the disease progression. However, is known to play a major pathogenic role in AD and has been a popular target in therapeutic strategies in the past decade (Mangialasche et al., 2010) Further, the fact that the Drosophila immune system is relatively simple compared to mice, has facilitated the study of mechanisms of rescue and pathogenesis at a more basic level, without the interference of a complex immune system. The need for improved safety and efficacy of AD therapeutics under development, coupled with advances in genetic and molecular engineering tools, has allowed the design of anti the s cFvs and the secHsp70. The scFvs are specific binding agents and they follow non mutually exclusive mechanisms of action against damage. They may disaggregate fibrillar activate the immune system to promote clearance of and change the equ ilibrium, as has been indicated in mouse AD models (Robert and Wark, 2012) On the other hand,

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37 secHsp70 and co localize, but we are yet to complete the co immunoprecipitation studies to test for their in vivo physical interaction. In vitro studies have also suggested a strong possibility of binding between Hsp70 and (Evans et al., 2006) Both the agents rescue the AD eye phenotype in our Drosophila model with secHsp70 sh owing superior neuroprotective activity when compared to the scFvs. We believe that this difference in rescue efficiency is not because of dosage since several independent lines expressing the scFvs and secHsp70 were tested. Most lines expressing secHsp70 showed better rescue than the individual scFvs or even the combination scFv. The compartmentalization of the molecular agent plays an important role in the rescue of the AD eye phenotype. The scFvs, secHsp70 and are all extracellular in our system, th us increasing the chances of direct interaction between these proteins. It is also possible for small quantities of cytosolic Hsp70 to move out of the cell, as has been suggested in cell systems, (Danzer et al., 2011) cell in a limited amount (Mohamed and Posse de C haves, 2011) leading to the Hsp70. The difference in rescue between the scFvs and secHsp70 may be due to differences in their binding affinity with The scFvs bind s pecifically to with high affinity in vitro whereas the secHsp70 probably binds to particular forms of that are more toxic. From previous studies, we understand that Hsp70 recognizes oligomeric forms of in vitro and has milder effects on fibril s (Evans et al., 2006) Hsp70 may bind to the pathogen ic forms of thereby not allowing them to interact

PAGE 38

38 with receptors in synapses and cause neuronal damage. Experiments to determine the binding affinity of secHsp70 to will confirm if this binding is stronger than that of the scFvs. Specific binding to all forms of may not be the most effective way to counter the AD neurotoxicity and I wonder if the usage of scFvs against oligomeric forms of would lead to a better rescue. There is no consensus in the scientific community about the neurotoxi c effector forms of 42. Some studies indicate that AD neurotoxicity involves the fibrillar/protofibrillar aggregate forms (Stphan et al., 2001) The deposition of insoluble fibrillar and cognitive decline are, however, not well correlated in many studies (Terry et al., 1991; Hsia et al., 1999; Koistinaho et al., 2001) A more recent interpretation is that the insoluble may be a less harmful sp ecies. Recent studies point towards the soluble oligomeric species as being the more pathogenic forms (Lacor et al., 2004; Ono et al., 2009; Marn Argany et al., 2011; Moreth et al., 2013) We also consider it a possibility that scFvs and secHsp70 may follow di fferent mechanisms of action against AD neurotoxicity. Studies in APPswe mouse models have documented a reduction in the load when subjected to the action of scFvs (Levites et al., 2006) and Hsp70 (Hoshino et al., 2011) Our studies in AD flies do not show a reduction in the levels of total 42 when expressing scFvs or secHsp70. I speculate that flies may not induce clearance of 42 by immune cells due to the lack of an adaptive immune system, thereby leading to no reduction in the levels of total 42. Our results indicate a possible rescue mechanism through the conversion of toxic (soluble) to non toxic (insoluble) forms. Preliminary data from flies expressing secHsp70 show an in crease in the amount of FA soluble thereby pushing the

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39 system from accumulating a highly toxic soluble form to what may be a less toxic form of aggregated Similar analyses with AD flies expressing the scFvs will help us understand if their mechanism against is similar to that of secHsp70. From the quantitative data of the SDS soluble I predict that as the disease progresses, there is an increased load of soluble oligomeric/protofibrillar that the scFvs are not able to access or act against, leading to an increase in the SDS soluble in older flies. The secHsp70 system seems to show no significant difference in levels of SDS soluble even in older flies which may be indicative of its robustness as an anti amyloid agent Future work includes analysis of the TBS and FA soluble fractions by sELISA. This could provide us with some clues to improve our understanding of the mechanism of action of these anti amyloid agents in flies. In addition to this, the quantitative analy sis of oligomeric via specific ELISAs or native gel systems will provide more insight into the dynamics. Experiments to study neurodegeneration in older flies will help us analyze the effects of the scFvs and secHsp70 in disease progression. Our studies in the flies, I believe will help find important pieces of the AD puzzle which when coupled with clues from the mouse models, can give us a comprehensive picture of the mechanisms of action of these anti amyloid agents.

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45 BIOGRAPHICAL SKETCH Swati Khare was born in India and did her schooling in a small township in Salem, Tamil Nadu. She o btained her b degree in b iotechnology from a c ollege in C oimbatore and thoroughly enjoyed that experience. She worked as a web programmer in Cognizant Technology Solutions for a year, before joining the University of Florida for her m aster s degree in b iomedical e ngineering. Through her two years in Gainesville, she has particularly enjoyed meeting new people and taking up new hobbies such as Argentine tango.