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1 QUALITY CONTROL PATHWAYS IN THE PERIPHERAL NERVE WITH DISEASE AND AGING By SUNITHA RANGARAJU 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 2010
2 2010 Sunitha Rangaraju
3 I dedicate this work t o my family for their unconditional love and support
4 ACKNOWLEDGMENTS I would like to thank my mentor Dr. Lucia Notterpek for her gui dance and support and for being a tremendous influence in my development as a scientist. I am extremely grateful for the encouragement and opportunities she has provided me, to work on interesting and challenging projects. I would like to thank my committee members Dr s.David Borchelt, Wil liam Dunn Jr, Susan Frost, Gerry Shaw and Matthew Sarkisian for all their critical input and intellectual contribution. I would also like to convey my thank s to our collaborators Dr s. Christiaan Leeuwenburgh, Christy Carter and Adeela Kamal for their time and resources I express my gratitude to Dr. Sue Semple Rowland for being a student friendly Program Director who has always had my best interest s. I am ever grateful to my parents for providing me with functional genes and unconditional love for the past 25 years. I thank my younger siblings Vidhya and Vengada Karthik for their love, affection, and most importantly, teaching me the virtue of patience. I thank all my late grandparents some of them whom I havent seen and some of them who couldnt see this day. My heartfelt thank s go to my best buddies in graduate school Karthik Bodhinathan and Mercedes Prudencio for always being there for me. I also thank my IDP and undergraduat e friends who have helped me along in this journey I sincerely thank the past and present memb ers of the Notterpek lab especially Irina, Stephanie, Jonathan and Katie for all their help. I greatly appreciate the services of the administration staff BJ, Kathy, Valerie, Teresa and Susan, who attend to me with a smiling face whenever I knock their doors. I express my thank s to Doug Smith for assistance with confocal microscopy. Finally I would like to acknowledge the N ational I nstitutes of H ealth and the M uscular D ystrophy A ssociation for financia l support
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION .................................................................................................... 16 Myelination in the Peripheral Nervous Sys tem ....................................................... 16 Myelin Constituents ................................................................................................ 17 The Proposed Functions for Peripheral Myelin Protein 22 ...................................... 19 PMP22 Associated Neuropathies ........................................................................... 21 Mouse Models of PMP22A ssociated Peripheral Neuropathies .............................. 23 Quality Control Mechanisms for PMP22 in TrJ and C22 Mouse Models ................. 25 Implications of Protein Aggregation in PMP22 Associated Neuropathy Models ..... 28 Chaperones in the processing of PMP22 aggregates ............................................. 30 Role of autophagy in PMP22 protein processing .................................................... 33 Contribution of Aging in Peripheral Neuropathies ................................................... 36 The Effect of Normal Aging on Peripheral Nerve Health ......................................... 37 Calorie Restriction Prevents AgeRelated Alterations and Neurodegeneration ...... 40 2 PHARMACOLOGICAL INDUCTION OF THE HEAT SHOCK RESPONSE IMPROVES MYELINATION IN A NEUROPATHIC MODEL ................................... 42 Introduction ............................................................................................................. 42 Materials and Methods ............................................................................................ 43 Mouse C olonies ................................................................................................ 43 Non Myelinating SC Cultures ........................................................................... 44 Cellular Toxicity Assay ..................................................................................... 44 DRG Explant Cultures and Compound Treatment Paradigms ......................... 45 Primary Antibodies ........................................................................................... 45 Immunolabeling Studies ................................................................................... 46 Biochemical Studies ......................................................................................... 47 Quantification of Myelin Internode Lengths ...................................................... 47 Results .................................................................................................................... 48 Myelin Production in C22 Neuropathic Samples is Enhanced by Geldanamycin ............................................................................................... 48 SmallMolecule HSP90 Inhibitors Enhance Chaperone Expression in SCs ..... 49
6 Enhancement of HSPs Promotes Myelination in Explant Cultures from Neuropathic Mice .......................................................................................... 52 Discussion .............................................................................................................. 56 3 ENHA NCEMENT OF AUTOPHAGY BY RAPAMYCIN IMPROVES MYELINATION IN SCHWANN CELLS FROM NEUROPATHIC MICE ................... 69 Introduction ............................................................................................................. 69 Materials and Methods ............................................................................................ 70 Mouse Colonies ................................................................................................ 70 Primary Mouse SC Cultures ............................................................................. 71 Autophagic Flux Measurement ......................................................................... 72 Metabolic Labeling and 35S Pulse Studies ....................................................... 72 DRG Explant Cultures and RM Treatment Paradigms ..................................... 73 Lentiviral Transduction of Mouse DRG Explants .............................................. 73 Biochemical S tudies ......................................................................................... 74 Immunolabeling Studies ................................................................................... 74 Quantification of Myelin Internode Abundance and Lengths ............................ 75 Results .................................................................................................................... 75 Rapamycin Activates Autophagy in Primary Mouse SCs ................................. 75 Myelin Production in C22 Neuropathic Samples is Enhanced by Rapamycin .. 77 Activation of Autophagy Improves PMP22 Trafficking ...................................... 79 The Positive Effect of RM is not Genotype Specific ......................................... 80 Autophagy is a Critical Pathway for the MyelinPromoting Effect of Rapamycin .................................................................................................... 80 Discussion .............................................................................................................. 82 4 MOLECULAR ARCHITECTURE OF MYELINATED PERIPHERAL NERVES IS SUPPORTED BY CALORIE RESTRICTION WITH AGING ................................. 102 Introduction ........................................................................................................... 102 Materials and Methods .......................................................................................... 104 Animals and Diets .......................................................................................... 104 Primary Culture of Schwann Cells from Young and Old Rats ......................... 105 Biochemical Studies ....................................................................................... 106 Immunochemistry ........................................................................................... 107 Nuclei Count ................................................................................................... 108 Results .................................................................................................................. 108 Chaperone Response of Schwann Cells Isolated from Nerves of Aged Rats 108 The Autophagic Response of Glial Cells is Altered with Age.......................... 109 Protein Homeostatic Mechanisms are Maintained in Nerves of Diet Restricted Rats ............................................................................................ 112 The Expression of Myelinated Schwann Cell Proteins in Diet Restricted Rats ............................................................................................................. 114 Axonal Constituents in Myelinated Peripheral Nerves of Diet Restricted Rats ............................................................................................................. 115 Discussion ............................................................................................................ 117
7 5 LIFE LONG CALORIE RESTRICTION ALLEVIATES AGE RELATED OXIDATIVE DAMAGE IN PERIPHERAL NERVES .............................................. 134 Introduction ........................................................................................................... 134 Materials and Methods .......................................................................................... 136 Animals and Diet ............................................................................................ 136 Biochemical Analyses .................................................................................... 136 Immunolabeling of Nerve Samples ................................................................. 138 Di8 ANEPPS Labeling ................................................................................... 139 Results .................................................................................................................. 139 A Calorie Restricted Diet Slows Protein Damage within Peripheral Nerves During Aging ............................................................................................... 139 Lipid OxidationMediated Protein Damage within Myelinated Peripheral Nerves ......................................................................................................... 141 Age related activation of proinflammatory pathways is attenuated by a lifelong CR diet ................................................................................................ 142 Discussion ............................................................................................................ 144 6 CONCLUSIONS ................................................................................................... 155 LIST OF REFERENCES ............................................................................................. 166 BIOGRAPHICAL SKETCH .......................................................................................... 192
8 LIST OF TABLES Table page 3 1 Primary antibodies us ed in this study. .............................................................. 101 4 1 P rimary antibodies used in this study. .............................................................. 133 5 1 Primary antibodies used in this study. .............................................................. 154
9 LIST OF FIGURES Figure page 2 1 Myelin production is stimulated by geldanamycin. .............................................. 61 2 2 Cellular toxicity and chaperone expression for the HSP90 inhibitors. ................. 62 2 3 EC137 stimulates chaperone production in nonmyelinating SCs in dose and time dependent manner. .................................................................................... 63 2 4 Treatment with EC137 activates HSF1.. ............................................................. 64 2 5 Myelin production by neuropathic explant cultures is enhanced by EC137. ....... 65 2 6 Induction of chaperones aids the processing and tra fficking of PMP22.. ............ 67 2 7 Working model: HSP90 inhibitor aids PMP22 processing and improves myelination in neuropathic samples. ................................................................... 68 3 1 Rapamycin effectively induces autophagy in cultured mouse SCs. .................... 87 3 2 Rapamycin treatment has minimal effect on the Akt pathway in mouse SCs. .... 88 3 3 Enhancement of autophagy reduces the levels of ubiquitinated substrates. ...... 89 3 4 Protein synthesis in rapamycin treated SCs.. ..................................................... 90 3 5 Rapamycin promotes myelination by SCs. ......................................................... 91 3 6 Late treatment of rapamycin improves myelin protein expression better than early treatment in C22 samples. ........................................................................ 92 3 7 The abundance and length of myelin internodes are increased by rapamycin. .. 93 3 8 Activation of autophagy by rapamycin is negli gible in DRG neurons. ................. 94 3 9 The processing of PMP22 in myelinating SCs.. .................................................. 95 3 10 Rapamycin enhances myelination in cultures fro m TrJ mice.. ............................ 96 3 11 Exposure to rapamycin reduces the accumulation of poly ubiquitin substrates in TrJ mouse SCs. ............................................................................................. 97 3 12 Dete rmination of lentiviral transduction efficiency. TrJ DRG explants were transduced with GFP lentiviral particles. ............................................................. 98 3 13 Atg12 is necessary for the myelin improvement by rapamycin. .......................... 99
10 4 1 The chaperone response of Schwann cells from aged rats.. ............................ 122 4 2 The response of glial cells to Stv stimulus. ...................................................... 123 4 3 The fusion of autophagosomes with lysosomes in Schwann cells. ................... 125 4 4 Age associated alterations in chaperones and autophagic proteins in mye linated peripheral nerves.. .......................................................................... 127 4 5 CR preserves myelin protein expression and myelinating Schwann cell phenotype. ........................................................................................................ 129 4 6 The e xpression of axonal proteins is supported by CR diet.. ............................ 131 4 7 The expression and localization of Na+ and K+ channel proteins in myelinated nerves of aged rats.. ....................................................................... 132 5 1 A calorie restricted diet minimizes accrual of damaged proteins within peripheral nerves during aging.. ....................................................................... 149 5 2 Accumulation of lipofuscin is curtaile d by CR diet with aging. .......................... 150 5 3 Lipid peroxidationassociated modifications of proteins with age are relieved in sciatic nerve with a CR diet. .......................................................................... 151 5 4 Age related increase in proinflammatory mediators is attenuated by a lifelong CR diet. ..................................................................................................... 152 5 5 A calorie restricted diet diminishes macrophage infiltration of peripheral nerves with age. ............................................................................................... 153
11 LIST OF ABBREVIATION S AL ad libitum Atg autophagy related homolog; Atg12 a utophagy related gene 12 CMT1A Charcot Marie Tooth disease type 1A CNS central nervous system CQ chloroquine CR calorie restriction di 8 ANEPPS 4 (2 (6 dibutylamino) 2 naphthalenyl)ethenyl) 1 (3 sulfopropyl) hydroxide DIV days in vitro DMSO dimethylsulfoxide DRG dorsal root ganglion EC137 experimental compound 137 ER endoplasmic reticulum ERAD ER associa ted degradation GA Geldanamycin GAPDH glyceraldehyde 3phosphate dehydrogenase HNE 4 hydroxynonenal HS heat shock HSF1 heat shock factor 1 HSP heat shock protein phosphoI B Kv v oltage gated potassium channel
12 LAMP1 lysosome associated membrane protein 1; LV lentivirus MAG mye lin associated glycoprotein MAP LC3 Microtubule associated protein light chain 3 MBP myelin basic protein MDA malondialdehyde m o month mUb mono ubiquitin Nav voltage gated sodium channel NF neurofila ment NF P postnatal day P0 protein zero pHH3 phosphorylated histone 3 PMP22 peripheral myelin protein 22 PNS peripheral nervous system pS6 phosphorylated S6 pUb poly ubiquitin RM rapamycin ROS reactive oxygen species SC Schwann cell Stv sta rvation TNF
13 UP S ubiquitin proteasome system Wt wild type
14 Abstract of Dissertation Presented to the Graduate Sc hool of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy QUALITY CONTROL PATHWAYS IN THE PERIPHERAL NERVE WITH DISEASE AND AGING By Sunitha Rangaraju May 2010 Chair: Lucia Notter pek Major : Medica l Sciences Neuroscience Peripheral myelin protein 22 ( PMP22 ) is a hydrophobic membrane glycoprotein expressed predominantly by myelinating Schwann cells (SCs) A heterozygous gene duplication of or point mutations in PMP22 in humans lead to the most common demyelinating peripheral neuropathies, namely Charcot Marie Tooth disease Type 1A (CMT1A). PMP22 overproducer C22 transgenic mice and spontaneous point mutation (L16P) mouse Trembler J (TrJ) model the disease and display the clinical phenotypes. A ffected nerves from these mouse models contain abnormally localized intracellular PMP22 inclusions associated with severe demyelination, similar to the human disease. P revious studies from our lab demonstrate that activation of protein homeost asis pathways, namely, chaperones and autophagy are able to reduce PMP22 aggregates in a toxininduced cellular model. Furthermore, intermittent fasting, a dietary approach to activate chaperone and autophagy pathways substantially improves myelination and locomotor functions in neuropathic mice. In this study we have assessed whether pharmacological stimulation of chaperone and autophagy pathways can aid the processing of PMP22 and alleviate the related myelin defects in neuropathic mice. For activation o f chaperones and autophagy pathways, small molecules EC137 ( from
15 Biogen Idec, Cambridge, MA ) and rapamycin (RM), respectively were employed. Exposure of myelinating dorsal root g anglion (DRG) explants from neuropathic mice to EC137 (50nM) or RM (25nM) res ults in the correct processing and trafficking of PMP22 as well as an overall improvement in myelination. The onset of this disease is ~35 years in humans and is progressive with age. Hence, we determined the contribution of normal aging to disease progres sion in a longitudinal study with rats. First, we confirmed previous findings that there is an age related decline in myelin and axonal proteins in rats. Concomitantly, we found that there are abnormal age related changes in key proteins tha t are part of t he chaperone and autophagy pathways along with increase in proteasomal substrates, oxidatively damaged proteins, proinflammatory mediators and immune cells Interestingly, our studies show that a life long calorie restriction (CR) diet dramatically minim iz es adverse agerelated changes in the chaperone and autophagy pathways and maintains myelination with age. Furthermore, diet restriction diminishes age related oxidative damage to the peripheral nerve, as well as the hostile immunological and inflammator y components to improve peripheral nerve health. Together, these results demonstrate that quality control mechanisms such as chaperones and autophagy activated by pharmacological as well as dietary means are pathways with therapeutic potential to tackl e the ageand disease related abnormalities in PMP22 associated neuropathies
16 CHAPTER 1 INTRODUCTION Myelination in the Peripheral Nervous System Myelin sheath is a multi lamellar, lipid rich structure deposited in segments along the axons of the centr al and peripheral nervous systems (CNS and PNS). The principal role of the myelin sheath is the insulation of axons which allow s faster transmission of the nervous impulse. In the PNS, myelin sheath is formed by the differentiation of the plasma membrane o f Schwann cells (SC s). During the development program in the PNS, SC precursors migrate from the neural crest and come in close contact with the peripheral axons (Kamholz et al., 1999) A lar ge bundle of naked axons become ensheathed by a single layer of SCs. The establishment of axonal contact triggers SC proliferation (Salzer, 1997) As the SCs undergo rapid proliferation, t he axons are segregated by radial sorting, sending their processes deeper into the bundle of axons A 1:1 relationship is established between a SC a nd an isolated axonal segment to be myelinated (Bunge et al., 1989) The SCs that do not form 1:1 relationships with the axons mature into a nonmyelinating type that embed multiple axons within them (Jessen and Mirsky, 2005) After the formation of a 1:1 relationship with the axon, the promyelinating SC subsequently undergoes a pronounced lateral elongation along the axon, and simultaneously secretes a continuous mucopolysacch aride basal lamina at the abaxonal (external) surface of the SC/axon unit The basal lamina establishes connections with the extracellular matrix. This enables the SC to begin wrapping around the axon inwardly (Bunge et al., 1989) The ability of SCs to elaborate a basal lamina correlates with their capacity to proceed to myelination (Bunge et al., 1989) and is one of the key events that drives SC differentiation towards myelination (Carey et al., 1986;
17 Eldridge et al., 1987) As the SC membrane wraps around the axon the many layers compress and the majority of the cytoplasm is extruded. This results in the formation of compact (myelin lamellae) and noncompact (cytoplasmic regions including the Schmidt Lantermann incisures, paranodal loops and the outer and inner mesaxons) co mpartments of mature PNS myelin. Each segment of the axon myeli nated by a single SC is termed internode and the uncovered axonal portions in between the internodes are the nodes of Ranvier. The se are higly specialized regions enriched with ion channels that facilitate nervous impulse (Garbay et al., 2000) The presence of axons is not only required for the expression of the myelin genes during development, but also for the maintenance of a myelinating phenotype (Wood and Bunge, 1975; Uyemura and Kitam ura, 1991) Myelin Constituents In both mammalian and nonmammalian species, lipids account for 72 78% of the dry mass of PNS myelin (Garbay et al., 2000) Proteins represent between 20 and 30% of the dry mass of myelin. The major PNS myelin proteins are myelin protein zero (P0, 5070%), myelin basic protein (MBP, 515%), myelin associated glycoprotein (MAG, 1%), periaxin (5%) and peripheral myelin protein 22 (PMP22, 25%). Th e myelin proteins that constitute <0.5% are E cadherin and connexin (Cx32). In addition to these well known proteins, r ecently, a novel myelin protein, MP11 ( myelin protein of 11 kDa) has been identified that is preferentially expressed in the PNS compared to CNS (Ryu et al., 2008) P0 is a 28kDa integral membrane glycoprotein, which is specific to PNS myelin. The putative role for the P0 protein is to function as a membrane adhesion molecule and to promote and maintain tight compaction of the myelin structure by homophilic
18 interactions via the L2/HNK 1 adhesion epitope (Filbin et al., 1990) In contrast to P0, MBP is present in both the PNS and the CNS myelin, which contains a high percentage (about 25%) of basic residues distributed throughout i t s polypeptide chain. In the PNS, P0, to gether with the MBP, is thought to be involved in the compaction of the myelin sheath via electrostatic interactions with acidic lipids in the membrane (Omlin et al., 1982; Martini et al., 1995) MAG and Cx32 are located in the noncompact myelin. MAG is localized in the periaxonal SC membrane, the external and internal mesaxons, the paranodal loops of the nodes of Ranvier and the Schmidt Lanterman incisures (Martini and Schachner, 1986; Trapp, 1990) MAG is believed to participate in axonal recognition and adhesion, intermembrane spacing, signal transduction during glial cell differentiation, regulation of neurite outgrowth, and in the maintenance of axonmyelin integrity (Trapp, 1990; McKerracher et al., 1994; Mukhopadhyay et al., 1994; Fruttiger et al., 1995) The extracellular domain of MAG possesses the L2/HNK 1 epitope similar to P0 (McGarry et al., 1983; Inuzuka et al., 1984; Kruse et al., 1985; Snipes et al., 1993) Cx32 is a gap junction protein that allows the movement of ions and small molecules to move through the multilamellar myelin membrane (Scherer et al., 1999) MP 11 is a glycoprotein specifically localized to the Schmidt Lanterman incisures and paranodal loops of the peripheral nerve (Ryu et al., 2008) PMP22 is a 22 kDa, tetraspan, hydrophobic, integral membrane glycoprotein, expressed mainly by myelinating SCs (Snipes et al., 1993) PMP22 also contains t he L2HNK 1 adhes ion epitope in its fi rst putative extracellular loop, to which a carbohydrate moiety linked to asparagine 41 is present (Sn ipes et al., 1993) The
19 carbohydrate residue in PMP22 may be involved in conferring adhesive properties (Snipes et al., 1993) s uggesting that the role of PMP22 in myelin may be similar to that of the P0 glycoprotein (Pareek et al., 1993) Furthermore, PMP22 P0 and MBP genes are coexpressed (Kuhn et al., 1993) and their protein expression is coreg ulated (Hagedorn et al., 1999; Notterpek et al., 1999a) in myelinating SCs. A lso, there is evidence for direct physical interaction between PMP22 and P0 proteins (D'Urso et al., 1999; Hasse et al., 2004) Although, it has been almost two decades since its discovery (Snipes et al., 1992) t he ex act function of PMP22 has not been eluci dated, yet The Proposed Functions for Peripheral Myelin Protein 22 The human PMP22 gene, located on chromosome 17, spans a 40 kbp region and contains six exons (Patel et al., 1992) In mouse and rat genomes, PMP22 gene is located on chromosome 11. In both humans and rodents, PMP22 expression is regulated by two alternatively used promoters, producing two mRNAs (1A and 1B) (Suter et al., 1994) which encode the same protein but differ only by their 5 untranslated region (Bosse et al., 1994; B osse et al., 1999) The expression of the 1A mRNA predominates in peripheral myelin, while the 1B mRNA is the major species expresse d in nonneural tissues. In addition to myelinating SC s in the PNS, PMP22 mRNA transcripts have been found in the embryo nic and adult stages in both neuronal and nonneuronal tissues (Baechner et al., 1995; Parmantier et al., 1995; Notterpek et al., 2001; Roux et al., 2004) The post transcriptional regulation of PMP22 in SCs during myelination, by microRNAs, has been recently demonstrated (Verrier et al., 2009) In the PNS, PMP22 is largely confined to the compact portion of myelin (Snipes et al., 1992; Haney et al., 1996) PMP22 has been proposed to have an important role in PNS myelin synthesis and assembly based upon the observations that point mutations
20 in the PMP22 g ene, leads to a PNS specific dysmyelination (defects in the formation of myelination) (Garbay et al., 2000) As a step towards understanding the function of PMP22, extensive studies have been carried out in PMP22 knock out mice (PMP22 / ) PMP22 d eficient mice display a delay in the onset of myelination (Adlkofer et al., 1995; Adlkofer et al., 1997b; Amici et al., 2006) PMP22 / animals have few myelinated axons in which the myelin is too thick with respect to the axonal diameter. These structures, called tomacula, result from a focal hypermyelination, and consist of redundant myelin l oops with normal spacing (Adlkofer et al., 1995; Amici et al., 2006) Thus, point mutations of PMP22 lead to dysmyelination while the complete disruption of the PMP22 gene leads to focal hypermyelination and demyelination (loss of my elin sheaths) Based upon these results, it has been proposed that PMP22 is involved in the initiation of myelination, determination of mye lin thickness and stabilization of the myelin sheath (Naef and Suter, 1998) In addition to its structural role in compact myelin, PMP22 is a binding partner in the integrin/laminin complex (Amici et al., 2006) This finding correlates with numer ous S C axon profiles showing loose basal lamina in PMP22 deficient (Amici et al., 2006) and overexpressing (Magyar et al., 1996) phenotypes I n the PMP22 / mice loose basal lamina might correspond to the missing PMP22 int egrin connection, suggesting the involvement of PMP22 in mediating interactions of Schwann cells with the extracellular matrix The PMP22 integrin interaction has proposed functions in the regulation of cellular growth (Zoidl et al., 1995; Zoidl et al., 1997) cell spreading, differentiation and migration of SCs (Niemann et al., 2000; Nobbio et al., 2004) cellular adhesion (Suter and Snipes, 1995; Hasse et al., 2004) and apoptosis (Fabbretti et al., 1995; Brancolini et al., 1999; Brancolini et al., 2000; Sancho et al.,
21 2001; Roux et al., 2005) Overa ll, it appears that PMP22 is a dosagesensitive gene, with two functional alleles being required for proper myelin formation and maintenance in the PNS (Adlkofer et al., 1995; Huxley et al., 1996; Adlkofer et al., 1997b; Adlkofer et al., 1997a; Perea et al., 2001) Additionally, PMP22 has been identified as an early component of the developing bloodnerve and bloodbrain barrier at intercellular junctions (Notterpe k et al., 2001; Roux et al., 2004) PMP22 also modulates epithelial morphology and monolayer permeability in cell culture (Roux et al., 2005) This property of PMP22 can be attributed to some similarities in its primary amino acid sequence with the claudin family of tight junction proteins (Van Itallie and Anderson, 2006) Finally, the fact that SCs expressing a truncated version of PMP22 protein proliferates at a significantly higher rate than those expressing the Wt (Johnson et al., 2005) suggests a possible role for PMP22 in cell cycle regulation and SC differentiation. PMP22 Associated Neuropathies Hereditary peripheral neuropathies comprise a heterogeneous group of disorders termed Charcot Marie Tooth diseases (CMT) among which, Charcot Marie Tooth disease type 1A (CMT1A) is the most prevalent form. It affects approximately one in 5,000 people which constitutes about 50% of all CMT cases however, there is currently no cure for this disease (Shy et al., 2008) CMT1A is a demyelinating neuropathy mainly associated with a 1.5megabase duplication in human chromosome 17 t hat includes the PMP22 locus (Lupski et al., 1991) The main hallmark of CMT1A is demyelination and, consequently, a marked reduction in nerve conducti on velocity together with slowly progressive distal muscular atrophy and weakness (Gabreels Festen and Wetering, 1999) On the other hand, the deletion of t he 1.5megabase region in human chromosome 17 or truncation of the protein results in a milder variant known as
22 Hereditary Neuropathy with liability to Pressure Palsies (HNPP) (Chance et al., 1993) Patients diagnosed with HNPP exhibit a clinically heterogeneous recurrent focal neuropathy following minor nerve trauma that is characterized mainly by segmental demyelination and focal myelin thickening (tomacula) (Pareyson et al., 1996; Chance, 1999) Compared to CMT1A, the reduction in nerve conduction velocity in HNPP is lesser, and in general, t he symptoms exhibited are not as severe (Gabreels Festen and Wetering, 1999) In addition to deletion and duplication, a variety of single point mutations in the PMP22 gene have been identified in a small percentage of CMT1A cases and in another type of related neuropathy, known as DejerineSottas Syndrome (DSS) (Roa et al., 1993) Most mutations are dominantly inherited, affect ing hydrophobic regions of the protein and are generally ass ociated with severe phenotypes. DSS is chronic motor and sensory neuropathy with early onset, marked reduction in nerve conduction velocity and more severe clinical pathology than CMT1A (Gabreels Festen and Wetering, 1999) Not only mutations in PMP22, but, mutations in other essential constituents of peripheral myelin proteins such as P0 and Cx32 lead to other types of CMT diseases, namely CMT1B and CMTX respectively (Scherer and Wrabetz, 2008) Furthermore, P0 physically interacts with PMP22 and the genedosage sensitivity of both proteins is consistent with the hypothesis that the two proteins are required in correct stoichiometric amounts in myelin. Overall, the phenotypes resulting from most point mutations are more critical than the duplication or the deletion paradigms (Gabreels Festen et al., 1995; Tyson et al., 1997; Boerkoel et al., 2002) In this context, independent mouse studies of the disease
23 have suggested that secondar y to the SC damage, the phenotype severity is influenced by impaired SC neuronal interaction, aberrant expression and reorganization of axonal ion channels, pronounced damage to axonal cytoskeleton and transport as well as recruitment of macrophages (Kohl et al., ; Kohl et al., ; Maier et al., 2002; Misko et al., 2002; Devaux and Scherer, 2005; Martini et al., 2008) Mouse Models of PMP22 Associated Peripheral Neuropathies Animal models display similar behavioral and morphological abnormalities found in human patients (Notterpek and Tolwani, 1999) Extensive studies in genetically engineered PMP22 overexpressor rats (Sereda et al., 1996; Niemann et al., 1999) and mice (Huxley et al., 1996; Perea et al., 2001; Robertson et al., 2002a) as well as PMP22 deficient mice (Adlkofer et al., 1995; Maycox et al., 1997; Fortun et al., 2006) have underscored the importance of adequate levels of PMP22 for myelin formation and stability (Huxley et al., 1996; Huxley et al., 1998; Niemann et al., 1999; Perea et al., 2001) Out of the several transgenic rodent CMT1A models that are based on the overexpression of the wild type ( Wt) PMP22, the transgenic mouse termed C22, which has seven copies of the human PMP22 integrated in its genome and expresses ~1.7 times more PMP22 than Wt serves as an accurate model for heterozygous PMP22 gene duplication (Huxley et al., 1996) The C22 mice mimics some of the morphological and behavioral phenotypes of the human disease (Huxley et al., 1996; Huxley et al., 1998; Fortun et al., 2006) Similar to the disease manifestation in humans diagnosed with CMT1A, C22 mice display slowed nerve conduction velocity a reduction in the percentage of myelinated fibers and demyelinating axonal profiles in the adult (Huxley et al., 1996; Huxley et al., 1998; Robertson et al., 2002b) Phenotypically, heterozygous C22 mice show unsteady gait, muscle weakness and progressive paralyses of the hind
24 limbs (Huxley et al., 1996) In a conditional mouse model with regulatable PMP22 overexpression, the disease phenotype was corrected when the expression of the exogenous pmp22 was shut down (Perea et al., 2001) This demonstration opened an avenue for therapeutic intervention to reverse CMT1A linked with PMP22 duplication. This fact is furth er supported by independent studies with progesterone antagonists or ascorbic acid treatment where the neuropathy associated with PMP22 overexpression in CMT1A models is ameliorated by a mechanism likely involving a reduction in PMP22 expression (Sereda et al., 2003; Passage et al., 2004; Meyer zu Horste et al., 2007) Mouse models that acc urately mimic CMT1A arising from point mutations in PMP22 include the Trembler (Tr) and Trembler J (TrJ) mice, which are spontaneous mutations identified (Suter et al., 1992) The fact that the exact same point mutations have been identified in patients diagnosed with CMT1A validates these mouse models (Valentijn et al., 1992) In the Tr mouse, Glycine150 is substituted by Aspartic acid (G150D), resulting in a new negatively charged amino acid in the fourth transmembrane domain of PMP22 (Snipes et al., 1993) Similarly, the TrJ mouse carries a point mutation that substitutes Leucine for Proline at positi helix breaking amino acid (Snipes et al., 1993) that leads t sheet (Yamada et al., 2003) These m utations give rise to similar, but not identical neuropathies that have been used to model CMT1A, as well as DSS (Suter et al., 1992; Notterpek and Tolwani, 1999) Based on the observation that both single gene deletion (HNPP) and duplication of PMP22 are associated with demyelinating peripheral neuropathies, it has been propos ed that these disorders are caused by a genedosage effect (Gabriel et al.,
25 1997) Furthermore the demonstration that disease is more severe in the duplication and point mutation than the deletion paradigms (Gabreels Festen et al., 1995; Adlkofer et al., 1997a; Boerkoel et al., 2002) strongly indicates a toxic gainof function of the point mutated or duplicated PMP22. The n ature of the toxic gain of function has not been elucidated; however, impaired intracellular trafficking of mutant PMP22 in cellular quality control compartments and failure of PMP22 to incorporate into myelin have been proposed (Sanders et al., 2001) Qu ality Control Mechanisms for PMP22 in TrJ and C22 Mouse M odels Extensive studies on the trafficking of Wt PMP22 reveals two checkpoints in the synthesi s and processing of the proteinthe endoplasmic reticulum (ER) and the Golgi compartments (Snipes et al., 1993) In normal SCs ~ 20% of the newl ysynthesized PMP22 is properly folded, glycosylated and trafficked via the ER and Golgi to the cell membrane. The other 80% of the protein is rapidly degraded within 30 minutes ( min ) via ER associated degradation ( ERAD ) by the proteasome machinery (Pareek et al., 1997; Notterpek et al., 1999a; Ryan et al., 2002) Unlike other membrane proteins, PMP22 has a very short half life, which may result from the inability of such a highly hydrophobic protein to fold correctly in the ER compartment. Alternatively, it is also possible that an intracellular function of PMP22 requires this rapid turnover (Naef and Suter, 1998) Ult imately, only a small fraction of the newly synthesized PMP22 traffic ks through the Golgi and acquires its correct N glycosylation modification. Once it has trafficked past the medial Golgi, upon an unidentified signal derived from SC axonal contact, PMP22 translocates to the plasma membrane and incorporates into myelin membrane (Pareek et al., 1997) In SC s f rom PMP22 neuropathic mice, the ratio of PMP22 degradation and pr oper folding is greatly altered, resulting in the retention of
26 PMP22 in cytosolic aggregates (Fortun et al., 2003; Fortun et al., 2006) Infact, this phenomenon is also observed in nerve biopsies from CMT1A patients with PMP22 gene duplication or point mutations Patient tissues show PMP22 like immunoreactivity wit hin the SC cytoplasm representing the accumulation of PMP22, in addition to thinmyelin like staining (Nishimura et al., 1996; Hanemann et al., 2000) Mammalian c ells are endowed with three levels of defense mechanisms against the potentially toxic effects of protein aggregates. They are the quality control pathways namely, m olecular chaperones (also known as heat shock protein (HSPs)) which suppress protein misfo lding and aggregation (Broadley and Hartl, 2009) the ubiquitinproteasome system (UPS) which degrades misfolded proteins that are unable to fold (Tai and Schuman, 2008) and macro autoph agy which is involved in eliminating proteins that have escaped the surveillance of the other two systems (Jaeger and Wyss Coray, 2009) While the proteasomal degradation is a selective process, which recognizes and degrades protein substrates conjugated to a poly ubiquitin (pUb) chain (Tai and Schuman, 2008) autophagy is a less selective clearance mechanism (Jaeger and Wyss Coray, 2009) Lysosomes could be involved in t he degradation of the aggregated proteins, most likely through the the autophagy lysosomal pathway (Tai and Schuman, 2008) In response to PMP22 overexpression, these quality control system s appear to be overwhelmed and protein aggregates are formed in vitro (Chies et al., 2003; Fortun et al., 2006) in animal models as well as in CMT1A human patients (Nishimura et al., 1996; Hanemann et al., 2000) In the C22 overexpression mouse model, a reduced PMP22 turnover and presence of PMP22 aggregates is associated with impaired
27 proteasome activity (Fortun et al., 2005; Fortun et al., 2006) Also the presence of spontaneous protein aggregates in C22 mice is concomitant with the activati on and recruitment of components of the autophagy lysosomal pathway to the site of protein aggregation (Fortun et al., 2003; Fortun et al., 2006; Fortun et al., 2007) In addition, PMP22 aggregates recruit molecular chaperones and components of proteasome machinery, which further alter the hom eostatic balance of the cell (Fortun et al., 2006) In terms of membrane protein trafficking, overexpression of PMP22, but not a point mutation in PMP22, in cell culture induces the formation of actin/phosphatidylinositol (4,5) biphosphate (PI 4,5 P2) positive vacuoles and PMP22 accumulates in these vacuoles (Chies et al., 2003) Whether, these PMP22 containing actin/PI 4,5 P2positive pool of vacuoles c ross talk with the late endosomal and autophagy lysosomal pathways, is not clear. With regards to the L16P point mut ation in TrJ mice mutant PMP22 and Wt PMP22 form homoand heterodimers and multimers (Tobler et al., 1999; Tobler et al., 2002) The L16P mutant PMP22 proteins are retained in the intermediate compartment and ER (Tobler et al., 1999) In addition, the TrJ mutant interacts with the ER chaperone, calnexin for a longer time than the normal protein (Dickson et al., 2002) in a glycan independent manner and this correlates with a reduced diffusion rate within the ER membrane (Fontanini et al., 2005) The retained mutant PMP22 protein traps the Wt PMP22 in the intermediate compartment between the ER and Golgi and thus may block part of its transport to the cell membrane by a dominant negative mechanism. The mutant PMP22 protein causes a deleterious effect even in the absence of Wt PMP22 indicating a toxic gainof function (Adlkofer et al., 1997a) Different in vitro studies have
28 suggested that TrJ and other point mutated PMP22s are not incorporated into plasma memb rane (D'Urso et al., 1998; Naef and Suter 1999; Tobler et al., 1999; Colby et al., 2000; Sanders et al., 2001) S tudies in our lab show that, in SC s from TrJ mice, the turnover rate of PMP22 and the activity of the proteasome are reduced, concomitant with the assembly of protein aggregates (Fortun et al., 2003; Fortun et al., 2005) The endosomal lysosomal pathway (Notterpek et al., 1997) and ubiqui tination (Ryan et al., 2002; Fortun et al., 2005) are also upregulated in SCs from TrJ mice. PMP22 aggregates (Notterpek et al., 1999b; Ryan et al., 2002) and myelinlike figures (Dickson et al., 2002) reminiscent of autophagic vacuoles (Dunn, 1990) have been found in TrJ SCs. In addition, the cytosolic PMP22 aggregates recruit molecular chaperones, components of pr oteasome machinery and autophagy lysosom al pathway which further alter the homeostatic balance of the cell (Fortun et al., 2003; Fortun et al., 2005; Fortun et al., 2006; Fortun et al., 2007) Implications of Protein A ggregat ion i n PMP22 Associated Neuropathy M odels In response to pharmacological inhibition of the proteasome, cells in culture accumulate misfolded poly ubiquitinated (pUb) s ubstrates in aggregates, which are transported along the microtubules towards the centrosome t o form an inclusion, termed the aggresome (Kopito, 2000) It has been proposed that these inclusions form when the cells capacity to d egrade misfolded protein is exceeded (Kopito, 2000) Inclusion formation is associated with the pathogenesis of many neurodegenerative diseases (Kopito, 2000; Goldberg, 2003) The characteristic features of aggresomes include, perinuclear assembly at the centrosome in a microtubuledependent fashion, devoid of membrane structures encaged by vimentin and exclusion from ER and Golgi (Johnston et al., 1998)
29 In SC cultured from Wt mice, after a 16h treatment with a proteasome inhibitor, PMP22 accumulates in aggresomes and displays all the characteristic features of aggresomes (Notterpek et al., 1999b) These aggresomes have been shown to colocalize with ubiquitin and the HSPs and recruit lysosomes and autophagosomes (Fortun et al., 2007) Similar to the Wt the Tr and TrJ PMP22s also form aggresomes in response to proteasome inhi bition, in vitro although they do so with a higher propensity to spontaneously form high molecular weight oligomers (Ryan et al., 2002; Tobler et al., 2002) In SCs from C22 and TrJ m ice, PMP22 aggregates associate with chaperones, autophagosomes and lysosomes suggesting an endogenous activation of these pathways in response to the presence of m isfolded proteins (Notterpek et al., 1997; Ryan et al., 2002; Fortun et al., 2003; Fortun et al., 2006) Based on these results, it is likely that the formation of protein aggregates in both paradigms represents a common protective response of the cell to cope with the excess or mutated PMP22 (Ryan et al., 2002; Fortun et al., 2003; Fortun et al., 2007) The formation of large PMP22 aggregates may be a protective response of SCs to eliminate misfolded protein under conditions of compromised prot easome activity (Isaacs et al., 2002; Fortun et al., 2003) However, the implications of inclusion body formation within the cytoplasm are still controversial. I t has been proposed that the inclusion bodies in neurodegenerative conditions are protective, whereas the intermediate aggregates trigger neuronal toxicity by an unknown sequence of events (Muchowski and Wacker, 2005) However, t he incidence of cell death is not a prominent feature in cell and mouse models of CMT1A neuropathies (Fortun et al., 2003; Fortun et al., 2006) One possible mechanism by which aggregates could affect cellular biology is
30 by compromised proteasome activity (Bence et al., 2001) The excess of (C22) or missense mutation (TrJ) in PMP22 overload the proteasome pathway, leading to a reduction in the turnover rate of the protein (Fortun et al., 2003; Fortun et al., 2006) This leads to accumulation of PM P22, together with other unrelated ubiquitinated substrates, like MBP (Akaishi et al., 1996) to form aggregates (Fortun et al., 2005) Another possible mechanism of putative toxicity associated with aggresomes is by the recruitment of components of protein quality control machinery to the site of aggregation and their entrapment in nonfunctional complexes (Corboy et al., 2005) Indeed, in cultured SCs from TrJ and C22 mice, PMP22 aggregates are immunoreactive for components of the UPS machinery (Fortun et al., 2003; Fortun et al., 2005; Fortun et al., 2006) which if trapped within, could contribute to an additional loss of proteasomal activity (Tai and Schuman, 2008) In agreement to both th e possibilities, the presence of PMP22 aggregates in overexpression (C22) and point mutation (TrJ) models correlate with reduced proteasome activity (Fortun et al., 2005; Fortun et al., 2006) Hence, protein aggregation represents an autocatalytic loop by which, the presence of aggregates further triggers their formation by means of an impaired proteasomal activity (Kopito, 2000) Despite the initial protective nature of aggregates, they could contribute to cellular dysfunction if they are not cleared eventually. Chaperones in the processing of PMP22 aggregates C ytosolic chaperones play an important role in the disassembly of molecular aggregates and accelerate the refolding of insoluble molecules (Sherman and Goldberg, 2001) The heat shock response (HSR) and HSPs have been shown to have therapeutic potential and has been implicated in many neurodegenerative diseases based on their protective role against intracellular aggregates (Westerheide and
31 Morimoto, 2005) HSPs (molecular chaperones) might prevent toxicity by at least three ways i ncluding blocking inappropriate protein interactions, facilitating disease protein degradation or sequestration, and blocking downstream signaling events that lead to cellular dysfunction and death (Muchowski and Wacker, 2005) The heat shock protein family includes the HSP90, HSP70, HSP40, and small heat shock protein (sHSP) families crystallin The HSR is regulated at the transcriptional level by the heat shock transcription factor 1 (HSF1) (Pirkkala et al., 2001) HSF1 exists normally in a negatively regulated state as an inert monomer in complex with HSP90 and upon exposure to a variety of stresses, including HS and HSP90 inhibitors, HSF 1 is derepressed. This is followed by hyperphosphorylation, nuclear translocation and trimeriz ation of HSF1 leading to the transcription and translation of HSPs (Westerheide and Morimoto, 2005) Geldanamycin, a HSP90 inhibitor, binds to the ATP site on HSP90 and blocks it s interaction with HSF1, promoting HSF1 activation and the synthesis of HSPs (Prodromou et al., 1997; Zou et al., 1998) The for mation of protein aggregates is common among the protein conformational diseases such as Alzheimers (AD), Parkinsons (PD), Huntingtons (HD) amyotrophic lateral s clerosis (ALS) and prion di seases. The aggregation and the associated toxicity of the disease AD and synuclein in PD, huntingtin in HD, superoxide dismutase 1 in ALS and the prion protein (PrP) are suppressed by overexpression of HSP70 in vitro as well as in vivo in some diseases (Broadley and Hartl, 2009) Pharmacological molecules such as geldanamycin and its derivatives that can i nduce a family of HSPs as opposed to overexpression of only HSP70, offer a
32 viable and translational approach to suppress aggregation and cytotoxicity in these diseases (Kalmar and Greensmith, 2009) The reason for the endogenous recruitment of HSPs to PMP22 aggregates in neuropathic nerves of C22 and TrJ mice (Fortun et al., 2003; Fortun et al., 2006) is uncertain, but it might represent an attempt of refolding and/or the targeting of the misfolded proteins for degradation (Broadley and Hartl, 2009) Infact live cell imaging experiments show that HSP70 associates transiently with huntingtin aggregates, with association dissociation k inetics identical to chaperone interactions with unfolded polypeptides likely reflecting the efforts of this chaperone to direct the unfolding and dissociation of substrates from the aggregate (Kim et al., 2002b) .On the contrary, the protection observed in drosophila models of Parkinsons and polyglutamine expansion diseases due to the elevation of HSPs is not associated with a reduction in the number of inclusions and suppresses aggregate formation and/or cellular toxicity (Auluck and Bonini, 2002; Kazemi Esfarjani and Benzer, 2002) In the PMP22 aggregation model, a protective rol e for chaperones in preventing the accumulation of misfolded proteins is suppor ted by our previous study where HS preconditioning or GA treatment hinders the formation of PMP22 aggresomes (Fortun et al., 2007) Similarly, in culture models induction of HSPs results in the suppression of the intracellular aggregation of several unrelated proteins (Broadley and Hartl, 2009) Collectively, these observations have lead to the hypothesis that the elevated levels of HSPs reduce or dampen aggregate formation and cellular degeneration. When chaperones cannot repair misfolded proteins, chaperonemediated targeting to the UPS or to lysosomes results in their degradation (Muchowski and Wacker, 2005)
33 CHIP (carboxy terminus of HSC70 interacting protein) is a protein that binds heat shock cognate 70 (HSC70) or HSP70 in the mammalian cytosol (Ballinger et al., 1999) and acts similar to the E3 ligase enzyme in the UPS to facilitate the transfer of a pUb chain to the misfolded substrate to carry out the chaperoneassisted proteasomal degradation (Jiang et al., 2001) Thus, CHIP mediates crosstalk between molecular chaperones and the UPS and aids the degradation of specific chaperone substrates (Luders et al., 2000) The importance of HSPs in regulating ubiquitinated substrates is further emphasized by a HSF1 knock out mouse model ( hsf1 / ) which shows accumulation of ubiquitinated substrates in brain and primary astrocytes in response to the absence of HSF1 (Homma et al., 2007) The accumulation of ubiquitinated substrates in the embryonic fibroblasts from th e hsf1 / mice is associated with impaired proteasome activity More strikingly, the hsf1/ mice exhibit demyelination in the spinal cord and reduced steady state levels of myelin proteins in different regions of the brain (Homma et al., 2007) These results imply that myelination is a highly demanding process in terms of quality control pathway requirements such as HSPs (Homma et al., 2007) Together, these studies support the idea that HS pathway has a therapeutic potential for PMP22 associated neuropathies. Role of autophagy in PMP22 protein processing Macroautophagy, from hereon referred to as autophagy is a constitutive event involved in the bulk degradation of long lived cytosolic proteins and organelles. In this mechanism of degradation, cytosolic cargo is engulfed in double membrane structures called autophagosomes. After maturation, autophagosomes fuse with the lysosomes to enable degradation of the cargo by the lysosomal enzymes (Klionsky and Emr, 2000)
34 A utophagy is induced under conditions of physiological stress, such as starvation and th e process requires the coordinat ed function of proteins encoded by Atg (autophagy related) genes (Klionsky and Emr, 2000) Work from our lab and others have shown that autophagosomes associate within or adjacent to the surface of cytoplasmic protein aggregates formed upon proteasome inhibition (Wojcik et al., 1996; For tun et al., 2003; Rideout et al., 2004; Fortun et al., 2005; Komatsu et al., 2005; Fortun et al., 2006; Fortun et al., 2007) Furthermore, in the TrJ and C22 neuropathic models, concomitant to impairment of proteasome activity, autophagy proteins are recru ited to the PMP22 aggregates (Fortun et al., 2003; Fortun et al., 2006) These results suggest that when proteasome activity is compromised, to maintain sub cellular homeostasis and prevent the accumulation of potentially harmful protein aggregates, proteasome substrates are re routed to an alternative pathway, nam ely autophagy lysosomal degradation. Studies have shown that, in response to experimental proteasome inhibition, autophagosomes are formed indicative of enhanced macroautophagy, thus establishing a relationship between these two major protein degradation pathways (Iwata et al., 2005; Ding et al., 2007; Fortun et al., 2007) Our studies show that, experimental enhancement of autophagy hampers the formation of PMP22 aggresomes and prevents the accumulation of unrelated substrates of ubiquitin proteasome system, when the proteasome is inhibited (Fortun et al., 2003; Fortun et al., 2007) This underscores the therapeutic potential of this pathway in PMP22 associated neuropathies. The development of pharmacologic agents to stimulate autophagy therefore could provide a therapeutic approach for PMP22 neuropathies as well as other protein misfolding diseases associated with aggregate
35 formation. A ggresome assembly is a multi step process, by which small aggregates throughout the cell are transported along microtubules towards the centrosome, where they form the final inclusion (Johnston et al., 1998; Notterpek et al., 1999b) Thus, it is likely that upon activation of autophagy under experimental conditions, small aggregates are engulfed within autophagosomes, reducing the load of proteins being transported towards the centrosome. In the protein conformational diseases, autophagy is endogenously activated, but appears to be impaired i n conjunction with protein aggregat ion (Jaeger and Wyss Coray, 2009) Pharmacologic al activation of autophagy by rapamycin (RM) or other small molecule enhancers of autophagy promotes the clearance of misfolded proteins and suppresses toxicity in cell culture and animal models of AD, PD, HD, ALS and spinocerebellar ataxia (Jaeger and Wyss Coray, 2009) A utophagy is activated as a com pensatory mechanism in response to proteasome inhibition in protein misfolding disorders. The converse of this theory has been demonstrated in autophagy gene knock out models. The role of autophagy in the degradation of ubiquitinated substrates is supporte d by the accumulation of ubiquitinated aggregates in the liver of conditional Atg7 deficient mice, which have defective autophagy, but intact UPS activity (Komatsu et al., 2005) Two independent studies provide striking findings that, mice deficient in Atg5 ( Atg5 / ) or Atg7 ( Atg7 / ) genes specifically in CNS form pUb intracellular inclu sion s in the brain and develop neurodegenerative symptoms, even without the expression of any disease causing, aggregate prone proteins (Hara et al., 2006; Komatsu et al., 2006) In Atg7 / mice, pUb proteins accumulated and aggregated into inclusions despite apparently normal
36 proteasome function, which suggests that basal autophagy may be essential in clearing some misfolded proteins that are beyond the degradative capability of th e proteasome (Hara et al., 2006; Komatsu et al., 2006; Mizushima and Hara, 2006) Similar conclusions have been made in cell culture models, where chemical inhibition of autophagy at the autophagosome formation or autophagosome lysosome fusion stages is associated with enhanced protein aggregation and cell death (Ravikumar et al., 2002) In agreement to these findings, the simultaneous inhibition of the proteasomal degradation and autophagy in our SC model is associated with more pronounced accumulation of PMP22, as compared to proteasome inhibition alone (Fortun et al., 2007) Together, these studies suggest that in the case of pr oteasomal impairment, activating autophagy is beneficial and serves as an alternative route through which misfolded proteins caould be removed. Contribution of Aging in Peripheral Neuropathies It is wellestablished that protein aggregation diseases such as AD, PD, HD, ALS etc. are agerelated neurodeg enerative disorders. The contribution of the events of normal aging in these brain disorders is thought to trigger as well as further exacerbate the disease condition (Keller et al., 200 4) Similar to these CNS disorders, CMT1A is progressive with age (Shy et al., 2008) The onset of CMT1A in humans is ~35 years although the patient possesses the defective PMP22 gene since their birth (Shy et al., 2008) The rate of progression of CMT1A in patients is measured using neurological and electrophysiological parameters such as nerve conduction velocity, sensory nerve action potential (SNAP) and com pound muscle action potential (CMAP) to assess the defects in the nerve and muscle function (Shy et al., 2008) These measurements show a significant yearly decline with disease succession (Shy et al., 2008) The disease
37 progression appears to be faster in older patients; however, the exact reason for the age related progression has not yet been determined (Shy et al ., 2008) There are very few longitudinal studies that have been performed in humans. Such long term aging studies are relatively easier to perform in rodents. Similar to the human diseases, the mouse models of CMT1A also show progressive degenerative changes in pathological processes as well as locomotor functions (Madorsky et al., 2009) Unpublished results from our lab indicate that there is an agerelated impairment i n protein homeostatic pathways such as chaperones, autophagy, proteasome and increase in PMP22 aggregation in neuropathic mice (Narvaez et al, in preparation) In order to dissect and differentiate the molecular events in quality control pathways that take place due to disease and those that take place due to normal aging, we performed a longitudinal study with Wt rats (Chapter 4 and 5). The E ffect of N ormal A ging on P eripheral N erve H ealth Aging is associated with structural, functional and biochemical alt erations in the nervous system. Neurons with long processes such as the ones in the PNS, are particularly vulnerable to degeneration (Mattson and Magnus, 2006) and are at risk to age re lated modifications. The pathways /machineries that are susceptible to agerelated alterations are metabolic pathways including glycolysis and energy metabolism cellular cytoskeleton and transport as well as cellular stress responses such as chaperones, autophagy and UPS (Martinez et al., 2009) These pathways are known to regulate important functions in the neurons an d glia. Since myelination is a highly metabolically active pathway maintained by intricate signaling events between neurons and glia (Garbay et al., 2000) disease or agerel ated degenerative changes in either cell type have global influences on overall nerve structure and function. Furthermore,
38 similar to CMT1A disease, aging of the PNS, could lead to aberrant alterations in the quality control pathways due to buildup of dam aged proteins. Myelinated peripheral nerves from animals that undergo normal aging, show fiber loss and morphological irregularities (Verdu et al., 2000) as well as a notable reduction in the express ion of myelin and neurofilament genes and proteins (Parhad et al., 1995; Melcangi et al., 1999; Melcangi et al., 2000; Uchida et al., 2004) There is evidence for demyelination and occasional remyelination in ag ed rat sciatic nerves, associated with nerve fiber degeneration (Sharma et al., 1980; Grover Johnson and Spencer, 1981; Adinolfi et al., 1991) In response to the demyelination, SC s respond by hyperproliferating and increasing in number (Gregson and Hall, 1973) which leads to their and dedifferentiation (Zanazzi et al., 2001) Similar to the disease phenotype in CMT1A (Huxley et al., 1996; Huxley et al., 1998) normal aging is also associated with changes in functional measures such as decline in nerve conduction velocity and muscle strength, and decreases in sensory discrimination, autonomic responses and endoneurial blood flow (Verdu et al., 2000) Together, these alterations contribute to decline in neuromuscular function and affect physical strength Similar to the case s of diseases, r eduction in the expression of functional proteins and the accumulation of damaged and misfolded proteins have been observed in a variety of organisms wi th aging (Sitte et al., 2000; Squier, 2001; Calabrese et al., 2004; Keller et al., 2004; Grune et al., 2005) The extent to which damaged proteins accumulate with disease as well as aging is highly dependent upon the cells capacity to repair or remove them by subcellular homeostatic mechanisms (Stadtman, 2001) namely chaperones, autophagy and proteasomal degradation. With an agerelated
39 decline in the activity of these quality control m echanisms, damaged proteins such as undegraded pUb substrates and organelles can accumulate and lead to cellular dysfunction and cell death (Macario and Conway de Macario, 2002; Bergamini et al., 2004) Particularly, m etabolically active cells such as myelinating SCs and postmitotic cells such a s neurons (Boulton et al., 2004; Weissman et al., 2007) are sensitive to the accumulation of damaged proteins. In addition to impair ed protein homeostatic pathways a ging of organ systems is associated with the accumulation of oxidatively damaged polynucleotides, proteins, carbohydrat es and lipids which leads to protein aggregation and cytotoxicity (Keller et al., 2004) This concept is called the Oxidative Stress Theory of Aging whereby agerelated loss of pr oper physiological function is due to the accumulation of oxidative damage (Bokov et al., 2004) Particularly, oxidation of proteins promotes protein aggregation due to the increase in rate of misfolding and higher hydrophobicity acquired by the modific ation (Keller et al., 2004) The specialized form of intracellular aggregates seen with aging is termed lipofuscin. Lipofuscin is a dynamic assembly of protein, lipids and carbohydrates (Keller et al., 2004) Again, l ong lived postmitotic cells such as neurons and energy demanding SCs, whose functions rely on proper intracellular protein trafficking, are at gr eater risk and a mass greater amounts of oxidative damage (Agarwal and Sohal, 1994; Sohal et al., 1994; Keller et al., 2004) In addition, SC s are rich in polyunsaturated fatty acids (Garbay et al., 2000) which serve as substrates for reactive oxygen species (ROS) mediated lipid peroxidation (Smith et al., 1999; Blair, 2001) The accumulation of waste material is not only harmful due to its interference with biological functions, but also for imparting toxicity via lipid peroxidation products
40 (Levine and Stadtman, 2001; Grune and Davies, 2003) Together, the buildup of agerelated damaged material along with their inefficient removal by homeostatic mechanisms become a concern in the vulnerable neurons and SC s of peripheral nerves. Calorie Restriction Prevents AgeRelated Alterations and Neurodegeneration A well ac cepted approach to slow the aging process and prolong lifespan is through dietary modulation, such as calorie restriction (CR) and intermittent fasting (Johnson et al., 2006; Martin et al., 2006) Dietary restricti on can induce HSPs (Heydari et al., 1996; Selsby et al., 2005) and autophagy (Bergamini et al., 2003; Wohlgemuth et al., 2007) and therefore support the maintenance of healthy cells and organs. While much work concerning dietary modulation has focused on the central nervous syst em (CNS), peripheral organs and lifespan (Feuers et al., 1989; Mattson et al., 2001; Jolly, 2004) little is known about the effects of such approach on peripheral nerves. In the CNS, life long reduction in calorie intake has been shown to preserve long term potentiation (Hori et al., 1992) and ameliorate agerelated cognitive decline (Pitsikas and Algeri, 1992) In the periphery, the decline in muscle mass and strength with age is ameliorated with a lifelong CR diet (Marzetti et al., 2008; Xu et al., 2008) which in part might be underlined by im proved neural function. Furthermore, a 5month long intermittent fasting diet, which is another dietary modulation, improves myelination and locomotor behavior in neuropathic mice (Madorsky et al., 2009) Res triction of calorie intake is an effective method to lower levels of oxidative stress and slow ageassociated changes, as well as to extend lifespan in mammals (Sohal and Weindruch, 1996; Martin et al., 2006) Previous studies have shown that calorie restriction (CR) decreases mitochondrial ROS generation and oxidative damage
41 to DNA, protein and lipids (Lambert et al., 2004) CR has been found to reduce markers of agerelated chronic inflammation (Martin et al., 2006; Chung et al., 2009) likely by sustained activation of chaperones (Keller et al., 2004) Although there have been extensive studies on the ability of CR to reduce agerelated oxidative damage in protein homeostatic and oxidative stress pathways in the CNS (Martin et al., 2006) little is known about the beneficial effects of CR on ageassociated changes i n the PNS and its influence on myelination and peripheral nerve health The overall hypothesis tested in the studies described in this dissertation is that activation of quality control pathways such as HSR and autophagy by pharmacological or dietary means will be beneficial for the processing of PMP22 and maintenance o f myelin with disease and age. In Chapter 2 of this study, we have tested whether HSP90 inhibitor mediated induction of HSPs restore the sub cellular homeostasis and improve the myelination in a neuropathic mouse model. In Chapter 3 we have determined whet her increasing the robustness of autophagy, by pharmacological activation of this pathway by RM is beneficial for minimizing protein aggregation and correcting the associated myelin defects in C22 and TrJ neuropathic mouse models In Chapters 4 and 5 of th is study, we have assessed the agerelated alterations in the protein homeostatic mechanisms and oxidative stress pathway to investigate their possible contribution to myelin and neuronal defects with age. We have also examined the potential of the dietary intervention method, CR, to maintain the quality control pathways and peripheral nerve health.
42 CHAPTER 2 PHARMACOLOGICAL INDUCTION OF THE HEAT SHOCK RESPONSE IMPROVES MYELINATION IN A NEU ROPATHIC MODEL Introduction Hereditary peripheral neuropathies com prise a heterogeneous group of disorders, among which Charcot Marie Tooth disease type 1A (CMT1A) is the most prevalent form (Shy et al., 2001) CMT1A is a demyelinating neuropathy mainly associated with a 1.5 megabase duplication on human chromosome 17 that includes the PMP22 locus (Lupski et al., 1991) Transgenic rodent m odels based on the overexpression of the W t PMP22 reproduce features of the human condition and provide experimental models to study disease pathogenesis (Huxley et al., 1996; Magyar et al., 1996; Sereda et al., 1996; Perea et al., 2001; Robertson et al., 2002b) One of these transgenic lines termed C22, express approximately 1.7fold higher levels of the PMP22 mRNA and display slowed ner ve conduction velocity and a reduction in the percentage of myelinated fibers (Huxley et al., 1996; Huxley et al., 1998; Robertson et al., 1999; Robertson et al., 2002b) Eukaryotic cells maintain protein homeostasis by using a collection of quality control pathways known as the unfolded protein response (UPR). Induction o f UPR leads to the attenuated protein translation in the ER, ER assisted folding and ER associated degradation (ERAD) via the proteasome (Kincaid and Cooper, 2007) PMP22 folds with only a modest efficiency even under normal conditions (Sanders et al., 2001) as approximately eighty percent of the newly synthesized protein is degraded by the proteasome (Pareek et al., 1997; Notterpek et al., 1999a) In response to PMP22 overexpression, the quality control system appears to be overwhelmed and protein aggregates form. In the C22 mouse model, a reduced turnover of the newly synthesized
43 PMP22 is associated with the presence of cytosolic protein aggregates within SCs and impaired proteasome activity (Fortun et al., 2006) In myelinating DRG explant cultures, the retention of PMP22 within the SC cytosol decreases the amount of protein at the plasma membrane (Fortun et al., 2006), which could contribute to the observed myelin defects in affected mice. A promising therapeutic approach for protein misfolding disorders, such as PMP22 a ssociated neuropathies, involves the enhancement of chaperone expression (Muchowski and Wacker, 2005) Inhibitors of HSP90, including geldanamycin (GA) and its pharmacologically improved derivatives, 17DMAG and 17AAG, have been shown to suppress aggregation of mutant huntingtin and synuclein in cultured cells (Sittler et al., 2001; Hay et al., 2004; McLean et al., 2004; Herbst and Wanker, 2007) A family of small molecule HSP90 inhibitors significantly reduce tau protein levels and selectively clear specific phosphotau aggregates in association with an increase in the levels of HSP27, HSP40 and HSP70 (Dickey et al., 2005; Dickey et al., 2006) These studies underscore the importance of HSPs in the elimination of misfolded proteins in neurodegenerative diseases; however their potential application for hereditary peripheral neuropathies has not been tested. Here we show that out of fifteen small molecule inhibitors of HSP90, EC137 effectively enhances chaperone levels and improves myelination, along with the trafficking of PMP22, in DRG explant cultures from neuropathic mice. Materials and Methods Mouse C olonies The PMP 22 overexpressor (C22) (Huxley et al., 1996) mouse breeding colony is housed under SPF conditions at the University of Florida, M cKnight Brain Institute
44 animal facility. The use of animals for these studies has been approved by an Institutional Animal Care and Use Committee (IACUC). Genomic DNA was isolated from tail biopsies of mouse pups (less than 10 days old) and litters were genotyped by PCR (Huxley et al., 1996) Non Myelinating SC C ultures Primary SC cultures from genotyped postnatal day 6 (P6) Wt and C22 mouse pups, or neonatal rat pups, were prepared and maintained as described (Ryan et al., 2002) Cells were grown to 80% confluency in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Hyclone, L ogan, UT, USA), 2.5 (mouse) or 5 M (rat) forskolin (Calbiochem, La Jolla, CA, USA) and 10 g/mL bovine pituitary extract (Biomedical Technologies Inc, Stoughton, MA, USA) Cellular Toxicity A ssay Non myelinating rat SC cultures were treated with small mol ecule, synthetic HSP90 inhibitors at 50 nM and 500 nM (Dickey et al., 2005; Dickey et al., 2006; Dickey et al., 2007) concentrations for 16 h and then incubated in the MTT (3[4,5 dimethylthiazol 2 yl] 2,5 diphenyl tetrazolium bromide) solution (0.5 mg/ml) for 5 h at 37 C, producing the formazan product as a result of the cleavage of the tetrazolium salt MTT by the mitochondrial enzyme succinatedehydrogenase (Mosmann, 1983) The amount of blue formazan product is directly proportional to the number of viable cells present. The optical density (OD) of each well was measured using an automated plate reader (550 nm) (Heaton et al., 2004) The toxici ty of each test compound with respect to dimethylsulphoxide ( DMSO ) treated control cells from three independent experiments was determined and graphed as the percentage of cell death. Statistical significance was determined by using Students t test.
45 DRG E xplant Cultures and Compound Treatment Paradigms Mouse DRG explant cultures were established as described (Cosgaya et al., 2002) Pregnant Wt and heterozygous C22 mice were sacrificed according to guidelines of University of Florida Institutional Animal Care and Use Committee. DRGs were collected from embryonic day 1214 mice, digested with 0.25% trypsin (Gibco, Rockville, MD), dissociated and plated on rat tail collagencoated (Biomedical Technologies, Inc.) glass coverslips. DNA was isolated from each embryo for genotyping by PCR, as described above. Explants were maintained in minimum essential medium (MEM; Gibco) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 0.3% glucose (SigmaAldrich, St. Louis, MO), 10 mM HEPES (Cellgro; Mediatech, Inc., H erndon, VA), and 100 ng/ml nerve growth factor (Harlan Bioproducts for Science, Madison, WI) for 7 days. In the case of the GA paradigm, myelination was Aldrich) which was followed by GA exposure for 72 h. For exposure with EC137, cultures were maintained under myelinationpromoting conditions for 14 days and treated with compound for two 48 h periods, with a 48 h washout, in between. After the second 48 h treatment and a 16 h washout, the cultures were processed for immunostaining and Western blot analyses (Fig, 2 5A). For SC depleted neuronal cultures (Einheber et al., 1993) the explants were treated for 24 or 48 h w ith HSP90 inhibitors. Primary Antibodies Antibodies for protein chaperones included anti HSP70, HSP40, crystallin and calnexin (all polyclonal rabbit antibodies, from Stressgen, Victoria British Columbia,
46 Canada), rat anti HSP90 (Stressgen) and goat anti HSP27 (Santa Cruz, CA). To monitor the HS transcriptional response, a polyclonal rabbit anti HSF1 antibody (Stressgen) was employed. Antibodies for myelin proteins included monoclonal mouseanti myelin associated glycoprotein (MAG), rat anti myeli n basic protein (MBP) (both from Chemicon, Temencula, CA, USA), mouse anti P0 (Archelos et al., 1993a) To detect PMP22, a 1:1 mixture of two rabbit polyclonal antibodies, developed against a peptide corresponding to the second extracellular loop of the human or the rat PMP22, was used (Pare ek et al., 1997; Fortun et al., 2006) Monoclonal anti actin, tubulin (both from Sigma), or glyceraldehyde3 phosphate dehydrogenase (GAPDH) (clone 1D4, EnCor Biotechnology Inc., Alachua, FL, USA) served as protein loading controls. Polyclonal rabbit ant i ubiquitin (Dako, Carpinteria, CA) was purchased from the indicated supplier. Immunolabeling S tudies Non myelinating SCs and myelinating DRG explant cultures on glass coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 100% me thanol for 5 min at 20 C. After blocking with 10% normal goat serum, the samples were incubated with the indicated primary antibodies overnight at 4 C, followed by the appropriate secondary antibodies, including Alexa Fluor 594 goat anti rabbit IgG, Alex a Fl uor 488 goat anti rat IgG and Alexa Fluor 488 goat anti mouse IgG (all from Molecular Probes, Eugene, OR). Hoechst dye (Molecular Probes) was included in the secondary Pro Long Antifade kit (Molecular Probes). Samples were imaged with a Spot camera
47 attached to a Nikon Eclipse E800 microscope, or a Leica TCS SP2 AOBS Spectral confocal microscope and were formatted for printing by using Adobe Photoshop 5.5. Biochemical S tud ies Untreated control and compound treated cultures were lysed in sodium dodecyl sulfate (SDS) gel sample b uffer (62.5 nM Tris, pH 6.8, 10% glycerol, 3% SDS) and protein concentrations were determined using BCA assay (Pierce, Rockford, IL, USA). Samples were analyzed on polyacrylamide gels under reducing conditions (except for the determination of MAG), and transferred to nitrocellulose membranes (BioRad Laboratories, Hercules, CA, USA ). Membranes were blocked in 5% nonfat milk in PBS and incubated overni ght with primary antibodies. After washing, anti mouse, anti rabbit or anti rat HRP linked secondary antibodies were added for 2 h. Bound antibodies were visualized using an enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Boston, MA, U SA). Films were digitally imaged using a GS 710 densitometer (Bio Rad Laboratories) and were formatted for printing by using Adobe Photoshop 5.5. Densitometric analysis of Western blots was performed using Scion image software. Quantification of M yelin I nt ernode L engths DRG explant cultures were subjected to the treatment paradigms described above and immunostained with an anti MBP antibody to label internodal myelin segments (Amici et al., 2007) Internode lengths from Wt and C22 cultures from three independent experiments were measured with Spot RT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Measure ments were collected from three coverslips per genotype per treatment paradigm. Statistical significance was determined by using Students t test using GraphPad Prism software.
48 R esults Myelin Production in C22 Neuropathic Samples is E nhanced by G eldanamycin In the DRG explant model, sensory neurons and SCs from normal mouse embryos produce many myelinated segments, while samples from neuropathic mice only form a few shortened segments (Fortun et al., 2006; Amici et al., 2007) SCs cultured with DRG neurons from C22 neuropathic mice accumulate PMP22 within their cytosol (Fig. 2 1A, upper left, arrows) (Fortun et al., 2006) To test, whether enhancement of protein chaperone expr ession might be beneficial for myelination in these samples, DRG explants from C22 mice were treated with GA for 72 h and processed for immunolabeling and Western blots (Fig. 2 1). GA is a naturally occurring ansamycin antibiotic which inhibits HSP90, ther eby activating HSF1 and the expression of chaperones, including HSP70, HSP40 and HSP27 (McDonough and Patterson, 2003) Exposure of explant cultures fr om neuropathic mice to 50 nM GA for 72 h reduced the presence of cytosolic PMP22 aggregates (Fig. 2 1A, upper right), as compared to DMSO treated control samples (Fig. 2 1A, upper left, arrows). Treatment with GA also increased myelin production, as judged from immunolabeling with an anti MBP antibody (Fig. 2 1A, lower right). As shown in previous studies with GA (Nixon et al., 1994; Kim et al., 1999; Petrucelli et al., 2004) in total protein lysates we detected a prominent induction of HSP70 and HSP27, as compared to DMSO treated controls (Fig. 2 1B). In agreement with the enhanced myelination (Fig. 2 1A), we also detected an increase in the steady state level of PMP22 (Fig. 2 1B), which is known to correlate with compact myelin formation (Snipes et al., 1992) While additional studies of GA treated cultures indicate a consistent improvement of myelination in explants from neuropathic mice, prolonged exposure to t his compound is known to be toxic (Miy ata, 2005) Therefore,
49 we decided to test a class of synthetic HSP90 inhibitors, which cause less toxicity in cultured neuroglioma cells and have the potential for in vivo application (Dickey et al., 2005; Dickey et al., 2007) Small M olecule HSP 90 I nhibitors Enhance Chaperone E xpression in SCs To begin our studies with the synthetic HSP90 inhibitors (E C compounds), nonmyelinating primary rat SC cultures were treated for 16 h at 50 and 500 nM concentrations, followed by MTT cellular toxicity assay (Fig. 2 2A). We used rat SCs for the initial screening of the compounds due to the relative ease of obtaini ng a large number of homogeneous cell populations from neonatal rat nerves, as compared to mouse. In comparison to GA, w hich led to a significant 2025 % glial cell death (p<0.001), several EC compounds were less toxic to SCs. For example, EC137 at 50 nM h as significantly less cellular toxicity as compared to GA at 50 nM (p<0.001) (Fig. 2 2A). In parallel with the cellular toxicity studies, we have tested the ability of these compounds to induce HSP70 expression (Fig. 2 2B). Eight out of fifteen tested comp ounds induced HSP70 at 50 nM, while thirteen out of fifteen were effective at 500 nM (Fig. 2 2B). Furthermore, two negative controls, EC116 and EC117 that are structurally related inactive HSP90 inhibitors, failed to induce HSP70. The levels of calnexin, w hich is an ER chaperone rather than a HSP, are unaltered by exposure of the cells to the HSP90 inhibitors (Fig. 2 2B). From these results, we chose EC137, EC119, EC127 and EC139 for further studies at 50 nM concentrations. While all four of these compounds at 50 nM concentrations enhanced HSP70 expression in rat SCs, pilot studies with neuropathic samples identified EC137 as the most effective compound in reducing the levels of poly ubiquitinated proteins (Fig. 2 3A). In agreement with our previous studies (Ryan et al., 2002; Fortun et al., 2003) the basal levels of HSP70 are
50 elevated in SCs from neuropathic mice, which is further enhanced upon treatment with HSP90 inhibitor s. To characterize the effect of EC137 on glial gene expression, the dose dependent induction of a panel of chaperones was determined at five different concentrations, including 10, 50, 100, 250 and 500 nM (Fig. 2 3B). Compared to control levels, after a 1 crystallin are enhanced. However, the levels of these HSPs are elevated ~3fold higher at the 50 nM dosage and are comparable to HS ( 45 C for 20 min) preconditioning, followed by an 8 h chase (Fig. 2 3B). In agreement with known molecular targets of EC137, this concentration also enhanced the levels of HSP90 and HSP40 in the SCs. As treatment of the cells with higher dosages of EC137 did not appear to further stimulate chaperone expression, we chose the 50 nM concentration for subsequent studies. To optimize potential treatment paradigms for the myelinating samples, the induction of HSPs was analyzed after 4, 8, 16, 24 and 48 h incubation with 50 nM EC137 (Fig. 2 3C). As judged from the We stern blot, the peak expression of HSPs is observed at 16 h. As seen previously (Fig. 2 2B), the levels of the ER chaperone calnexin are unaffected by EC137 (Fig. 2 3C). To further characterize the kinetics of HSP induction by EC137, SC cultures were treat ed for 4 h, followed by wash out and chase time points at 4, 24 and 36 h (Fig. 2 3D). As shown in the Western blot, a 4 h treatment with EC137 is associated with a sustained expression of HSP70 up to 36 h (Fig. 2 3D). In comparison, the influence on the le vels of HSP27 is short lived. The effects of EC137 on HSP70 in nonmyelinating rat SCs were confirmed by immunostaining (Fig. 2 3E). As compared to DMSO controls, HSP70 like immunoreactivity is prominent and detected within the cell soma after EC137 treat m ent.
51 Inhibitors of HSP90, such as EC137 enhance the expression of chaperones by promoting the nuclear localization and phosphorylation of HSF1 (Westerheide and Morimoto, 2005) To investigate the activation of HSF1 in our SC model, cells were exposed to a brief HS (45 C for 20 min) (Fortun et al., 2007) or 50 nM EC137 for 2, 4, 8 or 16 h, followed by analysis with an HSF1 antibody (Fig. 2 4A). HS preconditioning leads to rapid phosphorylation of HSF1, as detected by a shift in the mobility of the protein on SDS gels (Fig. 2 4A, arrow). The activation of HSF1 by HS is transient, as the nonphosphorylated form (Fig. 2 4A, arrowhead) becomes the prominent protein after a 0.5 h chase. In comparison, EC137 exposure promotes HSF1 phosphorylation starting from 2 h post treatment and the active phosphorylated form remai ns for up to 8 h. The protein returns to baseline by 16 h (Fig. 2 4A). To corroborate the activation of HSF1 by EC137, we determined protein localization in cells post HS or after a 2 h compound exposure (Fig. 2 4B). In DMSO treated control cells, HSF1 is detected both in and around the nucleus (Fig. 2 4B, top panel, arrows). HS preconditioning of the cells promotes the translocation of HSF1 to the nucleus within 20 minutes (Fig. 2 4B, middle panel, arrows). Similarly, exposure of the cultures to EC137 for 2 h leads to prominent nuclear HSF1like immunoreactivity (Fig. 2 4B, bottom panel, arrows). Together, these studies indicate that synthetic inhibitors of HSF1 are well tolerated by primary peripheral glial cells and EC137 is an effective inducer of the HS response, as judged from the enhanced and sustained expression of HSPs post treatment. The results of the cellular toxicity and protein expression studies with EC137 from rat SCs (Figs. 2 2 to 24), were confirmed in mouse SC isolated from Wt pups, prior to the beginning of the studies with the explant cultures from transgenic mice.
52 Enhancement of HSPs P romotes Myelination in E xp lant Cultures from Neuropathic M ice Next, we asked whether induction of HSPs could assist in the processing of glial proteins and improve myelination in samples from neuropathic mice. To test the influence of EC137 on myelination, DRGs from Wt and C22 embryos were incubated with DMSO as a control, or EC137 (50 nM), according to the paradigm shown (Fig. 2 5A). The treatment with EC13 7 was initiated after a 14day period under myelinationpromoting conditions (Fig. 2 5A). The explants were exposed to EC137 (50 nM) for a total of 96 h according to the schedule shown. This treatment paradigm is based on the data obtained from our dosage and time course experiments (Fig. 2 3 panels B D). In order to maximize the chaperone response, we chose 48 h exposure, during which time HSP levels are elevated in both SC and DRG explant cultures (Fig. 2 3C, 2 6D). The 48 h washout was selected based on the maintenance of chaperone expression for over 36 h (Fig. 2 3D). For the detection of compact myelin, samples were immunostained with antibodies to MBP (Fig. 2 5B). As previously shown (Fortun et al., 2006; Amici et al., 2007) SCs in explant cultures from Wt embryos deposit numerous anti MBP antibody reactive myelin segments, with or without compound treatment (Fig. 2 5B, top panels). At higher magnification, MBP like immunoreactivity appears as the characteristic "rail road tracks" of compact myelin (Fig. 2 5B, top panel, insets on bottom right). In comparison, in DRGs from C22 embryos, the DMSO control samples contain few, short MBP positive myelin segments. Significantly, treatment of the neuropathic cultures with EC137 is ass ociated with a pronounced improvement in the abundance of myelin internodes (Fig. 2 5B, bottom right). As indicated in the inset, myelin formed in response to EC137 exposure appears similar to those formed in cultures from Wt mice. To
53 quantify the improvem ent in myelination in explant cultures from neuropathic mice, we measured MBP reactive internode lengths (Fig. 2 5C). The average internode length of Wt DRGs treated with DMSO is 161.1 4.818 m (mean SEM). In Wt DRGs treated with EC137 there is a small but statistically significant increase in internode lengths (178.8 4.797 m; p<0.05) (Fig. 2 5C). Strikingly, in DRGs from neuropathic mice, we found a 5fold increase in internode lengths after treatment with EC137 (increase from 22.60 1.384 m to 9 0.26 8.410 m; p<0.001) (Fig. 2 5C). These results indicate that activation of HSPs has a positive influence on myelination by peripheral glial cells, particularly those from neuropathic mice. To corroborate the positive influence of EC137 on myelin prot ein expression, total protein lysates of DRGs from Wt and C22 embryos at the end of the treatment paradigms were analyzed by Western blots (Fig. 2 5D). In agreement with the improvement in myelin internode formation in EC137treated samples (Fig. 2 5B and C), we found an increase in the steady state level of myelin proteins, including MAG, P0 and MBP (Fig. 2 5D). In samples from Wt mice, there was a ~1.31.7 fold increase in the above mentioned myelin proteins, as determined by densitometric analysis, an ef fect that is statistically significant (p<0.05 for all the myelin proteins in Wt samples, n=3). In comparison, in samples from neuropathic mice, we detected a pronounced ~14 fold increase in the levels of MBP (p<0.01, n=3), ~4fold increase in P0 (p<0.01, n=3) and ~3fold increase in MAG (p<0.05, n=3) (Fig. 2 5D), subsequent to EC137 treatment. As expected, EC137 exposure is associated with a pronounced induction in HSP70 in both Wt and neuropathic samples. The blots shown are representative of at least three independent experiments for each condition and were reprobed with an antibody to GAPDH to monitor protein loading.
54 In nerves of C22 neuropathic mice, by 35S pulse chase analysis we detected an accumulation of endo H sensitive PMP22 and an increase in P MP22 levels, when we used a combination of antibodies against the mouse and the human protein (Fortun et al., 2006) Therefore, rather than comparing the overall levels of PMP22 between our Wt and neuropathic samples, we asked whether the subcellular trafficking of PMP 22 is influenced by EC137 treatment (Fig. 2 6A). Accumulation of endo H resistant PMP22 within SCs correlates with myelination and represents the long lived membrane form of the protein (Pareek et al., 1997) The fraction of endo H resistant PMP22 in DMSO treated DRGs from Wt and C22 embryos is comparable to that seen in sciatic nerve lysates from 6 month old Wt and C22 mice, respectively (Fig. 2 6A) (also see Fortun et al., 20 06), thus mimicking the in vivo situation closely. With EC137 treatment, the endo H resistant fraction in Wt DRGs is slightly increased from 84 to 86% (p=0.12; n=3). In comparison, in DRGs from neuropathic mice the endo H resistant pool of PMP22 is significa ntly improved from 56.03 1.23% to 73.97 0.92% (mean SEM, p<0.001; n=3). Compare the levels of endo H resistant ~22 kDa (arrow) and endo H sensitive 18 kDa forms (arrowhead)] (Fig. 2 6A). The slowed mobility of the endo H resistant PMP22 in EC137 tr eated samples, as compared to DMSO controls, is consistent among independent samples and may reflect altered glycosylation and/or folding of the protein. The intracellular retention of PMP22 in samples from C22 mice is associated with an accumulation of poly ubiquitinated proteins and an impairment of proteasome activity (Fortun et al., 2006) To test if EC137 (50 nM) treatment impacts the accumulation of such slow migrating poly ubiquitinated proteins in SCs from C22 mice, cell lysates with or without compound treatm ent were analyzed with an ubiquitin antibody (Fig. 2 6B). In agreement with our initial studies (Fig. 2 3A), a 16 h treatment with EC137 is associated
55 with a reduction in high molecular weight poly ubiquitinated substrates in SCs from C22 mice, as well as Wt mice. This reduction in poly ubiquitinated substrates is statistically significant in SCs from C22 mice (p<0.01; n=3) but not in SCs from Wt mice (p=0.29; n=3) (Fig. 2 6B). GAPDH is shown as a protein loading control. Improvement in myelination by neuro pathic samples could be mediated by an influence of EC137 on glial, as well as neuronal genes. Therefore, we tested purified Wt and C22 mouse SCs (Fig. 2 6C), and DRG neurons with or without glia (Fig. 2 6D), for their response to EC137 treatment. As shown on the Western blots for treatment of DRGs from Wt and C22 mice (Fig. 2 5D), EC137 enhances the steady state levels of HSP70 and B crystallin in SCs from Wt and C22 mice, alike (Fig. 2 6C). In agreement with our previous studies (Ryan et al., 2002; Fortun et al., 2003) and also seen previously (Fig. 2 crystallin are elevated in neuropathic mouse SCs as compared to Wt (compare DMSO cont rols in Fig. 2 6C), likely as a response to accumulated poly ubiquitinated substrates. The basal levels of HSP27 and HSP40 in DMSO treated SCs from C22 mice are comparable to that of SCs from Wt mice and are enhanced in response to EC137 treatment. The steady state expression of HSP90 (Fig. 2 6C), calnexin and Bip/Grp78 (data not shown) are largely unaffected by EC137. DRG explants from Wt mice containing SCs respond to EC137 by induction of HSP70, HSP27 and B crystallin after a 48 h treatment (50 nM), whi le DRG neurons without SCs show an attenuated reaction (Fig. 2 6D). The same experiment was also done for 24 h treatment with EC137 (50 nM) and essentially showed a similar pattern of induction (data not shown). This result indicates that EC137 primarily i nfluences chaperone synthesis in peripheral glial cells, as compared to sensory neurons.
56 Discussion Enhancement of the HS response by natural or synthetic compounds is of therapeutic interest for protein misfolding disorders (Westerheide and Morimoto, 2005) Hereditary neuropathies linke d to the misexpression of PMP22 share characteristics with such disorders including the formation of cytosolic protein aggregates (Fortun et al., 2003; Fortun et al., 2006) Here we tested if enhancement of the chaperone pathway through inhibition of HSP90 would be beneficial for myelin formation by SCs from neuropathic mice with PMP22 misexpression. The chosen synthetic, small molecule HSP90 inhibitors offer a favorable approach as they exhibit low cellular toxicity, and induce sustained expression of HSPs (Dickey et al., 2005; Dickey et al., 2007) Our results indicate that nonmyelinating and myelinating glial cells respond to EC137 by crystallin. Sig nificantly, the enhancement of chaperones is associated with a pronounced improvement in myelination in neuronglia explant cultures from neuropathic mice, as compared to untreated controls. These results suggest that peripheral glial cells are amenable to pharmacologic modulation of the HS response and recommend further studies with these compounds. While the precise molecular mechanism by which inhibition of HSP90 aids myelin formation by SCs from neuropathic mice is unclear, it likely involves the assist ance of chaperones in the folding and processing of myelin proteins, including PMP22 (Fig. 2 7). Studies in cultured cells and neuropathic nerves indicate that PMP22 is prone to aggregation and accumulates in the cytoplasm of SCs when the proteasome is inhibited or the protein is misexpressed (Fig. 2 7A) (Notterpek et al., 1999b; Fortun et al., 2003; Fortun et al., 2007) These intracellular PMP22 aggregates retain cyt osolic chaperones
57 and MBP, which alters protein homeostasis within SCs. The sustained enhancement of the available pool of chaperones by EC137 likely aids the correct folding of newly synthesized PMP22 and other glial proteins, and promotes their trafficki ng to the plasma membrane (Fig. 2 7B). The observed increase in the endo H resistant fraction of PMP22 in EC137 treated cultures (Fig. 2 6A) indeed supports a primary influence of this compound on protein folding. The treatment with EC137 also decreased the levels of poly ubiquitinated proteins within SCs from C22 neuropathic mice (Fig. 2 6B), which may suggest an effect on protein degradation. Based on our current results, a potential role for chaperones in aiding the removal of misfolded PMP22 cannot be r uled out. A protective role for chaperones in preventing the misfolding and subsequent aggregation of PMP22 is supported by our previous in vitro studies (Notterpek et al., 1999a; Fortun et al., 2007) In normal nonm yelinating rat SCs, under conditions of proteasome inhibition, over ninety percent of the cells form PMP22 aggregates. When these studies were performed in conjunction with HS preconditioning or GA treatment, the misfolding of PMP22 was significantly reduc ed (Fortun et al., 2007) The decrease in protein aggregate formation in this pharmacologic model was likely due to the enhancement of cytosolic molecular chaperones, which aid the processing of newly synthesized PMP22 and/or refolding of small aggregates before the assembly of large inclusions. In the same assay, GA was more effective in preventing protein aggregate formation, as compared to HS (Fortun et al., 2007) GA binds to the ATP site on HSP90 and blocks its interaction with HSF1, and thus promotes HSF1 activation and the synthesis of HSPs (Prodromou et al., 1997; Zou et al., 1998) How ever, extended or high dose treatment with GA is associated with cellular toxicity which limits the potential therapeutic use of this compound (Miyata, 2005) EC137 a synthetic small molecule
58 inhibitor of HSP90 used in this study has suitable pharmacokinetic profile (Figs. 2 2 to 2 4) for potential therapeutic use and enhances the levels of HSP70 for over 36 h in SCs, as compared to vehicle control (Fig. 2 3D). Intraperitoneal injection of EC102, a small synthetic HSP90 inhibitor, and EC72, a derivative of GA, in mouse models of tauopathy and experimental autoimmune encephalomyelitis, respectively, induced high levels of HSP70 with low toxicity and was associate d with an amelioration of disease (Dello Russo et al., 2006; Dickey et al., 2007) It is yet to be determined if EC137 identified i n this study could be administered intraperitoneally to neuropathic mice. The modulation of the HSR in preventing the aggregation of cytosolic and nuclear disease linked proteins has been studied extensively (Westerheide and Morimoto, 2005) For example, live cell imaging experiments show that HSP70 associates transiently with huntingtin aggregates, with associationdissociation kinetics identical to chaperone interactions with unfolded polypeptides (Kim et al., 2002b) On the contrary, the protection observed in Drosophila models of Parkinsons and polyglutamine expansion in response to overexpression of HSP70 is not accompanied by a reduction in the number of inclusions (Warrick et al., 1999; Auluck et al., 2002; Kazemi Esfarjani and Benzer, 2002) A multidomain glycoprotein whose misfolding is associ ated with disease is cystic fibrosis transmembrane regulator (CFTR) (Amaral and Kunzelmann, 2007) CFTR and PMP22 share similarities in their high propensity for aggregation and they are both substrates for proteasomal degradation (Johnston et al., 1998; Notterpek et al., 1999b) In cells stably transfected with Wt CFTR, the overexpression of HSP70 and its co chaperone HSP40 was associated with an increased stability of the immature CFTR, but had no influence on the maturation of the protein (Farinha et al., 2002) Currently it is unknown if the beneficial effects of elevated HSPs on PMP22 processing
59 are mediated by direct or indirect interactions between these proteins. The only chaperone so far identified to interact with PMP22 is calnexin (Di ckson et al., 2002) In general, a critical role for chaperones in myelination is supported by multiple studies, in distinct model systems. The association of HSP70 with MBP in an ATP dependent manner in the normal human brain (Lund et al., 2006) implies that HSP70 is involved in the proper folding and trafficking (Hartl, 1996) of this cytosolic myelin protein. Indeed, constitutive expression of HSC70 appears to be essential for the correct expression of MBP during the differentiation of oligodendrocytes (Aquino et al., 1998) When HSC70 expression was shut down the steady state levels of MBP dramatically decreased (Aquino et al., 1998) In agreement, we observed elevated expression of MBP and significant increase in MBP positive myelin internodes in response to EC137 treatment, as compared to control samples (Fig. 2 5). This functional improvement correlates with induction of HSP70 and HSP27 within SCs (Fig s. 2 5D, 2 6C). In an independent study using the transgenic approach, the del etion of hsf1 resulted in a demyelinating phenotype, possibly due to defects in oligodendrocyte differentiation, or myelin synthesis and assembly (Homma et al., 2007) These results support our working model (Fig. 2 7), in which induction of HSPs via HSF1 activation in myelinating neuropathic SCs enhances the cytosolic chaperone pool, reduces the accumulation of poly ubiquitinated substrates, and aids the trafficking of PMP22. Studies in CMT1A pedigrees and genetically engineered neuropathic models underscore the importance of adequate levels of correctly folded PMP22 for myelin formation and stability (Kuhlenbaumer et al., 2002; Robertson et al., 2002b) In C22 mice, the secretory pathway appears to be overwhelmed as we detected an accumulation of newly synthesized PMP22 within the cytosol and a decrease in the
60 endo H resistant PMP22 at the plasma membrane (Fortun et al., 2006) In agreement, studies of sural nerve biopsies from CMT1A patients with PMP22 gene duplication or point mutations show PMP22like immunoreactivity in the myelin sheath, as well as within the SC cytoplasm (Nishimura et al., 1996; Hanemann et al., 2000) However, the PMP22 like myelin staining is thin (Nishimura et al., 1996) and there is a reduction in the number of PMP22positive myelinated fibres (Hanemann et al., 1994) implying that only a small fraction of PMP22 is incorporated into myelin. Previously published therapeutic approaches to correct the myelin defects in PMP22associat ed neuropathies include the use of progesterone antagonists and ascorbic acid (Sereda et al., 2003; Passage et al., 2004) In both of these studies, the neuropathic phenotype was subst antially ameliorated by the interventions, and ascorbic acid is now in clinical trial for CMT 1A (Pareyson et al., 2006) Our current findings, while posing to be promising in culture, await furth er testing in neuropathic mice where optimal bioavailability of these compounds in peripheral nerves can be established. Based on data presented here, the tested small molecule inhibitors of HSP90, particularly EC137, could potentially offer a new approach for the treatment of demyelinating neuropathies.
61 Figure 21. Myelin production is stimulated by geldanamycin. (A) DRG explant cultures from C22 mice under myelinating conditions, were treated with DMSO or GA (50 nM) for 72 h, and stained with polycl onal anti PMP22 (red) or anti MBP (green) antibodies. Enhanced PMP22like immunoreactivity is associated with the SC bodies (arrows) in the DMSO treated samples. Magnification, X40. (B) The steady state levels of HSP70, HSP27 and PMP22 were determined in t otal lysates of DRG explant cultures (20 g/lane) from C22 neuropathic mice after 72 h of GA treatment (10 and 50 nM), as compared to DMSO control (C). Actin serves as a protein loading control. Molecular mass in kDa.
62 Figure 22. Cellular toxicity and chaperone expression for the HSP90 inhibitors. (A) Non myelinating rat SCs were treated with DMSO (control) or HSP90 inhibitors at 50 and 500 nM concentrations for 16 h and then incubated in MTT (0.5 mg/ml) for 5 h at 37 C. The toxicity of the test compounds was determined from three independent experiments with respect to DMSO treated co ntrol cells, which was set at 0% cell death (***p<0.001 and #p<0.001 with respect to DMSO control). Error bars indicate SEM. (B) The levels of HSP70 and calnexin (CNX) were determined by Western blot analyses in total cell lysates (20 g/lane) after 16 h treatment. GAPDH is shown as a protein loading control. Molecular mass in kDa.
63 Figure 2 3. EC137 stimulates chaperone production in nonmyelinating SCs in dose and ti me dependent manner. (A) SCs from C22 neuropathic mice were treated with low toxicity HSP90 inhibitors (EC137, EC119, EC127, EC139; all at 50 nM) and GA (50 nM) for 16 h and the levels of poly ubiquitinated (pUb) substrates and HSP70 were analyzed by Western blots (20 g/lane). Arrowhead indicates monoubiquitin (Ub). (B) For the dosage response, the levels of HSPs after EC137 treatment of rat SCs (10, 50, 100, 250 or 500 nM) were analyzed in total cell lysates (20 g/lane). HS followed by 8 h chase at 37 C is included as positive control. (C) For timecourse studies, the levels of HSPs were analyzed after treatment with EC137 (50 nM) for 4, 8, 16, 24 or 48 h. (D) To assess the maintenance of chaperone expression, cells were treated with EC137 (50 nM) for 2, 3 and 4 h. After the 4 h treatment, EC137 was washed out and chaperone expression assayed at 4, 24 and 36 h chase time points. Molecular mass in kDa. Tubulin (A C) or GAPDH (D) is shown as a protein loading control. (E) In cells treated with EC137 (50 nM 16 h) the localization of HSP70 is detected with an anti HSP70 (green) antibody. SCs treated with DMSO (control) exhibit low levels of HSP70like immunoreactivity (upper left, inset). Hoechst dye was used to stain the nuclei. Scale bar, 10 m.
64 Figure 2 4. Treatment with EC137 activates HSF1. (A) The phosphorylation state of HSF1 after HS and EC137 (50 nM) treatment was assayed by Western blot (20 g/lane). Phosphorylated HSF1 is indicated by the arrow and the arrowhead marks the migration position of the non phosphorylated form. Molecular mass in kDa. (B) Cultured rat SCs were treated with HSP90 inhibitor, EC137 and the translocation of HSF1 from cytosol to the nucleus was monitored by staining with an anti HSF1 (red) antibody. In untreated cells, HSF 1 is predominantly cytosolic (upper panel). HS preconditioning (45 C for 20 min) leads to rapid (0 h) nuclear localization of HSF1 (middle panel, arrows). The localization of HSF1 within the nucleus is detected at 2 h after treatment with EC137 (50 nM) ( lower panel, arrows). Hoechst dye was used to stain the nuclei. Scale bar, 10m.
65 Figure 25. Myelin production by neuropathic explant cultures is enhanced by EC137. (A) Schematic of the treatment paradigm for DRGs from Wt and C22 mice with EC137. The black line indicates the timescale for days in vitro (DIV). The lines in green, bold and dashed, represent the expression profiles of myelin proteins in DRGs from Wt and C22 mice respectively. The blue line indicates the timescale (h) for EC137 treatment Starting on DIV21, a pulse treatment of EC137 (50 nM) (green arrows) was added for 48 h, followed by 48 h washout (wo, black arrows). This sequence was repeated and a second wash out (16 h) was followed by analyses of the samples (arrow head). (B) DRG ex plant cultures from Wt (top panel) and C22 neuropathic (bottom panel) mice, under myelinating conditions, were treated with DMSO (control) or EC137 (50 nM) for a total of 96 h as described (A), and stained with an anti MBP antibody. Insets show the outlined regions at 3X magnification. Hoechst dye was used to stain the nuclei. Scale bar, 40 m. (C) The lengths of the myelin internodes (n is at least 100 for each condition) were measured in explant cultures from Wt and C22 mice treated with DMSO or EC137 (50 nM), using Spot Advanced software. *p<0.05, ***p< 0.001. Error bars show SEM. (D) DRG explants from Wt and C22 mice were treated as described (A) and whole protein lysates (40 g/lane) were analyzed for the levels of myelin proteins MAG, P0 and MBP, and of HSP70 from at least three independent experiments. Arrows on the MBP blots indicate the 21.5, 18.5, 17 and 14 kDa isoforms. GAPDH serves as a loading control. Molecular mass in kDa.
66 Figure 25. Continued
67 Figure 26. Induction of chaperones aids the processing and trafficking of PMP22. (A) Sciatic n erve lysates (10g/lane) from 6month old Wt and C22 mouse (n=3) were treated with endo H (H) or PNGase F (N) and blotted with anti PMP22 antibody. Total lysates (20g/lane) from Wt (top) and neuropathic (bottom) DRG explants treated with DMSO or EC137 (50nM) were incubated with endo H (H) or PNGase F (N) and PMP22 was detected. R denotes endo H resistant and S denotes endo H sensitive fractions (%) of PMP22. (B) A representative blot for the levels of pol y ubiquitinated proteins in lysates (20g/lane) of SCs from Wt and C22 mice treated with DMSO and EC137 (50 nM). (C) The levels of HSP70, HSP40, HSP27, B crystallin and HSP90 in SC lysates (20g/lane) from Wt and C22 mice (n=3) treated with DMSO and EC137 (50nM) were assayed. (D) The levels of HSP70, HSP27 and B crystallin in DRG explants, and SC depleted DRG neurons from Wt mice, after treatment with DMSO or EC137 (50nM, 48 h) are shown. GAPDH serves as a constitutive marker for the blots. Molecular mass in kDa.
68 Figure 2 7. Working model: HSP90 inhibitor aids PMP22 processing and improves myelination in neuropathic samples. (A) In SCs from neuropathic mice, there is an accumulation of PMP22 in cytosolic aggregates, which is associated with an impairm ent of protein degradation by the proteasome. Only a small fraction of PMP22 is transported to the plasma membrane, which leads to defects in myelination. (B) Exposure of SCs to HSP90 inhibitors promotes the activation and nuclear translocation of HSF1. Th e induction of HS genes and the expression of HSPs prevent the aggregation and promote the correct folding and processing of PMP22, as well as other myelin proteins. Restoration of subcellular protein homeostasis improves myelin formation.
69 CHAPTER 3 ENHA NCEMENT OF AUTOPHAGY BY RAPAMYCIN IMPROVES MYELINATION IN SCHWANN CELLS FROM NEUROPATHIC MICE Introduction Aggregation of misfolded proteins and their sequestration into intracellular inclusions is a problem common to diseases of the CNS and PNS (Stefani and Dobson, 2003) CMT1A is a prevalent protein misfolding disease in humans characterized by progressive demyelination of peripheral nerves and associated neuromuscular deficits. During the last ten years there have been many advances with the molecular diagnosis of CMT neuropathies, however effective drug therapy is still not available (Pareyson and Marchesi, 2009) Duplications of, or point mutations within, the PMP22 gene are known to cause CMT1A (Shy et al., 2001) and other related neuropathies. PMP22 is a hydrophobic integral membrane glycoprotein that is mostly expressed by my elinating SCs (Pareek et al., 1997) Transgenic mice based on the overexpression of the human Wt PMP22 termed C22, and spontaneous mutant (L16P) Trembler J (TrJ), reproduce features of the human neuropathy and provide valuable experimental models to study disease pathogenesis (Suter et al., 1992; Huxley et al., 1996) PMP22 folds with onl y a modest efficiency under normal conditions (Sanders et al., 2001) and nearly eighty percent of the newly synthesized protein is rapidly turned over by the proteasome (Pareek et al., 1997; Notterpek et al., 1999a) In response to PMP22 overproduction and the L16P mutation, excessive or defective PMP22 polypeptides are targeted for degradation by the ubiquitinproteasome system and accumulate in cytosolic aggregates (Fortun et al., 2003; Fortun et al., 2005; Fortun et al., 2006) Autophagic and lysosomal components as well as HSPs are recruited to ubiquitin positive PMP22 aggregates in nerves of C22 and TrJ mice, likely reflecting an
70 attempt by the cells to clear them through alternate pathways. This sequence of events decreases the amount of PMP22 protein within the SC plasma membrane, and likely contributes to the pronounced demyelinating phenotype (Huxley et al., 1996; Notterpek et al., 1997; Fortun et al., 2003; Fortun et al., 2006) Promising therapeutic approaches for protein misfolding disorders, such as PMP22 associated neur opathies, include increasing the synthesis of HSPs (Muchowski and Wacker, 2005) and stimulating autophagic protein degradation (Sarkar et al., 2009) In previous studies, we demonstrated that the activation of autophagy by amino acid and serum deprivation (Fortun et al., 2003; Fortun et al., 2007) or intermittent fasting, suppressed the accumulation of misfolded proteins with in neuropathic SCs and improved myelination in TrJ mice (Madorsky et al., 2009) Since such dramatic dietary restriction is not suitable for therapy in humans, here we asked whether pharmacological activation of autophagy within myelinating SCs could offer similar benefits. Rapamycin (RM), a macrolide antibiotic, is a widely used inhibitor of the mammalian target of rapamycin (mTOR) and induces autophagy in a variety of cell types (Sabers et al., 1995; Sarkar and Rubinsz tein, 2008) In this study, we show that autophagy is a critical pathway for RM mediated myelin improvement in neuropathic SCs. Materials and M ethods Mouse C olonies PMP22 overexpressor (C22) (Huxley et al., 1996) and spontaneous mutant TrJ (Suter et al., 1992) neuropathic mouse breeding colonies are housed under SPF conditions at the University of Florida, McKnight Brain Institute animal facility. The use of animals for these studies has been approved by an Institutional Animal Care and Use
71 Committee (IACUC). Genomic DNA was isolated from tail biopsies of mouse pups (less than 10days old) and litters were genotyped by PCR (Hux ley et al., 1996; Notterpek et al., 1997) Primary M ouse SC Cultures SC cultures from genotyped postnatal day 6 Wt, C22 and TrJ mouse pups were prepared and maintained as described with slight modifications (Nicholson et al., 2001) N erves were dissected and enzymatically digested over a period of 2 h. The digestion medium consisted of Dulbeccos Modification of Eagles Medium/F12 (DMEM/F12) with GlutaMAX I (Gibco, Grand Island, NY), 15% Fetal Bovine Serum (FBS) (Hyclone, Logan, UT), penicillin streptomycin (Gibco) and an enzyme cocktail of 0.03% collagenase type III (Worthington, Lakewood, NJ), 0.1% hyaluronidase (SigmaAldrich, St.Louis, MI) and 1.25 units/mL dispas e (Worthington). Next, the cell suspensions were washed once and resuspended in culture medium (DMEM/F12 containing 15% FBS). Cells were then plated in drops on poly L lysine (SigmaAldrich) coated plastic petri dishes and allowed to adhere overnight. The next day, cells were washed with D arabinofuranoside (SigmaAldrich), to eradicate contaminating fibroblasts. After two 48 h periods of antimitotic treatmen ts, given on forskolin [Calbiochem, La Jolla, CA]) was added and the cells were allowed to pro liferate for 2 days to reach 80% confluency.
72 Autophagic Flux M easurement Rapamycin (RM) (Calbiochem, San Diego, CA) was dissolved in ethanol vehicle and 0.5 mM stocks were stored at 80 C. Chloroquine (CQ) (SigmaAldrich) was dissolved in water to a stock of 50 mM and aliquots were stored at 80 C SCs were treated with vehicle control (Ct) or RM at a final concentration of 25 nM, either in the presence or absence of CQ for 48 h. Following this, the cultures were lysed and analyzed for microtubuleassocia ted protein light chain 3 ( MAP LC3, from here onwards referred to as LC3) by Western blots. A statistically significant increase in LC3 II band intensity in RM+CQ compared to Ct+CQ samples indicates an enhanced autophagic flux and signifies that the pathway attains completion (Mizushima and Yoshimori, 2007) Metabolic L abeling and 35S Pulse S tudies SCs were treated with vehicle control (Ct), RM (25 nM) for 48 h or positive control cyclohexamide (Tocris, Bristol, UK) (Chx, 100 g/ml) for 12 h. During the last 1.5 h of incubation, t he cells were starved in methionineand cysteinefree m edia with or without RM for 1 h. Following this, a 30 min pulse of medi um containing 0.25 mCi/ml trans 35S (ICN Biochemicals, Costa Mesa, CA) was added. After lysis in RIPA buffer (Pareek et al., 1997) proteins were precipitated wit h 5% trichloroacetate and bound 35S radioactivity was counted in a scintillation counter (LS6500, Beckman Coulter, Fullerton, triplicate experiments. Simultaneously, equal amounts of proteins were separated by SDS PAGE and the gels were dried and treated with AMPLIFY (Amersham Biosciences, Arlington Heights, IL). Dried gels were exposed to Classic autoradiography film (Molecular Technologies, St.Louis, MO) at 80C. Densitom etry was performed on the trans 35S labeled protein bands using Scion Image software (Scion, Frederick, MD).
73 Quantification was performed from triplicate experiments and statisti cal analysis was performed by Students t test using GraphPad Prism. DRG Expla nt C ultures and RM Treatment P aradigms DRG explant cultures were established from Wt, C22 and TrJ mouse embryos as described (Fortun et al., 2006) Cultures were exposed to RM (25 nM) for the indicated time points (Fig. 3 3A), including the last 8 h prior to analysis by immunostaining and Western blots. SC depleted neuronal cultures (Einheber et al., 1993) were treated for 48 h with RM (25 nM) or vehicle. Lentiviral Transduction of Mouse DRG E xplants Pre packaged lentiviral particles were purchased from a comm ercial provider (Santa Cruz Biotechnology, Santa Cruz, CA) which either encoded a nontargeting shRNA (negative shRNA) (cat# sc 108080) or a sequence specifically targeting mouse Atg12 (cat# sc 72579V). For the Atg12 knock down experiments, negative (Neg.) or Atg12 shRNA lentiviral particles were combined with the DRGs from Wt or TrJ mouse embryos at the time of plating. Each individual culture was transduced with approximately 7.5 x 10^4 infectious units of virus. Following transduction, cultures expres sing either Neg. or Atg12 shRNA were subjected to the late treatment paradigm ( Fig. 3 3A ) with RM. Each shRNA transduction experiment was performed at least three times from independent DRG isolations. To monitor the lentiviral transduction efficiency and transgene expression for the duration of the experiments, we incubated additional subsets of DRG explants with lentiviral particles enc oding GFP (cat# sc 108084). T ransduction efficiency was obtained as the ratio of the number of GFP+ to total number of ce lls from eight random visual fields from three independent culture experiments
74 Biochemical S tudies Control and RM treated samples were lysed and processed for Western analyses. The processing of PMP22 was assessed by treatment of the protein lysates with PNGaseF (N) or endo H (H) (New England Biolabs, Ipswich, MA) (Pareek et al., 1997) For the detection of PMP22 and protein zero (P0) we used previously characterized rabbit polyclonal and mouse monoclonal antibodies, respectively (Archelos et al., 1993b; Pareek et al., 1997) All other commercially available primary antibodies are listed in Suppl. Table 1 For the Western blot s, bound primary antibodies were detected with appropriate HRP linked secondary antibodies and visualized using the enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Boston, MA). Films were digitally imaged using a GS 710 densitometer (B io Rad Laboratories) and densitometric analysis of scans from three independent experiments was performed using Scion image. Statistical significance was determined by Students t test using GraphPad Prism. Immunolabeling S tudies Myelinating DRG explant cu ltures on glass coverslips were fixed, permeabilized and processed for immunostaining with anti MBP antibodies (Amic i et al., 2007) Bound primary antibodies were detected with Alexa Fluor 594 goat anti rat IgGs (Molecular Probes, Eugene, OR). Hoechst dye (Molecular Probes) was included in the secondary antibody solution to visualize nuclei. Coverslips were mounted usi ng the ProLong Antifade kit (Molecular Probes). Samples were imaged with an Olympus 1X81DSU spinning disk confocal microscope and were formatted for printing by using Adobe Photoshop 5.5.
75 Quantification of Myelin Internode Abundance and L engths DRG explan t cultures were subjected to the treatment paradigms described above and immunostained with an anti MBP antibody to label internodal myelin segments (Amici et al., 2007) The abundance of myelin internodes was counted as the number of MBP+ internodal segments per 0.1 mm2 area from three independent experiments, and eight random visual fields per condition (Fig. 3 3A). Internode lengths from the same experiments were measured with Spot RT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Measurements were collected from three coverslips per genotype per treatment paradigm (n=130260). Statistical significanc e was determined by Students t test using GraphPad Prism software. Results Rapamycin Activates Autophagy in Primary M ouse SCs The activation of autophagy by dietary restriction is able to suppress the formation of PMP22 aggregates and stimulate myelin for mation in TrJ mice (Madorsky et al., 2009) To enhance autophagy by pharmacologic means, we exposed mouse SCs to varying doses (10500 nM) of RM (Sarkar and Rubinsztein, 2008) After six hour (h) incubation, mTOR complex 1 (mTORC1) activity was monitored by the dephosphorylation of phosphoS6 (pS6) ribosomal protein and the activation of autophagy via the lipidation of LC3 (Fig. 3 1A). Based on the observed response, we chose the 25 nM concentration for all subsequent studies as it was the lowest dosage that consistently activated autophagy within Wt cells (Fi g. 3 1A to 3 1 C). To optimize the frequency of the treatment, the dephosphorylation of pS6 and the conversion of LC3 I to LC3 II (lipidated form) were analyzed over 248 h period (Fig. 3 1B). As shown on the Western blot, RM exposure activates autophagy wi thin SCs for at least 48 h, leading to a pronounced
76 increase in the ratio of lipidated LC3 II/LC3 I (Fig. 3 1C, **p<0.01, n=3). As exposure of certain cell lines to RM at 100 nM concentration can lead to dephosphorylation of Akt through the mTOR complex 2 (mTORC2) pathway (Sarbassov et al., 2006) we analyzed the same cell lysates for phosphorylated Akt (pAkt) (Ser473) (Fig 3 2 A). While o ver the 48 h treatment period there was a slight decline in the levels of pAkt when we analyzed samples from three independent experiments this change was not statistically significant (Fig. 3 2 B). This data suggest that under the described conditions RM act s primarily via the mTORC1 complex with in primary mouse SCs. To assess autophagic flux, a subset of samples were cotreated with RM and CQ, which inhibits the degradation of LC3 II by lysosomal hydrolases (Maiuri et al., 2007; Mizushima and Yoshimori, 2007) As shown on the blot (Fig. 3 3 A ) and supported by semi quantitat ive analyses (Fig. 3 3 B), the simultaneous exposure of SCs to RM and CQ leads to a significant increase in LC3 II, as compared to CQ treatment alone (**p<0.01, n=4). In agreement with the elevated basal lysosomal activity i n neuropathic samples (Notterpek et al., 1997; Fortun et al., 2006), the response of C22 SCs to CQ alone is more robust than of Wt Still, we detected an increase in LC3 II levels upon cotreatment (Fig. 3 3 C) which is significant in three independent experiments (Fig. 3 3 D, *p<0.05, n=3) These data indicate that while the basal level of autophagy is distinct between Wt and neuropathic samples, RM enhances the response in both cell types. The intracellular retention of PMP22 in samples from C22 mice is as sociated with an accumulation of poly ubiquitinated proteins and an impairment of proteasome activity (Fortun et al., 2006) Hence, we asked whether RM activated autophagy could suppress the accumulation of pUb substrates with in these cells. While w e detected a slight
77 reduction in slow migrating pUb proteins in Wt samples, this change was more pronounced in cells from neuropathic mice (Fig. 3 3 E). As reported previously (Fortun et al., 2006) s emi quantitative analyse s confirmed the accumulation of pUb proteins in C22 SCs as compared to Wt (Fig. 3 3 F, *p<0.05, n=3) and its reduction by RM (Fig. 3 3 F, **p<0.01, n=3). To evaluate the effect of extended (48 h) RM treatment on protein synthesis in general within primary SCs, we performed 35S pulse labeling followed by autoradiography (Pareek et al., 1997) and scintillation counting. Analyse s of autoradiographs (Fig. 3 4 A ) and qu antification of protein bound 35S (Fig. 3 4 B ) reveal that 48 h exposure of the cells to RM does not significantly alter protein synthesis, as compared to vehicle control These results support the notion that RM promotes the clearance of pUb proteins from neuropathic SCs by activating autophagy, rather than by blocking protein synthesis. Myelin Production in C22 Neuropathic Samples is E nhanced by R apa mycin DRG explant cultures from C22 neuropathic mice are impaired in their ability to make myelin (Fortun et al., 2006; Rangaraju et al., 2008) therefore serve as an ideal model to test potential therapeutic compounds for enhancing myelination. C ontrol samples Ct* and Ct** were obtained on days in vitro (DIV) 9 and 15 respectively either two or eight days post ascorbate, which time points coincided with the initiation of early and late RM exposure s (Fig. 3 5 A). Ascorbate is necessary for the init i ation of myelination (Eldridge et al., 1987) and was included in all of our experiments. As expected from previous in vivo and in vitro studies (Huxley et al., 1996; Fortun et al., 2006) whole protein lysates of vehicle treated neuropathic c ultures harvested on DIV24 contain low levels of myelin proteins, as compared to Wt (Fig. 35B, C). However, in
78 response to RM they show an increase in the steady state expression of both MBP and P0 (Fig. 35 B, C). The effect is more pronounced when the c ultures were analyzed after the late treatment paradigm as seen from the quantification of three independent experiments (Fig. 3 6 ). This data also show that RM exposure enhanced myelin protein synthesis by Wt SCs, which is nearly significant in the late treatment paradigm. We confirmed the positive effect of RM on myelination by immunostaining DRG explant cultures with an anti MBP antibody (Fig. 3 7 ). C ultures from Wt mice contain numerous MBP positive myelin internodes, while C22 e xplants form only a few shortened internodes (Fortun et al., 2006) Significantly, RM treatmen t of affected cultures is associated with a dramatic improvement in the number and length of myelin internodes, as shown on the confocal images (Fig. 3 7 A). The characteristic, railroad like appearance of myelin tracks is displayed in the enlargements in e ach of the panels (Fig. 3 7 A, insets, 3X magnification). Quantifications of the number (Fig. 3 7 B) and length (Fig. 3 7 C) of myelin internodes reveal highly significant improvements in samples from neuropathic mice, after both early and late RM treatments (***p<0.001, n=3). Wt DRGs show a slight, but not significant increase in the number of myelin internodes with the intervention (Fig. 3 7 B, n.s, n=3); however a lengthening of internodal myelin is significant for both early and late treatment paradigms (Fi g. 3 7 C, p<0.05, n=3). As the myelinating cultures contain DRG neurons, we tested the response of SC depleted Wt and C22 cultures (Einheber et al., 1993) to RM. While RM is active in neur ons, as judged from the pronounced dephosphorylation of pS6, induction of autophagy is less apparent (Fig. 3 8 A). Quantification of the LC3 I I and LC3 I ratios
79 indicate that peripheral neurons are less responsive to RM as compared to SCs (Fig. 3 8 B, C). To gether, these results demonstrate that the exposure of myelinating SCs to RM is beneficial for myelin synthesis, particularly for cells from neuropathic mice. Activation of Autophagy Improves PMP22 T rafficking The accumulation of endo H resistant PMP22 at the SC membrane correlates with myelin synthesis (Pareek et al., 1997; Notterpek et al., 1999a) Therefore, besides measuring the overall levels of PMP22 in our samples, we determined the subcellular trafficking of the protein (Fig. 3 9 A). Consistent with the improvements in myelination ( Figs 3 3 and 3 4), the overall levels of PMP22 are increased in neuropathic samples after early and late RM exposure (Fig. 3 9 A). Signi ficantly, we detected an increase in the fraction of endoH resistant PMP22 in RM treated cultures, as compared to vehicle. Since PMP22 exists in various glycosylated forms, we used N glycosidase (PNGase F) to confirm that the identified protein bands are i ndeed PMP22 (Pareek et al., 1997) Semi quantitative analyse s of three independent experiments confirm ed the reduced levels of endo H resistant PMP22 in C22 neuropathic samples as compared to Wt (Fig. 3 9 B, ***p<0.001, n=34) (Fortun et al., 2006) and its improvement after RM exposure at both early and late RM treatment s (Fig. 3 9 B, **p<0.01, n=3). There is a slight increase in the endo H resistant fraction of PMP22 in the Wt samples after early and late RM treatment, however, it is not significant (p=0.0576, p=0.0855, n=4). These results indicate that enhancement of autophagy within C22 neuropathic SCs is associated with an improvement in myelin formation and the subcellular processing of PMP22 (Figs. 3 5, 3 6, 3 7 and 3 9 ).
80 The Positive Effect of RM is not Genotype S pecific A suitable therapeutic agent for hereditary neuropathies would not be genotypespecific, but rather show efficacy in multiple models. To assess whether RM could be beneficial for PMP22 point mutant neuropathies, we carried out studies with SCs and myelinating explant cultures from TrJ mice (Fig. 3 10). Similar to the C22 model, DRGs from TrJ mice were incubated with RM at early or late stages of myelination and analyzed for MBP and P0 (Fig. 3 10). Myelinating explants from TrJ mice respond positively to RM and show an increase in the steady state level s of MBP and P0 (Fig. 3 10A, B). In agreement, DRG cultures immuno labeled with an MBP antibody reveal a pronounced increase in MBP positive myelin segments in response to treatment (Fig. 3 10C). Quantification from three independent experiments shows a significant improvement in the number (Fig. 3 10D, ***p<0.001, n=3) and length (Fig. 3 10E, **p<0.01, n=3) of myelin internodes, as compared to vehicle. We confirm ed the autophagic response in TrJ SCs, by analyz ing protein lysates for pUb and LC3 after 48 h treatment with RM (Fig. 3 11A). As seen with the C22 sample (Fig. 3 3 E, F), treatment with RM promotes a significant reduction in slow migrating pUb substrates (Fig. 3 11B ) and an approximately twofold increase in LC3I I /LC3 I ratio s (F ig. 3 11C). Therefore, RM is an effective enhancer of autophagy in SCs from TrJ mice and improves their myelination capacity when in neuronal contact. Autophagy is a Critical P athway for the Myelin Promoting Effect of R apamycin While RM is an effective en hancer of autophagy in SCs (Figs. 3 1, 3 3 and 3 11 ), to confirm the contribution of this pathway to the observed positive effects we utilized lentiviralmediated shRNA gene ablation (Bolis et al., 2009) We transduced DRG
81 explants with lentivir us carrying GFP and visualized the efficiency of transduction by direct GFP fluorescence ( Fig. 3 12). We obtained high transduction efficiency which was nearly 65% ( 64.669.04) on DIV11 (Fig. 3 12A) and around 62 % (62.23 11.88 ) on DIV26 (Fig. 3 12 B). W e performed shRNA mediated inhibition of the vital autophagy gene, autophagy related gene 12 ( Atg12 ) that is required for the elongation of the autophagosome (Dreux et al., 2009) Scramble negative (Neg.) a nd Atg12 shRNA transduced Wt and TrJ explants were treated with vehicle control (Ct) or RM as described in Fig. 35 A. Upon analysis of the protein lysates at the end of the treatment, the steady state levels of Atg12 protein i s reduced in samples transduce d with Atg12 shRNA, both in the absence and presence of RM (Fig. 3 13A). We analyzed the levels of MBP and P0 within the same samples (Fig. 3 13A). Quantification from three independent experiments reveals that Atg12 protein levels a re reduced by ~50% in cultures expressing Atg12 shRNA, as compared to those transduced with Neg. shRNA. (Fig. 3 13B, *p<0.05). Analyses of MBP levels normalized to glyceraldehyde 3phosphate dehydrogenase (GAPDH) reveal that downregulation of Atg12 did not significantly decrease MBP in Ct and RM treated Wt samples (Fig. 3 13C, n.s, n=3). However, the effect of RM to slightly improve MBP levels in Wt DRGs (see Fig. 36 ) in this case transduced with Neg. shRNA, is reproducible (Fig. 3 13A, p=0.0569). The reduction in Atg12 did not lead to a significant decrease in MBP in Ct samples from TrJ mice (Fig. 3 13C, n=3). As seen previously (Fig. 3 10B) we detected i mprovement in MBP levels with RM treatment in Neg. shRNA transduced neuropathic samples (Fig. 3 13C, **p<0.01) which was then largely abolished by the inhibition of Atg12 (Fig. 3 13C, *p<0.05, n=3). Similarly, the RM mediated improvements in P0 levels are attenuated by
82 the knock down of Atg 12 (Fig. 3 13D). Together, these results indicate that an intact autophagy pathway is necessary for the myelinationpromoting activity of RM. Discussion Hereditary neuropathies are common among the population, yet treatment options for affected individuals are limited. Currently, there is no effective drug therapy for CMT1A neuropathies and supportive care is limited to rehabilitation and surgical treatm ents (Pareyson and Marchesi, 2009) A recent twelve month clinical trial with a scorbic acid did not produce positive results, as efficacy of this anti oxidant was not shown (Micallef et al., 2009) Here we tested RM, a widely used therapeutic compound for its ability to enhance autophagic response within neuropathic SCs, and improve t heir capacity to myelinate. We show that RM effectively activated autophagy in mouse SCs and lead to a significant reduction in pUb substrates. SCs from neuropathic mice responded to low concentrations (25 nM) of RM and produced more myelin, as compared to vehicle controls. Significantly, RM improved myelination in two genetically distinct neuropathic models including the gene duplication ( C22 ) and the point mutation ( TrJ ) genotypes K nockdown of a key autophagy gene Atg12 in myelinating SCs abolished the improvements in myelination by RM, suggesting a crucial role for autophagy in this beneficial effect. CMT1A neuropathies associated with PMP22 gene duplication or mutations share characteristics with protein misfolding disorders including the accumulation of ubiquitin reactive protein aggregates and alterations in chaperone levels (Winklhofer et al., 2008) Once protein aggregates form they can interfere with essential cellular functions such as myelination in the case of SCs. In this study, the reduction in pUb substrates with RM treatment is evident in SCs from both neuropathic models. The dec line in the
83 accumulation of pUb substrates by autophagy may help in allev iating the burden on the proteasomal pathway (Fortun et al., 2005) which is the main degra dative mechanism for the removal of newly synthesized PMP22 (Pareek et al., 1997) Restoration of protein homeostasis, including the improvement in the processing of PMP22 would likely provide a more permissive environment for myeli nation by S Cs f rom neuropathic mice. Since P0 and MBP genes are coexpressed with PMP22 in myelinating SCs (Kuhn et al., 1993) and at the protein level PMP22 and P0 interact (Hasse et al., 2004) the improvement in PMP2 2 processing by RM most likely contributed to the concomitant increase in the ex pression of P0 and MBP (Figs. 3 5, 3 7 and 310). Compounds that enhance the activity of intr acellular quality control mechanisms are possible treatment approaches for protein aggregation disorders. The roles of the chaperone and autophagy pathways in peripheral nerve biology have been less studied as compared to neurons and glia of the CNS nonet heless a decline in the activity and/or efficiency of protein folding and/or degradation mechanisms likely contribute to age associated degenerative changes in myelinated peripheral nerves. In a previous study, we chose the intermittent fasting regimen as proof of principle intervention to activate chaperones and autophagy in neuropathic mice (Madorsky et al., 2009) After a five month long intervention, diet restricted mice showed an improvement in locomotor performance and myelination, as compared to ad libitum (AL) fed littermates. Since such drastic dietary restriction is not suitable for humans, compounds that enhance chaperone production and/or autophagy will be more attractive for future therapies. Autophagy is a particularly appealing target for therapeutic development for neurodegenerative diseases which are progressive with age, as activation of autophagy
84 in normal aged mice appears to extend lifespan without negative side effects (Harrison et al., 2009) Upregulating autophagy by RM and other small molecules has been shown to suppress a ggregation of disease linked proteins, including huntingtin, alphasynuclein, and to reduce cellular toxicity (Jaeger and Wyss Coray, 2009) More recently, RM and its analog have been shown to confer neuroprotection and ameliorate disease symptoms in mouse models of Parkinsons disease (Malagelada et al.) and spinocerebellar ataxia type 3 (Menzies et al.) respectively. Independent studies using t he transgenic approach confirm the involvement of autophagy in the removal of misfolded proteins, as mice deficient in autophagy genes within the CNS form intracellular inclusions and develop neurodegenerative symptoms (Hara et al., 2006; Komatsu et al., 2006; Mizushima and Hara, 2006) Consistent with these findings, the presence of misfolded aggregated PMP22 within peripheral nerves is associated with the neuropathic phenotype and demyelination (Fortun et al., 2003; Fortun et al., 2006) In compar ison, aggregates are reduced and myelin is improved in response to activation of autophagy and chaperones by long term dietary restriction (Madorsky et al., 2009; Rangaraju et al., 2009) or by RM treatment as shown here. The results presented here are comparable to the increase in myelination in neuropathic samples by pharmacological activati on of the HS pathway (Rangaraju et al., 2008) and suggest that combined pharmacological activation of the two protein quality control mechanisms, HS and autophagy pathways might provide additive benefits. RM is a widely used therapeutic compound that can elicit unique responses in different model systems and at given doses (Sarkar and Rubinsztein, 2008) With regards to the neural biology, besides its influence on autophagy in protein aggregation-
85 linked neurodegenerative disorders (Ja eger and Wyss Coray, 2009) RM has been tested in models of brain tumors and neuronal cell death. For example, in a mouse model of tuberous sclerosis the exposure of young mice to RM was associated with improved phenotype, including enhanced myelination (Meikle et al., 2008) In our PNS models, RM has a pronounced positive ef fect on SC function, specifically on myelination. We detected an increase in both the abundance and the lengths of myelin internodes indicating that RM supported the differentiation of myelinating SCs and their capacity to expand myelin sheaths (Figs. 3 7 and 3 10). The Atg12 knockdown experiments indicate that an intact autophagy pathway is necessary for the myelin improvements in RM treated cultures (Fig. 3 13). This result supports our hypothesis that restoration of protein homeostasis within SCs improves the cells ability to myelinate axons. Nonetheless, contribution from other mechanisms such as the Akt pathway cannot be ruled out at this time. Akt is involved in neuregulinmediated SC survival (Li et al., 2001) and in regulating myelination by oligodendrocytes (Narayanan et al., 2009) Additionally, RM is known to affect the endocytic pathway which appears to be important for myelination and membrane wrapping (Saito et al., 2005; Winterstein et al., 2008) Therefore, while the rationale for our studies with RM as a therapeutic agent for PMP22 associated neuropathies stem from our investigations of PMP22 trafficking and protein aggregation, it is possible that besides autophagy, RM will benefit neuropathic nerves through other mechanisms. The observed improvement in samples from Wt mice supports such hypothesis. In addition, in mice, RM may suppress the immune component of the neuropathy, which could offer an additional benefit to affected nerves (Hartung et al., 1992)
86 Based on the positive results presented here, we propose that RM is a suitable compound for further testing in neuropathic mice. As shown by our cellular studies this drug is well tolerated by myelinating SCs as well as per ipheral neurons and is effective at the low nM range. A recent study in aged mice showed that RM fed late in life extends lifespan, without toxic side effects (Harrison et al., 2009) RM is an FDA approved drug that is widely used in immunosuppression following organ transplantation, however chronic use of RM can le ad to adverse sideeffects (Kahan, 2008) As SCs respond robustly to RM and the PNS is outside of the blood brain barrier, it is possible that peripheral nerves will respond to this compound more robustly than the CNS. In addition, small molecule enhancers of autophagy are being developed and identified and could offer alternatives for RM in the future (Sarkar and Rubinsztein, 2008) In summary, our results provide further support for the involvement of autophagy as a protective pathway in PMP22associated neuropathies and provide a positive step toward identifying pharmacologic agents for these disorders.
8 7 Figure 3 1 Rapamycin effectively induces autophagy in cultured mouse SCs. (A) For the RM dose response, the expression levels of the indicated proteins were monitored via Western blot. Lysates of v ehicle control (Ct) and 4 h st arvation ( Stv) medium treated cells were included as negative and positive controls respectively. (B) For the time course studies, the levels of the same proteins were analyzed after treatment with Ct or RM ( 25 nM ) for the indicated times. (A, B) GAPDH is the protein loading control. Molecular mass, in kDa. (C) The ratios of LC3 II to LC3 I band intensities measured from Western blots of cell lysates treated with vehicle ( ) or 25 nM RM (+) for 48 h are shown. (**p<0.01, t test, mean SEM, n=3)
88 Figure 3 2 Rapamycin treatment has minimal effect on the Akt pathway in mouse SCs. (A) T he levels of phosphorylated Akt (pAkt) and total Akt were analyzed after treatment with C t or RM at 25 nM for the indicated times. GAPDH is the protein loading control. Molec ular mass, in kDa. (B) Quantification of the ratio of pAkt/total Akt without ( ) or with (+) RM (25 nM, 48 h) is shown (p=0.0602, t test, meanSEM, n=4).
89 Figure 3 3 Enhancement of autophagy reduces the levels of ubiquitinated substrates. (A) Wt mouse SCs were treated with vehicle control ( ) or RM (+), without () or with (+) the lysosomotropic alkaline CQ. The level of autophagy markers LC3 I (arrow) and LC3 II (arrowhead) were determined by Western blots. (B) Quantification of LC3 I and LC3 II band intensities normalized to tubulin are shown. LC3 I and II values of cel ls treated with CQ was set as 1 (**p<0.01, t test, mean SEM, n=4 ), AU, arbitrary units. (C) Western blot showing LC3 I and LC3 II levels in C22 mouse SCs, treated as in (A). A longer ex posure (high exp.) blot for LC3 is shown to visualize LC3 I levels. (D) Quantification of LC3 I and LC3 II band intensities in C22 samples is shown. (*p<0.05, t test, mean SEM, n=3) (E) The steady state levels of slow migrating poly ubiquitinated proteins (pUb) in lysates of SCs from Wt and C22 mice treated with vehicle ( ) or RM (+) are shown. Monoubiquitin (mUb) is marked by concave arrowhead. GAPDH is a loading control. Molecular mass, in kDa. (F) Quantification of pUb/GAPDH in A. The value of pUb/GAPD H in Wt was set as 1 (*p<0.05, **p<0.01, t test, mean SEM, n=3), AU, arbitrary units.
90 Figure 34 Protein synthesis in rapamycin treated SCs. (A) Wt mouse SCs were (Chx) for 12 h followed by 35S pulse labeling. Equal amounts of protein were resolved by a SDS PAGE and autoradiographs were obtained. Quantification of autoradiographs is shown (p=0.0684, **p<0.01, t test, meanSEM, n=3). (B) In a subset of samples, the proteins were precipitated and counts per minute (cpm) of proteinbound 35S radioactivity was measured and normalized test, meanSEM, n=3).
91 Figure 3 5 Rapamycin promotes myelination by SCs. (A) Schematic representation of the RM treatment paradigms. The black line indicates DIV while t he green line s represent the expression profile of myelin proteins in Wt ( bold green) and neuropathic ( dashed green) samples. The blue line indicates the timescale for RM treatment in hours (h). Asterisks indicate the DIV at which the control samples Ct* and Ct** were obtained. For early RM treatment (a), starting on DIV9, or for late treatment (b) starting on DIV15, a pulse of RM (green arrows) was given for 48 h, followed by 48 h washout (wo, black arrows). This sequence was repeated and a third RM treatment (8 h) was given prior to analysis ( arrowhead). (B, C) DRG explants from Wt and C22 mice were treated with RM as indicated and whole protein lysates were analyzed for the myelin proteins, MBP and P0. The main isoforms of MBP are marked. Tubulin is shown as protein loading control. Molecular mass, in kDa.
92 Figure 3 6 Late treatment of rapamycin improves myelin protein expression better than early treatment in C22 samples. (A) Quantification of myelin protein levels (MBP and P0) in Wt and C22 DRG explants following early treatment with vehi cle ( ) or RM (+) in Fig.3B. MBP and P0 values after normalizing to tubulin in vehicle treated ( ) samples were set as 1. AU, arbitrary units. (p= 0.0654, p=0.0722, *p<0.05, **p<0.01, t test, meanSEM, n=34). (B) Quantification of MBP and P0 band intensit ies in Wt and C22 samples after late RM treatment paradigm in Fig. 3C. MBP and P0 values after normalizing to tubulin in Ct ( ) samples were set as 1. AU, arbitrary units. (p=0.0565, p= 0.0586, *p<0.05, **p<0.01, t test, meanSEM, n=3 4).
93 Figure 3 7 The abundance and length of myelin internodes are increased by rapamycin. (A) Explant cultures from Wt and C22 mice were treated with Ct or RM at late stage, as described in Fig. 3A, and stained for MBP (green) to visualize myelin. Scale bar, 40 m. Hoechs t dye stains the nuclei (blue). (B) The abundance of myelin internodes w as quantified in a fixed area (p>0.2, n.s; non significant; ***p<0.001, t test, mean SEM, n=3). (C) The lengths of the myelin internodes (n=130260) were measured and quantified in thr ee independent experiments (*p<0.05, **p<0.01, ***p<0.001, t test, mean SEM).
94 Figure 3 8 Activation of autophagy by rapamycin is negligible in DRG neurons. (A) SC depleted DRG neurons from Wt and C22 embryos were treated with vehicle ( ) or 25 nM RM f or 48 h (+) and analyzed for the protein levels of pS6, S6, LC3 I and II by Western blots. GAPDH, loading control. Molecular mass, in kDa. (B, C) The ratio of LC3 II to LC3 I steady state protein levels was measured from Western blots of Wt and C22 DRG neurons treated with vehicle ( ) or 25 nM RM for 48 h (+) is shown. (n.s, not significant, t test, meanSEM, n=3).
95 Figure 3 9 The processing of PMP22 in myelinating SCs. (A) Total protein lysates of DRG explants from Wt and C22 mice after treatment with vehicle (Ct) or RM were incubated with endo H (H) or PNGase F (N) and PMP22 was detected by Western blots E ndo H resistant (R) (square bracket, ~22 kDa) and endo H sensitive (S) (18 kDa) fractions of PMP22 are marked. Control (C) protein lysates for each sample without enzymatic treatment are also shown. (B) Quantitative analysis of data from three independent experiments are shown (p=0.0576, p=0.0855, **p<0.01, ***p<0.001, t test, mean SD).
96 Fig ure 3 10. Rapamycin enhances myelination in cultures fro m TrJ mice. (A, B) DRG explants from TrJ mice were treated as described in Fig. 3A and whole protein lysates analyzed for the levels of myelin proteins, MBP and P0. Tubulin is shown as loading control. Molecular mass, in kDa. (C) C ultures from TrJ mice were treated with vehicle Ct or RM at late stage as described in Fig. 3A, and stained with an anti MBP (red) antibody. Scale bar, 40 m. Hoechst dye stains the nuclei (blue). (D) The abundance (***p<0.001, t test, mean SEM) and (E) t he lengths of the myelin i nternodes (n=150200) were quantified in explant cultures from TrJ mice treated without ( ) or with (+) early or late RM treatment, (**p<0.01, t test, mean SEM, n=3).
97 Figure 3 1 1 Exposure to rapamycin reduces the accumulation of poly ubiquitin subs trates in TrJ mouse SCs. (A) A representative blot for the steady state levels of slow migrating pUb proteins and m Ub in lysates of SCs from TrJ mice treated with vehicle ( ) or RM (+). LC3 I and II and GAPDH loading control were detected. Molecular mass, in kDa. (B) Quantification of pUb/GAPDH in A. The value of pUb/GAPDH in Ct sample was set as 1. AU, arbitrary units. (**p<0.01, t test, meanSEM, n=3). (C) Quantification of the ratio of LC3 I I to LC3 I band intensities is shown. (*p<0.05, t test, meanSEM n=3).
98 Figure 3 12. Determination of lentiviral transduction efficiency. TrJ DRG explants were transduced with GFP lentiviral particles. (A) Live GFP fluorescence (left panel ) and phase contrast (right panel ) images of DRG explants in culture on DIV11 are shown. (B) Direct GFP fluorescence after fixation (left panel) and Hoechst (right panel) staining are shown on DIV26. Scale bars, 40 m.
99 Figure 3 13. Atg12 is necessary for the myelin improvement by rapamycin. (A) Wt and TrJ DRG explants were transduced with scrambled negative (Neg.) shRNA or Atg12 shRNA. The shRNA transduced DRGs were given a late treatment with vehicle control (Ct) or RM as indicated. Wester n blot analyse s for Atg12 and myelin proteins MBP, P0 are shown. GAPDH serves as protein loading control. Molecular mass in kDa. (B) Quantification of Atg12 protein levels in Wt (left panel) and TrJ (right panel) cultures after normalizing to GAPDH The value of Atg12/GAPDH in Ct samples transduced with Neg. shRNA w as set as 1 (*p<0.05, t te st, meanSEM, n=3 ) (C) Quantitative analysis of MBP protein levels in Wt (left panel) and TrJ (right panel) explants normalized to GAPDH The value of MBP/GAPDH in Ct samples transduced with Neg. shRNA w as set as 1 (n.s, not significant, p=0.0569, *p<0.05 **p<0.01, ttest, mean SEM, n=3 ) (D) Quantitative analysis of P0 protein levels in Wt (left panel) and TrJ (right panel) samples normalized to GAP DH. The value of P0/GAPDH in Ct samples transduced with Neg. shRNA w as set as 1 (n.s, not significant, p=0. 0540, *p<0.05, t test, mean SEM, n=3) ; AU, arbitrary units
100 Figure 3 13. Continued
101 Table 3 1. Primary antibodies used in this study. WB, Western Blot; IS, Immunostaining
102 CHAPTER 4 MOLECULAR ARCHITECTURE OF MYELINATED PER IPHERAL NERVES IS SUPPORTED BY CALORIE RESTRIC TION WITH AGING Introduction Aging is associated with structural, functional and biochemical alterations in the nervous system. Neurons with long processes are particularly vulnerable to degeneration (Mattson and Magnus, 2006) which makes peripheral nerves susceptible to agerelated modifications. Signal propagation along axons is facilitated by myelin, a lipid rich membranous structure formed by SC s. Distinct domains within the myelin and the axonal plasma membrane are maintained by complex signaling events between neurons and glia (Garbay et al., 2000) Therefore, degenerative changes in either cell type have global influences on overall nerve structure and function. Myelinated peripheral nerves from aged animals show fiber loss and morphological irregularities (Verdu et al., 2000) as well as a notable reduction in the expression of myelin and neurofilament genes and proteins (Parhad et al., 1995; Melcangi et al., 1999; Melcangi et al., 2000; Uchida et al., 2004) There is evidence for axonal demyelination and occasional remyelination in aged rat sciatic nerves, associated with nerve fiber degeneration (Sharma et al., 1980; Grover Johnson and Spencer, 1981; Adinolfi et al., 1991) In response to demyelination, SC s increase in number (Gregson and Hall, 1973) and dedifferentiate (Zanazzi et al., 2001) Ageassociated functional changes include decline in nerve conduction velocity and muscle strength, and decreases in sensory discrimination, autonomic responses and endoneurial blood flow (Verdu et al., 2000) Together, these alterations contribute to decline in neuromuscular function and impact physical performance.
103 Reduction in the expression of functional proteins and the accumulation of damaged and misfolded proteins have been obs erved in a variety of organisms with age (Sitte et al., 2000; Squier, 2001; Calabrese et al., 2004; Keller et al., 2004; Grune et al., 2005) The extent to which damaged proteins accumulate is highly dependent upon the cells capacity to repair or remove them by subcellular homeostatic mechanisms (Stadtman, 2001) namely chaperones and protein degradation. Chaper ones (also referred to as HSPs) transiently interact with proteins to aid their folding, trafficking and degradation (Frydman, 2001; Sherman and Goldberg, 2001) Cellular degradative pathways include the UPS and the autophagy lysosomal pathway (also referred to as autophagy). With an age related decline in the activity of these homeostatic mechanisms, damaged proteins and organelles can accumulate and lead to cellular dysfunction and cell death (Macario and Conway de Macario, 2002; Bergamini et al., 2004) Metabolically active and postmitotic cells (Boulton et al., 2004; Weissman et al., 2007) such as neurons and myelinating SC s are particularly sensitive to the accumulation of damaged proteins. One approach to slow the aging process and prolong lifespan is through dietary modulation, s uch as calorie restriction (CR) (Johnson et al., 2006) Dietary restriction can induce HSPs (Heydari et al., 1996; Selsby et al., 2005) and autophagy (Bergamini et al., 2003; Wohlgemuth et al., 2007) and therefore support the maintenance of healthy cells and organs. While much work co ncerning dietary modulation has focused on the CNS, peripheral organs and lifespan (Feuers et al., 1989; Mattson et al., 2001; Jolly, 2004) little is known about the effects of such approach on peripheral nerves. In the CNS, life long reduction in calorie intake has been shown to preserve long term
104 potentiation (Hori et al., 1992) and ameliorate agerelated cognitive decline (Pitsikas and Algeri, 1992) In the periphery, the decline in muscle mass and strength with age is ameliorated with a lifelong CR diet (Marzetti et al., 2008; Xu et al., 2008) which in part might be underlined by improved neural function. Here we examine the chaperone and autophagic responses of SC s isolated from young and aged nerves, and relate the findings to agerelated biochemical and cellular alterations in peripheral nerves. Our results indicate that a lifelong CR regimen supports the maintenance of the molecular architecture of myelinated axons, including the expression of essential axonal and glial proteins. Materials and Methods Animals and D iets To establish rat SC cultures, we used male Fischer 344 rats from National Institute on Aging colony at Harlan Sprague Dawley Inc (Indianapolis, IA) (Norris et al., 1996) For dietary modulation studies, Male Fisher 344 x BN (Brown Norway) rats of four distinct ages 8, 18 29 and 38months (mo) and specified diets were obtained from the National Institute on Aging colony. AL fed rats had free access to NIH 31 average nutrient composition pellets, whereas the calorie restricted (CR) group received fortified pellets once daily, 1 h before the onset of the dark period. The dietary restriction was initiated at 14 weeks of age with 10% restriction, increased to 25% at 15 weeks, and continued at 40% from 16 weeks of age. Both the AL and CR rats had access to water at all times. The use of animals in this study was approved by an Institutional Animal Care and Use Committee of the University of Florida.
105 Primary Culture of Schwann Cells from Young and Old R ats SC cultures were established from the sciatic nerves of postnatal day 2 (P2) 5mo and 25mo old rats using established procedures (Notterpek et al., 1999a) Nerves were dissected and gently stripped of connective tissue and epineurium, chopped into small pieces and digested over a period of 1 h for P2 (young) and 5 h for the adult samples, at 37 C in a humidified atmosphere of 5% CO2. The digestion medium consisted of Dulbeccos Modification of Eagles Medium (DMEM) (Gibco, Grand Island, NY ), 15 % Fetal Bovine Serum (FBS) (Hyclone, Logan, UT), penicillin streptomycin (Gibco) and an enzyme cocktail of 0.03% collagenase type III (Worthington, Lakewood, NJ ), 0.1 % hyaluronidase (SigmaAldrich), 1.25 units/mL dispase (Worthington). Following digestion, cell suspensions were washed once and resuspended in cul ture medium (DMEM containing 10% FBS). Cells were then plated in small drops on poly L lysine (Sigma Aldrich) coated glass cover slips or on plastic petri dishes and allowed to adhere overnight. The next day, cells were washed with DMEM followed by addition of DMEM containing 10% FBS, 10 M of antimitotic agent cytosine D arabinofuranoside (Sigma Aldrich), to eradicate contaminating fibroblasts. After four 24 h periods of antimitotic treatments, given on alternate days, standard growth medium (DMEM containing 10% FBS, 10 g/mL bovine pituitary extract [Biomedical Techn ologies, Inc., Stroughton, MA] and 5 M forskolin [Calbiochem, La Jolla, CA]) was added and the cells were allowed to proliferate for 78 days. For the HS treatments, the cells were incubated at 45 oC for 20 min, followed by a recovery for 6 h (Cristofalo et al., 2004) Stv medium (amino acidand serum free) was used to stimulate autophagy (F ortun et al., 2003; Fortun et al., 2007) To estimate the autophagic flux, CQ ( 50 M) was added
106 simultaneously with Stv condition for 4 h. After the specified treatments, samples were processed for biochemical or immunochemical studies. Biochemical S tudie s Sciatic nerves isolated from AL and CR rats (n=3) were frozen immediately and stored in liquid nitrogen. Cultured SC s were washed twice with ice cold PBS followed by lysis. Cell or nerve lysates were prepared in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 3% SDS) supplemented with phosphatase inhibitors, PMSF (both from SigmaAldrich, St. Louis, MO) and complete protease inhibitor (Roche, Indianapolis, IN). Protein concentrations were determined using the BCA protein as say kit (Pierce Chemicals, Rockford, IL). Protein samples (525 g/lane) were separated on 7.5 % 10 % 12.5 % or 15 % SDS polyacrylamide gels under reducing conditions and proteins were transferred to nitrocellulose or Polyvinylidene Flouride (PVDF) membranes (for LC3) (both from BioRad Laboratories, Hercules, CA). Membranes were blocked in 5% non fat milk in PBS and incubated overnight with primary antibodies (See Table 1). After washing, anti mouse, anti rabbit (both from Cell Signaling Technology), anti ch icken or anti rat (both from SigmaAldrich) HRP linked secondary antibodies were added for 2 h. Bound antibodies were visualized using an enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Boston, MA). Films were digitally imaged using a GS 710 densitometer (BioRad Laboratories) and were formatted for printing by using Adobe Photoshop 5.5. Semi quantitative analysis of protein levels was performed using Scion image software. The relative densitometric units were determined after normalizi ng with a protein loading control. For the nerves, the value of 8mo old AL sample was set to 1 and the values of other agediet combination were determined with respect to this. One way ANOVA followed by Fishers
107 PLSD analysis was performed using the Stat View program, to compare the normalized densitometric values of proteins (dependent variables) between AL and CR diet with age (factor). To determine statistical significance between samples, unpaired t test was performed using GraphPad Prism v5.0 software. Graph pad Prism software. Immunochemistry SC s on glass coverslips were fixed with 4% paraformaldehyde (10 min) and permeabilized with ice cold 100% methanol (5 min). Sciatic nerves from rats were dissected and frozen by immersion in liquid nitrogen. Froz en nerves were longitudinally Superfrost/Plus microslides (Fisher, Pittsburgh, PA) for 1 h. Dried tissue sections were fixed with 4% paraformaldehyde for 1 h, permeabilized with ice cold 100% methanol (5 min) or acetone (2 min) and processed for immunostaining (Ryan et al., 2002) After an overnight inc ubation at 4 C, bound antibodies (see Table 1) were detected using Alexa Fluor 594conjugated (red) anti rabbit and Alexa Fluor 488conjugated (green) anti mouse secondary antibodies (Molecular Probes, Eugene, OR). To visualize the nuclei, Hoechst dye (Molecular Probes) was included with the secondary antibodies. Control samples without primary antibodies were processed in parallel. Cover slips were mounted using the Prolong Antifade kit (Molecular Probes). Images were acquired with a SPOT digital camera attached to a Nikon Eclipse 800 microscope Leica TCS SP2 confocal laser scanning microscope and processed for printing by using Adobe Photoshop 5.5. Colocalization of LC3 and LAMP1 For analysis of colocalization of LC3 and LAMP1, i mages were acquired on a 63X water immersion lens using the Leica confocal microscope and z stack images
108 were captured. The 2D cytofluorograms in Figure 4 3C were created using Leica software with the same exposure settings for red and green channels for P2 and 25mo samples. The software quantifies the extent of colocalization by creation of a binary mask for all of the pixels that are double positive for both LC3 and LAMP1 fluorescence so that colocalizing elements will appear in the diagonal of the cytofluorogram (Fig. 4 3C, out lined in green). Colocalization of LC3 and LAMP1 was then quantified by 2), normalized per cell, between P2 and 25mo samples treated with Stv+CQ from three independent experiments (n=8 random visual fields per condition). Statistical significance was determined by unpaired t test using GraphPad Prism v5.0 software. Nuclei C ount The nuclei (stained with Hoechst) of non epineurial and nonendoneurial cells were counted in longitudinal sections ( 5 m thickness) of sciatic nerves from two different depths and eight random visual fields (0.1mm2) per animal (n=3). Statistical significance was determined by unpaired t test using GraphPad Prism v5.0 software. Results Chaperone Response of Schwann Cells Isolated from Ner ves of Aged R ats We isolated SC s from postnatal day 2 (P2) and aged (25mo) rats to investigate the subcellular mechanisms underlying the agerelated alterations in the molecular architecture of myelinated peripheral nerves (Verdu et al., 2006). SC s from y oung (P2 6) animals respond to in vitro manipulation of protein degradative and chaperone pathways and allow for mechanistic studies (Fortun et al., 2003; Fortun et al., 2007) We examined the chapero ne response of the cells to Stv and HS stimuli ( Fig. 4 1A). Under control culture conditions (Ct), both cell populations express similar levels of HSP90,
109 HSP70 and HSP40. Incubation of the cells in Stv medium is associated with a notable increase in HSP70 (PlakidouDymock and McGivan, 1994) particularly in young cells. Similarly, stimulation of HSPs by incubating the cells at 45 C for 20 min (Cristofalo et al., 2004; Fortun et al., 2007) leads to a pronounced increase in the expression of HSP70 and a slight increase in HSP90 and HSP40 ( Fig. 4 1A). Quantifications from three independent experiments indicate a nearl y 5 fold greater HSP70 induction in P2 cells, as compared to cells from 25mo old rats (*p<0.05). GAPDH levels are similar among the cells and paradigms tested. To further investigate the muted chaperone response of the aged cells, we performed immunostaining with anti HSP70 antibodies ( Fig. 4 1B). As suggested by the biochemical data ( Fig. 4 1A), SC s cultured from P2 nerves show a robust induction of HSP70 ( Fig. 4 1B, green), which is nearly uniform among all the cells. The low level of HSP70 like immunore activity in control cells is shown in the inset. In comparison, cells isolated from the 25mo old rats exhibit notable heterogeneity in their ability to respond to HS, with only a fraction of the cells showing elevated HSP70 expression ( Fig. 4 1B, green). Quantification of three independent experiments indicates that 42.48 6.14% of 25mo cells respond to HS in comparison to over 90.45 2.47 % of young (P2) cells (mean SEM; ***p<0.001). Therefore, the reduced level of HSP70 on the Western blots ( Fig. 4 1A) i s a reflection of the inability of a fraction of aged cells to respond to stress stimuli. The Autophagic Response of Glial Cells is Altered with A ge Next, we tested SC s isolated from P2 and 25mo old rats for their autophagic capacity ( Fig. 4 2). To stimul ate autophagy, the cells were incubated in medium
110 deprived of amino acids and serum (Stv medium) (Fortun et al., 2003) for 4 h followed by lysis. In a subset of samples, we simultaneously added CQ ( 50 M) to evaluate differences in the autophagic flux under conditions that inhibit lysosomal enzyme activity (Mizushima and Yoshimori, 2007) CQ is a lysosomotropic alkaline that increases the pH within lysosomes thereby inhibiting their proteolytic activity (Rocca et al., 2001) As a biochemical measure of autophagy, we evaluated the levels of Atg7 and lipidation of LC3 ( Fig. 4 2A). The young (P2) cells respond to nutrient deprivation (Stv) by activ ating the autophagic pathway, as revealed by an increase in Atg7 and the conversion of LC3 I to LC3 II (lipidated form) (Kabeya et al., 2000; Mizushima and Yoshimori, 2007) In the old cells, the response to Stv is lessened, which is particularly notable for Atg7. Upon co treatment with Stv medium and CQ, the levels of LC3 II further increase, indicating ongoing autophagic flux (Mizushima and Yoshimori, 2007; Klionsky et al., 2008) This trend is significantly (*p<0.05) more robust in cells from young, as compared to those from aged rats ( Fig. 4 2B). Furthermore, in young cells (P2), the phosphorylated form of ribosomal protein S6 (pS6) disappears completely upon Stv (Blommaart et al., 1995) whereas in 25m o old cells it does not ( Fig. 4 2A). As expected, compared to Stv alone, the levels of pS6 slightly increase in the Stv+CQ co treatment paradigm in both samples, confirming the specificity of this marker for the Stv induced autophagy. Both young and old ce lls respond to Stv as seen by significant decline in the ratio of pS6 and S6 ( Fig. 4 2C), a value that is used as a marker of autophagic activity (Blommaart et al., 1995) However, the ratio in 25mo old samples is significantly hi gher (**p<0.01) than in P2 cells, indicating defective autophagy ( Fig. 4 2C).
111 We also examined the expression of lysosomal proteins within the same samples and detected a slowed mobility of LAMP1 in aged cells ( Fig. 4 2D, asterisk) To obtain insight into lysosomal proteolytic potential (Fusek and Vetvicka, 2005) we studied the expression of cathepsin D (cath D). The levels of procathepsin D (procath D) and active cath D under control conditions are lower in 25mo cells as compared to P2 ( Fig. 4 2D, E). There is a decrease in the active cath D upon CQ treatment in both ages, as this lys osomotropic agent increases the secretion of procath D and prevents the processing of the active cath D (Samar el et al., 1989) Under Stv conditions, the active form of cath D is notably higher in young, as compared to 25mo cells ( Fig. 4 2E, **p<0.01). This active form is depleted when Stv is combined with CQ treatment, thereby confirming the specificity of the Stv response in processing cath D. These results are consistent among three independent experiments ( Fig. 4 2E) and suggest alterations in the autophagy lysosomal activity of SC s isolated from nerves of aged rats. To examine the fusion of autophagic vacuol es with lysosomes, we double immunostained SC s with LC3 and LAMP1 antibodies ( Fig. 4 3). SC s from P2 rats contain small LAMP1positive lysosomes and relatively few LC3reactive autophagosomes dispersed within the cytosol ( Fig. 4 3A, panel on left, Ct). Su bjecting the young cells to Stv increases the size of lysosomes ( Fig. 4 3A, Stv, arrow) and the number of autophagosomes. On the other hand, SC s from 25mo rats show visibly large lysosomes under control conditions ( Fig. 4 3A, panel on right, Ct, arrows) and only a modest increase in LAMP1positive lysosomes upon Stv. In agreement with the biochemical results ( Fig. 4 2A), the abundance of LC3positive autophagosomes remains low in Stv treated 25mo SC s (insets on right). To evaluate the autophagic flux,
112 we analyzed the Stv+CQ treated cells by confocal microscopy ( Fig. 4 3B). On representative single plane images, there are several fusion events of LC3positive autophagosomes and LAMP1positive lysosomes in SC s from P2 rats ( Fig. 4 3B, yellow spots). The fusi on of lysosomes (green) with autophagosomes (red) is resolved in more detail on single plane sections ( Fig. 4 3B, x and y sections). In agreement with the defective autophagosomelysosome fusion hypothesis in old cells (Cuervo et al., 2005) there are fewer colocalization of LAMP1 and LC3 (yellow spots) within SC s isolated from the old rats. 2D cytofluorograms reveal more yellow pixels in young, as compared to old cells ( Fig. 4 3C). Quantification of colocalization mask area shows a significant (*p<0.05) reduction in autophagosomelysosome fusion events in 25mo cells ( Fig. 4 3D). We com pleted a similar set of studies in cells isolated from the peripheral nerves of 5 mo old rats which behave similar to the P2 rats (data not shown). Together, these results indicate that SC s isolated from aged nerves respond less vigorously to HS and Stv st imuli, as compared to neonatal (P2) or young adult (5mo) cells. Protein Homeostatic Mechanisms are Maintained in Nerves of Diet Restricted R ats Protein homeostatic mechanisms that maintain tissue health and repair damage are known targets of agerelated alterations (Rattan, 2004) To examine how age and diet affect the steady state expression of protein chaperones within myelinated nerves, we focused on the HSP90/HSP70 network ( Fig. 4 4A). HSF1, a key regulator of this pathway, is held in an inactive state in the cytosol by HSP90. (Ohtsuka and Suzuki, 2000; Voellmy and Boellmann, 2007) and upon release it translocates to the nucleus, where it promo crystallin (Pirkkala et al., 2001) The levels of HSF1 and HSP90 gradually increase with age in the AL group, a
113 trend which is s ignificant at 38mo ( Fig. 4 4A, B). The CR diet attenuates this pattern and is associated with sustained low level of HSF1. Upon analysis of the corresponding chaperones, we observe a significant increase in HSP90like reactivity, while the steady state le crystallin appear to decline with age ( Fig. 4 4A). Quantification of the data confirms the changes for HSP90 ( Fig. 4 4B), but not for crystallin (data not shown). The steady state expressions of HSP40 and of the small chap erone HSP27 are sustained across the studied samples. Demyelination of nerves and accumulation of damaged proteins within SC s elicits a proteolytic response, which is reflected by activation of the autophagy lysosomal pathway (Notterpek et al., 1997; Fortun et al., 2003) In agreement with the known degenerative changes in peripheral nerves of aged rodents (Melcangi et al., 1999; Verdu et al., 2000; Uchida et al., 2004) we found a gradual increase in the steady state levels of LAMP1 and the autophagic protein Atg7 with age ( Fig. 4 4C). The semi quantitative analysis shows that this trend is lessened by the intervention, leading to balanced expression of both these proteins ( Fig. 4 4C, D). Similarly, the age associated increase in the levels of pS6 and total S6, are diminished by the intervention. The ratio of these two species (Blommaart et al., 1995) is significantly higher in the samples from the ALfed 18 mo and 38mo rats as compared to CR ( Fig. 4 4E ). However, the differences in the pS6/S6 ratios between AL and CR samples at 8and 29mo ages are not significant (p=0.823 and p=0.526, respectively). Overall, these data suggest that the degenerative changes are muted with CR, which decreases the demand on protein homeostatic mechanisms, including the autophagy lysosomal pathway.
114 The Expression of Myelinated Schwann Cell Proteins in Diet Restricted R ats Efficient functioning of peripheral nerves is supported by myelinforming SC s. To examine the influenc e of the dietary restriction on peripheral nerve health, we prepared tissue lysates from sciatic nerves and studied the expression of glial and axonal proteins by Western blots ( Fig. 4 5). Our results confirm earlier findings (Melcangi et al., 1999; Melcangi et al., 2000) in a wide age group, where we detect gradual decline in three structural myelin proteins, including protein zero (P0), PMP22 and MBP ( Fig. 4 5A ). The decreases are pronounced by 29mo of age, and reach statistical significance for all three proteins at 38mo (Fig. 4 5B). In comparison, in nerve lysates from diet restricted animals, the levels of these proteins are maintained ( Fig. 4 5A, B). We pr eviously reported that rats kept on the CR diet display significantly better functional performance in the grip strength task (Ingram et al., 2007; Xu et al., 2008) Within the AL fed group, there is a positive correlation between the decline in forelimb grip strength and MBP expression (p<0.001), and an emerging trend for strength and PMP22 (p=0.096). Neither association is present in the CR fed group (p>0.05). These data suggest that agerelated loss of myelin is correlated with a decline in strength, and interventions such as CR, which slow myelin loss, may also preserve functional performance (Ingram et al., 2007; Xu et al., 2008) In response to demyelination SC s re enter the cell cycle and prol iferate (Jessen and Mirsky, 2005) Change in the differentiation state of SC s is reflected by the reexpression of the p75 neurotrophin receptor (p75NTR) (Jessen and Mirsky, 2005) In agreement, immunoblotting the nerve lysates with an anti p75NTR antibody shows higher levels of this protein in the samples from 38mo old ALfed rats ( Fig. 4 5C, D), as compared to the younger ages. This increase in expression is attenuated by the CR
115 regimen, supporting the maintenance of t he differentiated SC phenotype ( Fig. 4 5D). In accordance, the levels of the mitotic marker, phosphorylated histone3 (pHH3) (Ribalta e t al., 2004) are higher in the ALgroup as compared to CR ( Fig. 4 5C, D). We also labeled nerve sections with Hoechst dye and counted the number of supernumerary SC nuclei, excluding epineurial and endoneurial cells ( Fig. 4 5E). The average number of nuc lei within fixed tissue area of 18mo old AL fed rats is 42.80 0.98 (n=3, mean SEM) whereas there is a small but statistically significant decrease in response to the intervention (39.88 1.24 nuclei, *p<0.05). Significantly, there is a nearly 3fold increase in the number of nuclei in the oldest samples (117.9 6.08 vs. 42.80 0.98), which is remarkably alleviated by CR ( Fig. 4 5E). Together, these results indicate that a lifelong dietary restriction supports the maintenance of the differentiated S C phenotype, which is beneficial for myelin and axonal structure. Axonal Constituents in Myelinated Peripheral Nerves of Diet Restricted R ats SC s provide support for the functional integrity of myelinated axons (Nave and Trapp, 2008) Therefore, under conditions of myelin loss as seen in the aged nerves ( Fig. 4 5A) alterations in axonal and structural proteins are expected. We corroborated the biochemical analysis of myelin proteins ( Fig. 4 5) by double immunolabeling longitudinal nerve sections from 18and 38mo old rats with anti MBP and neurofilament light chain (NF L) protein antibodies ( Fig. 4 6A). In agreement with the pronounced reduction in MBP in the nerve lysates ( Fig. 4 5A) in samples from AL 38mo old animals there are NF L antibody reactive axons devoid of myelin ( Fig. 4 6A, arrowheads). In contrast, nerves of 38mo old CR rat show a remarkable maintenance of MBP positive internodes ( Fig. 4 6A, red) and only few axons without myelin (arrowheads). Immunostaining for NF heavy (NF H) and medium (NF M) chain protein
116 shows similar pattern (data not shown). Nerve sections from 18mo old rats are included for comparison ( Fig. 4 6A, insets in top panels). Since there seem to be fewer NF reactive fibers in ALfed 38mo old samples ( Fig. 4 6A), we analyzed the levels of the three major NF proteins ( Fig. 4 6B). In agreement with previous r eports (Parhad et al., 1995; Uchida and Brown, 2004; Uchida et al., 2004) nerves from 38mo old rodents show reduction in the steady state expression of NF H, M and L (trend for NF H and signif icant for NF M and NF L) (Fig. 4 6B, C). Strikingly, their levels are maintained in rats on the intervention ( Fig. 4 6C), corresponding to the immunolabeling studies ( Fig. 4 6A). In sciatic nerve samples from our oldest ALfed rats, vimentin, an intermediate filament protein expressed by SC s and neurons (Toth et al., 2008) appears to undergo proteolytic degradation ( Fig. 4 6B, square bracket). This aberrant phenotype, which may be a reflection of enhanc ed caspase activity in aged nerves (Byun et al., 2001) is absent from independent samples on the dietary regimen ( Fig. 4 6B). Together these results show that the CR intervention supports maintained expression of glial and axonal gene products, including myelin and neurofilament proteins. Demyelination of nerve fibers with age and disease leads to spreading of voltagegated ion channels in the axonal membrane (Adinolfi et al., 1991; Verdu et al., 2000; Amici et al., 2007) To determine if the CR diet supports the expression of channel proteins at nodes of Ranvier, we performed bioc hemical and immunohistochemical analyses of panvoltage gated sodium channels (Nav) and voltage gated potassium channels (Kv1.1) ( Fig. 4 7). While the steady state levels of both Nav and Kv1.1 dramatically increase with age in the ALfed group, especially at 38 mo (6 fold increase
117 for Nav and 7fold increase for Kv1.1), in nerves from CR animals such changes are muted (*p<0.05 for both Nav and Kv1.1) ( Fig. 4 7A). Furthermore, proteolytic cleavage of the subunit of Nav channels (Zwerling et al., 1991; Benz et al., 1997) most notable in the 38mo sample ( Fig. 4 7A, arrowhead), is lessened by the intervention. We confirmed the biochemical results by co immunolabeling nerve sections with antibodies against Nav or Kv1.1 channels, and MBP ( Fig. 4 7B, C). As suggested by the higher expression level of the channel proteins ( Fig. 4 7A) and the abundance of unmyelinated fibers ( Fig. 4 6A), N av channel like immunoreactivity is prominent and diffuse along unmyelinated axons in nerves from 38mo old ALfed rats ( Fig. 4 7B). Furthermore, there is wider distribution of Nav at nodes of Ranvier ( Fig. 4 7B, asterisks and insets in top panel), likely due to segmental demyelination. In comparison, focal localization of Nav channel is maintained in samples from 38mo old rodents on the regimen ( Fig. 4 7B, asterisks and insets in bottom panel). Similarly, the Kv1.1 channel like immunoreactivity is spread along axons in nerve sections from 38mo AL rats (Fig. 4 7C), instead of being confined to the paranodal region. Again, in response to dietary restriction, spatial localization of Kv1.1 is maintained to the paranodal region ( Fig. 4 7C). Together, these res ults show a beneficial effect of CR on the molecular architecture of myelinated peripheral nerves, including the expression and localization of glial and axonal proteins (Figs. 5 5 to 57). Discussion Glial cells isolated from aged rats have muted response to stress stimuli, which may in part underlie the degenerative changes observed with age in myelinated peripheral nerves. Reduction in calorie intake, the most widely accepted and effective method of defying agerelated alterations (Everitt et al., 2006) preserves the molecular
118 architecture of myelina ted axons likely by supporting SC function. The benefits are evident by maintained expression and correct localization of glial and axonal molecules, including myelin, neurofilament and ion channel proteins. Constituents of protein homeostatic mechanisms r emain leveled in nerves from diet restricted rats, potentially reflecting lower demand on these pathways. While CR diet affects specific physiologic parameters and cellular pathways in different organs (Mattson et al., 2001) here we focused on two protein homeostatic mechanisms, namely chaperones and autophagy. W e isolated SC s from myelinated nerves of young and old rats and found notable differences in their chaperone responses ( Fig. 4 1). Th e muted response of old glial cells at least in part is reminiscent of the findings from the nerves of ALfed aged animals ( Fig. 4 4). In sciatic nerve lysates, we detected a prominent increase in the steady state expression of HSF1 at 38mo ( Fig. 4 4), wh ich may indicate a compensatory attempt for compromised signaling of the HSP90/HSF1 pathway (Ohtsuka and Suzuki, 2000; Voellmy and Boellmann, 2007) In agreement, while there is an increase in HSP90like reactivity, the steady state levels of HSP70 and crystallin are low. Age related increase in HSF1 levels have been previously reported in isolated rat hepatocytes and is associated with a decline in binding activity of HSF1 with heat shock elements (HSE) (Heydari et al., 2000) A crucial role for chaperones in myelinated nerves is supported by the ATP dependent interaction of HSP70 with MBP (Lund et al., 2006) Constitutive expression of HSC70 appears to be also necessary for the correct expression of MBP during the differentiation of oligodendrocytes (Aquino et al., 1998) suggesting HSP70 is involved in the proper folding and trafficking of this myelin protein. Furthermore, hsf1 knock out mice exhibit a
119 demyelinating phenotype in the CNS, likely due to defective oligodendrocyte different iation, or myelin synthesis and assembly (Homma et al., 2007) Complimentarily, the enhancement of HSPs improves myelination in a neuropathic model (Rangaraju et al., 2008) Thus, t he decline in the ability of SC s to induce chaperones may impair their ability to sustain and repair myelin with age. CR diet has the ability to prevent the aforementi oned decrease in HSF1 binding activity (Heydari et al., 1996) and support the expression of chaperones. In aged cells, macroautophagy is known to be defective both in the formation and clearance of autophagosomes (Cuervo et al., 2005) Poor elimination of autophagosomes could result from a decrease in lysosomal enzyme activity and/or impaired fusion of ly sosomes with autophagosomes (Cuervo et al., 2005) Our data support these hypotheses, as SC s from old rats show low levels of the lysosomal endoprotease, cath D with Stv, and prominent swollen lysosomes and few fusion events of autophagosomes and lysosomes (Figs. 5 2 and 5 3). A lthough the overall levels of LAMP1 are unchanged by the modulation, the mobility of LAMP1 is slowed in old cells, possibly due to altered glycosylation, or compromised pathway activity. Indeed, SC s isolated from old rats contain visibly larger lysosomes under basal conditions ( Fig. 4 3A), as compared to young cells. In addition, the described alterations in biochemical markers of the autophagy lysosome pathway (Atg7, LC3 and pS6/S6 ratios) suggest that autophagy becomes less efficient with age. It has been previously shown that phosphorylation of S6 and inhibition of autophagy have a linear relationship in rat hepatocytes (Blommaart et al., 1995) In our model, based upon the levels of pS6 in response to Stv the activation of autophagy is minimal in SC s from 25mo old rats ( Fig. 4 -
120 2A). The maintenance of autophagy with diet restriction in sciatic nerves is likely mediated via S6 kinase (Klionsky et al., 2005) which is reflected upon the lower levels of pS6 in 38mo CR rat nerves as compared to AL. Peripheral nerves serve as long cables connecting the CNS with distal targets, such as skeletal muscle. The function of peripheral nerves is affected by age and is associated with morphologic (Verdu et al., 2000) and biochemical myelin abnormalities ( Fig. 4 5 to 4 6). D efective myelin and segmental demyelination are coupled with dedifferentiation of SC s (Gregson and Hall, 1973; Saito et al., 1990; Zanazzi et al., 2001) We observed this switch in phenotype in the aged nerves from ALfed rats by reexpression of p75NTR ( Fig. 4 5C), which is detected only at low levels when the cells are in a myelinating state (Jessen and Mirsky, 2005; Amici et al., 2007) In nerves from rats on the diet regimen, myelin maintenance is associated with subdued expression of p75NTR and the mitotic marker, pHH3 ( Fig. 4 5). Prevention of demyelination and hyp erproliferation of SC s with age suggest better axoglial communication in diet restricted animals, which supports the maintenance of the major structural proteins of axons. Together, the prevention of glial and axonal changes preserves the functional archi tecture of myelinated nerves (Figs. 5 5 to 5 7) and is associated with improved motor performance (Xu et al., 2008) Demyelination of axons leads to a decrease in nerve conduction velocity and reorganization of voltagesensitive ion channels has been suggested as an underlying cause (Adinolfi et al., 1991; Verdu et al., 2000) The crosstalk between axons and glia is responsible for maintaining the proper localization of ion channels (Novakovic et al., 2001; Hinman et al., 2006) In this study, we found segmental demyelination in nerves
121 from AL fed aged rats and a corresponding redistribution of ion channels ( Fig. 4 7). In transgenic mice, when SC s are selectively killed by diphtheria toxin, a demyelinating phenotype is associated with overexpression and redistribution of sodium channels (Vabnick et al., 1997) Similarly, a knock out mouse with deletion of MBP shows a strikingly hi gh density of Na channels along hypomyelinated axons (Noebels et al., 1991; Westenbroek et al., 1992) likely as a compensatory mechanism to support signal propagation. Nonetheless, the redistribution in ion channel proteins associated with defective myelination is not an effective replacement of myelinated internodes and can lead to sensory and motor dysfunction (Novakovic et al., 2001) Our study shows that the CR regim en has the ability to minimize the changes in expression and organization of the Nav and Kv channel at the node of Ranvier (Fig. 5 7), likely by preservation of internodal myelin segments. Together, t he muted responsiveness of stress induced pathways in ag ed SC s might exacerbate the observed molecular and structural defects in myelinated peripheral nerves. Approaches to limit the demand on protein homeostatic pathways, such as dietary restriction appear to provide benefits for maintenance of nerve health.
122 Figure 4 1. The chaperone response of SC s from aged rats. (A) SC s isolated from P2 and 25mo rats were treated with amino acid and serum deficient (Stv) medium, or subjected to HS and allowed to recover at 37 C for 6 h. Steady state expression of HSP90, HSP70 and HSP40 were analyzed in whole cell lysates (15 g/lane) of untreated control (Ct), Stv or HS treated samples. Blots were reprobed with anti GAPDH antibody as a protein loading control. Molecular mass at the left, in kDa. (B) SC s isolated from P2 and 25mo old rats were subjected to HS ( 45 C; 20 min) and allowed to recover at 37 C for 6 h (HS+6h). Control untreated cells (Ct) and HS+6h samples were immunolabeled with anti HSP70 (green) antibody. Hoechst dye (blue) was used to visualize nuclei Scale bar, 10 m.
123 Figure 4 2. The response of glial cells to Stv stimulus. (A) Primary rat SC s from postnatal day 2 (P2) and 25mo old rats were maintained in normal (Control, Ct), or amino acid and serum deficient Stv medium for 4 h, with (+) or wi thout ( ) CQ The levels of autophagy markers, Atg7, LC3 I and II, pS6 and S6 were determined by Western blots (15 g/lane). (B) Quantification of LC3 I and LC3 II band intensities in the presence of CQ normalized to GAPDH from three independent experiment s are shown. LC3 I and II values of P2 cells treated with CQ was set as 1. AU:arbitrary units. (*p<0.05, unpaired t test, mean SEM, n=3). (C) Levels of pS6 and S6 from three independent experiments were quantified and the values are represented as ratio of pS6/S6. The value of P2 Ct sample was set as 1. (D) The expression of lysosomeassociated membrane protein 1 (LAMP1) and cathepsin D were determined by Western blots. The shift in LAMP1 mobility in 25mo samples is indicated by an asterisk. The bands re presenting procathepsin D (procath D) and active form of cathepsin D (active cath D) are marked with arrows. (E) Semi quantitative analysis of procath D and activecath D protein levels after normalization to GAPDH from three independent experiments. Th e values of pro and activecath D in P2 Ct sample were set as 1. AU:arbitrary units. (**p<0.01, ***p<0.001, unpaired t test, mean SEM). (A and D) GAPDH is shown as a protein loading control. Molecular mass at left, in kDa.
124 Fig ure 42. Continued
125 Figure 4 3. The fusion of autophagosomes with lysosomes in SC s. (A) Composite confocal images of control (Ct) and Stv treated (Stv) SC s from postnatal day 2 (P2) and 25mo old rats, double immunostained with anti LC3 (red) and anti LAMP1 (green) antibodies. Insets show LC3like (red) staining alone. Enlarged lysosomes positive for LAMP1 (green) are indicated by arrows. (B) Mid z stack images of cells after treatment with CQ, in Stv medium. Single x and y plane sections are shown at the left to reveal the i nteraction of autophagosomes (red) with lysosomes (green), in yellow. In cells from 25mo old rats, swollen LAMP1positive lysosomes (green) are indicated by arrows. Nuclei are visualized by Hoechst dye (blue). Scale bars, 10 m. (C) 2D cytofluorograms for LC3 and LAMP1 colocalization in Stv+CQ treated P2 and 25mo samples in which the interaction between the red channel (x axis) and the green channel (y axis) is highlighted in green in the diagonal region. (D) The area of colocalization (mask area) per cel l was estimated using Leica software from P2 and 25mo cells (n=200) treated with Stv+CQ from three independent experiments, and eight random visual fields per condition ( *p<0.05, unpaired t test, mean SEM).
126 Figure 43. Continued
127 Figure 4 4. Age associated alterations in chaperones and autophagic proteins in myelinated peripheral nerves. (A) Total sciatic nerve lysates (25 g/lane) from the indicated ages and diet were analyzed with anti HSF1 antibody. The same nerve lysates (20 g/lane) were als o probed with antibodies against HSP90, HSP70, HSP40, HSP27 and B crystallin. (B) Quantification of HSF1 and HSP90 protein levels normalized to GAPDH from three independent experiments (##p<0.01, ###p<0.001, Fishers PLSD, mean SEM), AU: arbitrary units The effect of CR on these proteins was analyzed by comparing HSF1 or HSP90 protein values with agematched AL counterparts ( *p<0.05, **p<0.05, unpaired t test, mean SEM). (C) Steady state levels of LAMP1, Atg7, pS6 and S6 proteins in sciatic nerves from AL and CR rats were analyzed by Western blots. Blots were reprobed with anti GAPDH antibody as protein loading control. Molecular mass at the left, in kDa. (D) Quantification of LAMP1 and Atg7 protein levels normalized to GAPDH from three independent exp eriments (#p<0.05, ###p<0.001, Fishers PLSD analysis, *p<0.05, unpaired t test, mean SEM). (E) Blots of pS6 and S6 from three independent experiments were quantified and the values are represented as ratio of pS6/S6. The pS6/S6 ratio of 8mo old AL sample was set as 1 ( *p<0.05, unpaired t test, mean SEM). A D, n=3 rats per condition.
128 Figure 44. Continued
129 Figure 4 5 CR preserves myelin protein expression and myelinating SC phenotype. (A) Total sciatic nerve lysates (10 g/lane) from AL and CR rats at 8, 18, 29 and 38mo ages were analyzed with antibodies against protein zero (P0), P MP22 and MBP by Western blots. The arrow and arrowhead on the right indicate the 18.5 and 14 kDa of isoforms of MBP. GAPDH serves as a protein loading control. (B) Densitometric analysis of myelin proteins P0, PMP22 and MBP normalized to GAPDH (#p<0.05, ##p<0.01 [Fishers PLSD], *p<0.05 [unpaired t test], mean SEM). (C) Total sciatic nerve lysates (20 g/lane) from rats under AL and CR diet were analyzed by Western blotting with polyclonal anti p75NTR and monoclonal anti pHH3 antibodies. The blots were reprobed with anti tubulin to monitor protein loading. Molecular mass at left, in kDa. (D) Quantification of p75NTR and pHH3 normalized to GAPDH (###p<0.001 [Fishers PLSD], *p<0.05, *p<0.01, ***p<0.001 [unpaired t test]). (E) The nuclei of SC s were counted in longitudinal sections of sciatic nerves from two different depths and eight random visual fields (0.1mm2) per animal. (*p<0.05, ***p<0.001, unpaired t test, mean SEM). A E, n=3 rats per condition.
130 Figure 45. Continued
131 Figure 4 6. The expression of axonal proteins is supported by CR diet. (A) The severe reduction in MBP like (red) as well as NF L like (green) staining is shown on longitudinal sections o f sciatic nerves of 38mo old ALfed rats. Arrowheads indicate nerve fibers positive for neurofilaments but devoid of MBP reactive myelin. Nerves from 18mo old AL and CR rats are shown in the insets on the right. Nuclei are stained with Hoechst dye (blue) Scale bars, 40 m. (B) Total sciatic nerve lysates (10 g/lane) of AL and CR rats from the indicated ages were analyzed with antibodies against neurofilament proteins, NF H, NF M, NF L and the intermediate filament protein, vimentin. The arrow indicates the fulllength form (60 kDa) and the bracket shows the proteolytic cleavage products of vimentin. Tubulin serves as a protein loading control. Molecular mass at left, in kDa. (C) Densitometric analysis of neurofilament proteins NF H, M and L normalized to tubulin from three independent sets of blots (#p<0.05, ##p<0.01 (Fishers PLSD), *p<0.05 (unpaired t test), mean SEM, n=3 rats). AU:arbitrary units.
132 Figure 4 7. The expression and localization of Na+ and K+ channel proteins in myelinated nerves o f aged rats. (A) Lysates of sciatic nerves (25 g/lane) from the indicated ages and diet regimen (AL and CR) were assayed by Western blots for the expression of panvoltage gated sodium channels (Nav) and a subtype of voltage gated potassium channel (Kv1.1) with polyclonal anti pan Nav and anti Kv1.1 antibodies, respectively. The arrow indicates the subunit of Nav and the arrowhead points to its proteolytic cleavage product. Tubulin serves as a protein loading control. Molecular mass at left, in kDa. (B) Longitudinal sciatic nerve sections were co immunolabeled with anti pan Nav (red) and anti MBP (green) antibodies (panel on right). The node of Ranvier, as marked by clustered Nav channel like staining (asterisk) is enlarged 4X in the inset. (C ) Localization of Kv1.1 channel was visualized in longitudinal sciatic nerve sections from 38 mo old AL and CR rats by co immunolabeling with anti Kv1.1 (red) and anti MBP (green) antibodies (panel on right). The nodes of Ranvier are marked by asterisks. Scale bar, 10 m.
133 Table 4 1. Primary antibodies used in this study. WB, Western Blot; IS, Immunostaining
134 CHAPTER 5 LIFE LONG CALORIE RESTRIC TION ALLEVIATES AGERELATED OXIDATIVE DAMAGE IN PERIPHERAL NERVES Introduction Aging of organ systems is assoc iated with the accumulation of oxidatively damaged polynucleotides, proteins, carbohydrates and lipids which compromise cellular function. This is considered the Oxidative Stress Theory of Aging whereby agerelated loss of proper physiological function i s due to the accumulation of oxidative damage (Bokov et al., 2004) Long lived postmitotic cells such as neurons are at higher risk and accrue greater amounts of damaged waste than short lived cells ( Agarwal and Sohal, 1994; Sohal et al., 1994) In addition, SC s, the myelinating glia of the PNS, are rich in polyunsaturated fatty acids (Garbay et al., 2000) which serve as a substrate for ROS mediated lipid peroxidation (Smith et al., 1999; Blair, 2001) Together, the buildup of age related damaged material along wit h their inefficient removal by homeostatic mechanisms become a concern in the vulnerable neurons and SC s of peripheral nerves. Intracellular proteolytic mechanisms, including the UPS and the autophagy lysosomal pathway (macroautophagy) are responsible for degradation and removal of damaged cellular material. The accumulation of waste material is not only harmful due to its interference with biological functions, but also for imparting toxicity via lipid peroxidation products such as malondialdehyde (MDA), 4hydroxynonenal (HNE) and nitrotyrosine (Levine and Stadtman, 2001; Grune and Davi es, 2003) Although the precise mechanisms underlying the Oxidative Stress Theory of Aging are still largely unknown, there is accumulating evidence to support the involvement of the inflammatory response (Chung et al., 2009) An increase in the
135 levels of serum cytokines, such as TNF 6 and interferon are commonly associated with senescence and play integral roles in activating inflammation and innate immunity (Spaulding et al., 1997) Free radicals are known to modulate this stress induced activation of inflammation through the regulator of innate immunity and of the (Seo et al., 2006; Salminen et al., 2008; Wang et al., 2008) Activation of NF oxidizing agent, inducible nitric oxide synthase (iNOS) (Seo et al., 2006; Chung et al., 2009) Restriction of calorie intake is a widely accepted approach to lower levels of oxidative stress and slow ageassociated changes, as well as to extend lifespan in mammals (Sohal and Weindruch, 1996; Martin et al., 2006) Previous studies have shown that calorie restriction (CR) decreases mitochondrial ROS generation and oxidative damage to DNA, protein and lipids (Lambert et al., 2004) CR has been found to activate the autophagic protein degradative pathway in aging rats (Wohlgemuth et al., 2007; Rangaraju et al., 2009) and to reduce markers of agerelated chronic inflammation, like TNF B and iNOS (Martin et al., 2006; Chung et al., 2009) Although there have been extensive studies on the ability of CR to reduce agerelated oxidative damage in the CNS (Martin et al., 2006) little is known about the beneficial effects of CR on ageassociated oxidative stress and inflammation within the PNS. I n this study, we asked whether CR can relieve the oxidative stress placed upon the PNS during aging. We found that a lifelong CR diet decreases the steady state levels of undegraded poly ubiquitinated substrates and oxidative damage markers of
136 proteins an d lipids in myelinated peripheral nerves. Furthermore, CR relieves the chronic inflammation commonly associated with age. Materials and Methods Animals and D iet The use of animals in this study was approved by the Institutional Animal Care and Use Committe e (IACUC) of the University of Florida. AL fed and calorierestricted (CR) male Fischer 344 x BN (Brown Norway) rats (n=3 per group) of ages 8, 18 29 and 38mo were obtained from the National Institute on Aging colony at Harlan Sprague Dawley Inc. (Ind ianapolis, IN). The animals were housed in a temperature and light controlled environment and had water available at all times. AL fed rats had free access to NIH 31 nutrient composition pellets, while the CR group received fortified pellets once daily 1 h before the onset of the dark cycle. Calorie reduction began at 14 weeks of age with 10% restriction, increased to 25% at 15 weeks, and was maintained at 40% from 16 weeks of age until sacrificed at 8, 18 29 and 38mo of age. Data on survival character istics and physical performance of the same colony of rats have been previously reported (Turturro et al., 1999; Xu et al., 2008) The survival percentages of the male Fischer 344 x BN rats in the AL group for the ages 8, 18 29 and 38 mo are, 100, 98, 7 0 and 30% respectively. In comparison, those for the CR group for the same ages are 100, 100, 90 and 70% respectively (Turturro et al., 1999) Bio chemical Analyses Rats of above mentioned ages kept under AL or CR diets were sacrificed as per IACUC protocols. The proximal ends (approximately 5 cm long piece) of the left and right sciatic nerves were surgically removed within 5 minutes of decapitation and frozen
137 immediately and stored in liquid nitrogen. The nerves from the right sides were used for biochemical analysis. Whole nerve lysates (including myelin and axonal proteins) were prepared separately from individual nerves in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 3 % SDS) supplemented with phosphatase inhibitors, PMSF (both from SigmaAldrich, St. Louis, MO) and complete protease inhibitor (Roche, Indianapolis, IN). The lysates were assayed for protein content using the BCA kit (Pierce, Rockford, IL) and then separated on polyacrylamide gels and transferred to nitrocellulose membranes for Western blotting. Primary antibodies in blocking buffer (5% milk in phosphate buffered saline (PBS) were applied to the memb ranes overnight at 4C (see Table 1). Membranes were then incubated with horseradish peroxidase conjugated secondary antibodies directed against the primary antibody, including anti rabbit, anti mouse (Cell Signaling technology, Inc., Danvers, MA), anti ra t or anti goat (Sigma Aldrich, St. Louis, MO). Membranes were then reacted with an enhanced chemiluminescent substrate (Perkin Elmer, Boston, MA). A GS 800 densitometer (BioRad, Hercules, CA) was used to digitally image the films. In each experiment the densitometric value of AL 8mo sample was set as 1 and the values of other agediet combination were determined with respect to this. One way ANOVA followed by Fishers PLSD analysis was performed using the StatView program, to compare the normalized densit ometric values of proteins (dependent variables) between AL or CR diet with age (factor). For each analysis, the pvalue and significance (#, ##, ###) for Fishers PLSD analysis was determined by comparing the densitometric values of 8mo old AL samples wi th the older ages (18, 29 or 38 mo) of the AL group. Similarly in the CR group, the 8mo old CR sample was compared with older ages. To
138 determine statistical significance between samples, unpaired t test was performed using GraphPad Prism v5.0 software. D ifferences were considered significant at p<0.05. For each analysis, nerve samples from 3 individual rats were used. Immunolabeling of Nerve S amples The proximal ends of each left sciatic nerve (n= 3 animals for each age and diet group) were sectioned at 5 Superfrost/Plus microslides (Fisher Scientific, Pittsburg, PA). Sections for HNE staining were fixed and permeabilized with 1% paraformaldehyde and 90% ethanol for 2 min at 25C. Sections analyzed for lysosome associated membrane protein 1 (LAMP1), pUb, CD11b and NF permeabilization with ice cold methanol for 5 min (Rangaraju et al., 2009) Following fixation, slides were blocked in 20% normal goat serum in PBS, and then incubated with primary antibodies overnight at 4C (see Table 1). Bound antibodies were detected using Alexa Fluor 594 or Alexa Fluor 488 goat anti rabbit or anti mouse IgG secondary antibodies (Molecular Probes, Eugene, OR). Hoechst dye (Molecular Probes) was included with the secondary antibodies t o visualize nuclei. Slides were mounted with coverslips using the ProLong Antifade kit (Molecular Probes) and imaged with a SPOT camera attached to a Nikon Eclipse E800 microscope (Melville, NY). Images were formatted using Adobe Photoshop 5.5. For quantif ication of macrophages, CD11bpositive cells were counted per field of view in longitudinal sections ( 5 m thickness) of sciatic nerves from two different depths and eight random visual fields (0.1mm2) per animal (n=3) To determine statistical significanc e between samples, unpaired t test was performed using GraphPad Prism v5.0 software. Differences were considered significant at p<0.05.
139 Di 8 ANEPPS L abeling For labeling of accumulated oxidized material, 4(2 (6 dibutylamino) 2 naphthalenyl)ethenyl) 1 (3 sulfopropyl) hydroxide (di 8 ANEPPS), a lipophilic dye that recognizes lipofuscin adducts (Grune et al., 2004) was used. Frozen nerve sections (as above) were fixed with 4% paraformaldehye in PBS for 10 min followed by permeabilization with 0.2% Triton X 100 in PBS for 15 min at 25oC. Samples were blo cked in 20% normal goat serum in PBS then incubated with 2 nM di 8 ANEPPS in PBS for 30 min at 25C. Slides were mounted with coverslips and imaged, as above. Mean di 8 ANEPPS pixels per fixed area (0.1mm2) were determined using the mean gray value tool in the NIH Image J software. Statistical analysis was performed by unpaired t test using GraphPad Prism software v5.0 and differences were considered significant at p<0.05. Results A C alorie R estricted D iet S lows P rotein D amage within Peripheral N erves Durin g A ging As a marker of damaged and/or aggregated proteins we compared the steady state levels of anti ubiquitin reactive molecules between sciatic nerve lysates from 818, 29 and 38 mo old rats (Fig. 5 1A). There is a gradual accumulation of slow migrat ing poly ubiquitinated substrates in the nerves of AL fed rodents, which becomes prominent by 29mo (Fig. 5 1A). In comparison, the levels of ubiquitinreactive proteins remain remarkably low in nerves of CR fed rats, even at 38 mo (Fig. 5 1A). Quantificat ion of blots after correcting for GAPDH from three independent experiments reveals a significant increase in poly ubiquitin reactive proteins by 29mo of age in the AL group, which is absent from the CR animals (Fig. 5 1B).
140 To examine if lysosomes are recr uited to ubiquitin aggregates in aged nerves, we coimmunolabeled longitudinal sciatic nerve sections for ubiquitin and LAMP1 (Fig. 5 1C). As suggested by the Western blots (Fig. 5 1A), nerves samples from 38mo AL fed rats contain numerous ubiquitinpositi ve protein aggregates (Fig. 5 1C), as well as enlarged lysosomes and vacuoles (Fig. 5 1C, green, arrows) (Rangaraju et al., 2009) Many of the ubiquitinreactive aggregates are surrounded by LAMP1 positive lysosomes, which are revealed by yellow color on merged images (Fig. 5 1C, 3X magnifications). On sections from 38mo old CR animals, the abundance and apparent size of ubiquitinpositive aggregates are dramatically lower (Fig. 5 1C, red, arrows), which agree with the biochemical data (Fig. 5 1A). Correspondingly, these nerves show an overall decrease in LAMP1like immunoreactivity and fewer vacuoles (Fig. 5 1C, bottom panel). The CR diet also attenuates the agerelated increase the number of nuclei per fixed area of nerve tissue (Fig 5 1C, blue), as shown previously (Rangaraju et al., 2009) In aged postmitotic mammalian tissues, besides undegraded ubiquitintagged substrates, there is a buildup of oxidized and cros slinked proteins and lipids, known as lipofuscin (Jung et al., 2007) In accordance, sciatic nerve sections from 38mo old AL fed rodents stain prominently with di 8 ANEPPS (Fig. 5 2A, arrows), a lipophilic dye that recognizes lipofuscin adducts (Grune et al., 2004) as compared to samples from 18mo old rats (Fig 5 2A). Nerve tissues of diet restricted rats contain fewer and apparently smaller di 8 ANEPPSpositive structures at both ages examined (Fig. 5 2A, arrowhead). Quantification of the mean di 8 ANEPPS pixels per fixed area shows a statistically significant increase with age from 18to 38mo in samples from AL rats (Fig.
141 5 2B). When compared across ages and diet groups, th e accrual is significantly reduced by CR diet (Fig. 5 2B, *p<0.05, ***p<0.001). Together, these results emphasize that a life long CR diet is effective in reducing the accumulation of oxidized and cross linked substances in peripheral nerves, either by low ering damage across lifespan and/or enhancing the activity of protective mechanisms, such as the ubiquitinproteasome and/or autophagy lysosomal pathways. Lipid O xidationMediated Protein Damage within Myelinated Peripheral N erves Oxidation of polyunsaturated fatty acids of myelin lipids (Garbay et al., 2000) results in the formation of hydroperoxides and hydroxyalkenals, like malondialdehyde (MDA) and 4 hydroxynonenal (HNE), whi ch then can react with proteins and alter their surface hydrophobicity (Adams et al., 2001; Davies, 2001) To examine lipid modification during aging of myelinated nerves, we analyzed tissue lysates with antibodies to MDA (Fig. 5 3A). As shown on the Western blot, there is a gradual increase in MDA adducted proteins with age, detected as a smear, in AL samples (Fig. 5 3A, square bracket). In addition, the mobility of MDA positive proteins is slowed in the AL 38 mo old lysate likely representing aggregates (Fig. 5 3A). In comparison, we find a marked decrease in MDA adducted proteins within nerves from animals on CR diets (Fig. 5 3A, square bracket). Quantification after correction for tubulin, reveals a significant increase (###p<0.01) in MDA adducted proteins in sciatic nerves of AL fed animals from 29to 3 8 mo, a trend that is attenuated by the CR intervention (Fig. 5 3B). Furthermore, the differences are significant when comparing across diet groups at the 38mo time point (Fig. 5 3B, *p<0.05). We also measured the levels of protein nitrosylation within wh ole nerve lysates and found a similar increasing pattern across the ages in AL samples (Fig. 5 3C, D, ##p<0.01). In contrast, significantly lower levels
142 (*p<0.05) of nitrosylated proteins are detected in nerves of animals maintained on a lifelong CR (Fig. 5 3D). As both axonal and glial proteins are vulnerable to HNE modification (Gard et al., 2001) we also quantified HNE adducts by biochemical analysis and found a significant increase from 29to 38 mo in the AL f ed group (Fig. 5 3E, F, ##p<0.01). While the CR diet lowers the levels of HNE reactive proteins across lifespan, statistical analysis between AL and CR 38mo samples did not yield significance (p=0.0697) (Fig. 5 3F). To examine the distribution of oxidativ ely damaged proteins within peripheral nerves, we immunostained sciatic nerves with the same anti HNE antibody that was used for the Western blots (Fig. 5 3G). There is a prominent increase in HNE like immunoreactivity with age within the nerve tissues of AL fed rats, with diffuse localization throughout axonal and glial compartments (Fig. 5 3G). As suggested by the Western blots, there are fewer HNE positive areas within the 38mo CR counterpart. While the anti MDA and anti nitrotyrosine antibodies are uns uitable for immunolocalization studies, we predict that those antibodies would show a similar diffuse immunoreactivity. Age related activation of pro inflammatory pathways is attenuated by a lifelong CR diet The inflammatory response appears to play a cri tical role in decreasing redox status and increasing oxidative damage observed with aging (Beckman and Ames, 1998; Salminen et al., 2008) Therefore, we analyzed sciatic nerve lysates for the levels of markers of chronic inflammation, including TNF p50 and p65 subunits of proinflammatory NF and for phosphoI B. For each of these proteins, we found an increasing trend with age, which is attenuated by the CR intervention (Fig. 5 4A).
143 Quantification of blots from three different group of animals revealed a significant increase in TNF from 8 mo to 38mo (##p<0.01) in animals receiving an AL diet (Fig. 5 4B). In contrast, the expression of TNF significant increase in the oldest animals (##p<0.01). Additionally, diet restriction significantly reduced TNF and 38mo old nerves when compared to their AL fed counterparts (Fig. 5 4B, *p<0.05). Similar to TNF there is an increase in the expression of both p50 and p65 subunits of proinflammatory NF ###p<0.001 between 8mo and 38mo, Fig. 5 4A, C, D) in the AL group. Strikingly, the steady state levels of p50 subunit of NF in sciatic nerve of 38mo old CR rats is significantly lower (*p<0.05) as comp ared to age matched ALfed rats. An analogous trend is observed for the p65 subunit of NF protein in nerves of AL rats with age, and the attenuation of this rise with diet restriction, ( Fig. 5 4D *p<0.05). We analyzed the steady state levels of phospho (phospho5 4E). In the CR group the increase is still significant at the oldest age examined (##p<0.01), however a marked reduction in normalized phosphowhen compared across diet groups (**p<0.01) (Fig. 5 4E). The main source for inflammatory mediators within myelinated peripheral nerves are infiltrating macrophages (Hartung et al., 1992) Hence we tested whether a CR diet would influence the number of infiltrating immune cells associated w ith aging in sciatic nerves. We used an anti CD11b antibody to stain for macrophages (Misko et al., 2002) which appear to increase in numbers from 18 to 38mo on longitudinal nerve sections from AL fed rats (Fig. 5 5A). Quant ification of CD11bpositive immune cells reveals a
144 significant agerelated increase (***p<0.001) in the AL group from 18 to 38mo old (Fig. 5 5B). On the other hand, the number of macrophages is low in nerves of the CR rats, particularly when compared at the 38 mo time point (***p<0.001) (Fig. 5 5B). Accordingly, Western blot analysis of whole nerve lysates reveals a pronounced increase in CD11b protein levels in 38mo old AL samples as compared to 8mo (Fig. 5 5C), while the levels remain low across the t ested ages in the diet restricted group (Fig. 5 5C, D). In agreement with the increased inflammatory response, biochemical studies with an anti rat IgG antibody detect a marked increase in endogenous nerve immunoglobulins, specifically IgG heavy chain ( H C) and light chain ( LC) with age. The dietary modulation notably alleviates the increase in the levels of endogenous nerve IgGs (Fig. 5 5C, E, F). D iscussion As physical barriers against circulating toxic oxidants such as capillary endothelia and the chor oid plexus are absent within the periphery (Samson and Nelson, 2000) the PNS is a vulnerable target for oxidative damage. H ere we show a pronounced accumulation of ubiquitinated and oxidatively damaged proteins with age within myelinated peripheral nerves, which prompts an immunologic response from the host. In comparison, nerves of rats kept on a lifelong CR diet accumulate lower levels of modified proteins and the inflammatory response is muted. While a lifelong CR might not be practical for humans, this study clearly shows the power of this intervention in preventing ageassociated damage in peripheral nerves. Based on the findings presented here, we hypothesize that the PNS would also respond to a more acute, short term CR, or a long term mild CR. Both of these approaches have been explored in
145 other organ systems with success (Seo et al., 2006; Ingram et al., 2007) but their effects on peripheral nerves is not known. Proteomics studies of aging rat brain have revealed a decrease in proteins required for proper ubiquitinproteasomal degr adation, resulting in a buildup of undegraded substrates (Klionsky et al., 2008) While due to sample size limitations we were unable to test the levels of proteasomal constituents, we detected t he accumulation of slow migratin g poly ubiquitinated substrates within nerves of ALfed aged rats, suggestive of compromised proteasome activity. Significantly, the levels of such ubiquitin reactive proteins are reduced in nerves of CR rats (Fig. 5 1). This effect could be due to less pr otein damage throughout lifespan, and/or sustained proteasomal processing of ubiquitinated substrates and/or their removal by an alternative mechanism, such as autophagy. Although we do not know yet which of these possibilities are influenced by the lifel ong diet restriction, a contribution from the autophagy lysosomal pathway is likely (Rangaraju et al., 2009) With normal aging, lysosomes overwhelmed with lipofuscin still receive proteolytic enzymes, which leads to their depletion and decrease in overall autophagic capacity of the cell (Brunk and Terman, 2002) Diet restriction is known to activate the autophagy lysosomal pathway and assist in removal of damaged proteins (Wohlgemuth et al., 2007; Madorsky et al., 2009) Accordingly, transgenic mice with compromised autophagic activity accumulate high levels of aggregated proteins that are reactive for ubiquitin (Komatsu et al., 2005; Mizushima and Hara, 2006) These findings together with our previous reports ( Wohlgemuth et al., 2007; Madorsky et al., 2009; Rangaraju et al., 2009) suggest that dietary restriction is
146 beneficial for the activity of protein degradative pathways, such as the UPS and the autophagy lysosomal system. High levels of HNE, nitrotyrosine, and other irreversible protein adducts have been observed in agerelated neurodegenerative conditions of the CNS (Beal, 2002) yet their contribution to peripheral nerve disease is unclear. Given that lipids are sensitive t o free radical damage (Pamplona et al., 2000) myelina ted nerves provide an opportune environment for lipid peroxidation mediated oxidative damage to organelles and proteins. Indeed, we observed a gradual increase in lipid peroxidation products in our AL samples with age, including MDA, HNE and nitrotyrosine (Fig. 5 3). Oxidative modifications in cellular proteins lead to increased proteolytic susceptibility due to the hydrophobicity (Davies, 2001; Grune and Davies, 2003; Grune et al., 2003) This increase in proteolytic recognition, however, functions only for mildly oxidized substrates as extensively oxidized protei ns tend to aggregate and covalently cross link (Davies, 2001; Merker et al., 2001; Farout and Friguet, 2006) Such changes are suggested by the accumulation of slow migrating proteins in nerve samples from old AL rodents (Fig. 5 1 and 53). Beside protein degradation mechanisms, cells utilize antioxidant cascades to modulate oxidative damage. With age, both of these processes become less efficient, which can lead to further accumulation of lipid peroxidation, protein oxidation and other deleterious cellular modifications (Merker et al., 2001; Grune et al., 2004) Our results suggest that a CR diet attenuates lipid peroxidation (Fig. 5 3) and thereby decreases oxidative damage within peripheral nerves with age. Age associated oxidant stress is known to activate the inflammatory process via the NF (Chung et
147 al., 2009) In human and rodent studies, the circulating levels of this proinflammatory cytokine TNF nts is significantly attenuated by CR (Phillips and Leeuwenburgh, 2005; Bruunsgaard, 2006) In agreement with t hese findings, we found age associated increase in TNF trend that was attenuated up to 38mo of age by CR (Fig. 5 4). One of the upstream modulator of NF nd oxidative stress (Chung et al., 2009) B, which then leads to the activation of NF (Zandi et al., 1997; Karin, 2006) Studies in different organ systems have shown an increase in NF (Chandrasekar and Freeman, 1997; Chung et al., 2009) Corresponding to the increase in NF the levels of p65 a nd p50 subunits of NF (Kim et al., 2002a) a pat tern that is reproduced in myelinated peripheral nerves (Fig 5 4). The upregulation of NF the severe demyelination (Rangaraju et al., 2009) in combination with an inflammatory response (Fig. 5 5) (Andorfer et al., 2001) In inflammatory demyelinating neuropathies, macrophag es infiltrated into the nerves are the primary expressors of NF (Andorfer et al., 2001) and are the likely source for this cytokine in aged nerves, as well. While due to antibody binding sp ecificities we could not perform colocalization studies for macrophages and NF macrophages in CR samples in association with a decrease in proinflammatory NF levels supports this theory.
148 Infil tration of peripheral nerve tissue by CD11bpositive macrophages in inflammatory nerve disease and hereditary demyelinating neuropathies take part in the pathogenic cascade (Ceballos et al., 1999; Andorfer et al., 2001; Misko et al., 2002) Upon analyses of longitudinal sections of sciatic nerves, we observed a signifi cant increase (*** p <0.001) in the number of CD11bpositive macrophages in AL fed animals from age 18to 38 mo (Fig. 5 5), which could contribute to the pronounced morphologic changes observed (Rangaraju et al., 2009) Indeed, ultrastructural studies of senescent paranodal junctions of rat peripheral nerves have detected myelin fragment filled macrophages between the ages of 24and 31 mo (Sugiyama et al., 2002) In addition to a possible role in myelin damage, a pronounced inflammatory response is also evidenced by accumulation of immunoglobulins within nerves, targets of which antibodies are unknown. Together, our studies support the activation of the inflammatory response through oxidative damage within aged peripheral nerves, a detrimental cascade that is muted in nerves of calorierestricted r odents. In summary, our studies show evidence of pronounced oxidative damage within myelinated peripheral nerves with age and associated activation of proinflammatory events. In comparison, lifelong CR thwarts such detrimental changes likely by attenuati ng the levels of damaging molecules and allowing the cells to maintain endogenous protective mechanisms (Martin et al., 2006; Rangaraju et al., 2009) These findings offer novel insights into possible mechanisms of ageassociated decline in neural function and provide targets for therapeutic interventions.
149 Figure 5 1. A calorie restricted diet minimizes accrual of damaged proteins within peripheral nerves during aging. (A) Western blot analysis of whole sciatic CR diets probed with an anti ubiquitin antibody. Slow migrating poly ubiquitinated (pUbi) protein substrates are marked by a square bracket. GAPDH is shown as a protein loading control. Molecular mass in kDa, on the left. (B) Quantification of pUbi band intensity normalized to GAPDH is shown (#p<0.05, ###p<0.001, Fishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). In this and subsequent figures, the pvalue for Fishers PLSD analysis is determined by comparing the 8mo old sample with the older ages (18 29 or 38mo) for each diet group. (C) Longitudinal sciatic nerve sections from 38 mo old animals fed AL or CR diets were analyzed for ubiquitin (red) and LAMP1like (green) immunoreactivity. Arrows point to ubiquitin positive protein aggregat es adjacent to LAMP1positive lysosomes (magnified 3X in insets). Asterisks indicate intracytoplasmic vacuoles. Nuclei
150 Figure 5 2. Accumulation of lipofuscin is curtailed by CR diet with aging. (A ) Sciatic nerve sections from 18 mo and 38mo old animals fed AL or CR diets were stained with di 8 ANEPPS dye, which labels lipofuscin content (red). Large 8 ANEPPS positive adducts are marked with arrows and arrowheads, r espectively. Nuclei are labeled with Hoechst 8 ANEPPS dye positive pixels per fixed area is shown (*p<0.05, ***p<0.001, unpaired t test, meanSEM, n=3).
151 Figure 5 3. Lipid peroxidationassocia ted modifications of proteins with age are relieved were probed with a rabbit anti malondialdehyde (MDA) antibody to detect MDA adducts to proteins (square bracket). Tubulin is shown as a protein loading control. (B) Quantification of MDA adduct band intensities normalized to tubulin is shown (###p<0.001, Fishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). (C) Biochemical analysis of tyrosine residues (Nitros) on proteins (square bracket) using an anti nitrosylation antibody in the same lysates as in A.. Tubulin is shown as a protein loading control. (D) Quantification of nitrotyrosine band intensities from Western blots normalized to tubulin is shown (##p<0.01, F ishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). (E) Western blot analysis of 4hydroxynonenal (HNE) in the same lysates used in A and C. Tubulin is shown as a protein loading control. In A, C and E molecular mass is in kDa, on the left. (F ) Quantification of HNE adduct band intensities from Western blots normalized to tubulin is shown (###p<0.001, Fishers PLSD analysis; p=0.0697, unpaired t test, meanSEM, n=3). (G) Immunohistochemical staining of sciatic nerve from 18mo and 38mo old ani mals fed AL and CR diets with an antibody against 4hydroxynonenal (HNE). Nuclei are labeled with Hoechst dye (blue). Scale bar,
152 Figure 5 4. Age related increase in proinflammatory mediators is attenuated by a lifelong CR diet. (A) Total scia tic nerve lysates (20 g/lane) from the indicated subunits of NF Molecular mass is in kDa, on the left. (B) Quantification of T intensities after normalization to tubulin is represented (##p<0.01, Fishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). (C) Quantification of the p50 subunit of NF (##p<0.01, ###p<0 .001, Fishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). (D) Quantification of the p65 subunit of NF intensities normalized to tubulin is shown ( p=0.0546, ##p<0.01, ###p<0.001, Fishers PLSD analysis; *p<0.05, unpaired t test, meanS EM, n=3). (E) Phosphoquantification of band intensities normalized to tubulin was performed (p=0.0830, ##p<0.01, ###p<0.001, Fishers PLSD analysis; **p<0.01, unpaired t test, meanSEM, n=3).
153 F igure 5 5. A calorie restricted diet diminishes macrophage infiltration of peripheral nerves with age. (A) Cryosections of sciatic nerves from 18mo and 38mo old rats either maintained on AL or CR diet were immunostained with an antibody against CD11b (g reen). Nuclei are stained with Hoechst dye (blue). Scale bar, 20 positive cells per 0.1 mm2 of nerve tissue area is shown for 18and 38mo old animals fed AL or CR diets (***p<0.001, unpaired t test, meanSEM, n=3). (C) Western blot analysis of sciatic nerve lysates (20 g/lane) for ste ady state levels of CD11b and endogenous IgG [heavy chain ( HC) and light chain ( LC)] from AL and CR rats is shown. GAPDH is used as a protein loading control. Molecular mass in kDa, on the left. (D) Quantification of the CD11b band intensities normalized to GAPDH from Western blot analyses from whole sciatic nerve lysates is shown (#p<0.05, ###p<0.001, Fishers PLSD analysis; **p<0.01, unpaired t test, meanSEM, n=3). (E) Quantification of the IgG HC band intensities normalized to GAPDH from whole sciatic nerve lysates of AL and CR rats of the indicated ages is shown (##p<0.01, Fishers PLSD analysis; *p<0.05, unpaired t test, meanSEM, n=3). (F) Quantification of band intensities of IgG LC normalized to GAPDH was performed (##p<0.01, ###p<0.001, Fishers PLSD analysis ; *p<0.05, unpaired t test, meanSEM, n=3).
154 Table 51. Primary a ntibodies used in this study. WB, Western Blot; IS, Immunostaining
155 CHAPTER 6 CONCLUSIONS Misexpression and intracellular retention o f PMP22 is associated with hereditary n europathies in humans, namely, CMT1A (Fig. 6 1A [ 1]) In neuropathic SC s from both the mouse models of this disease, C22 and TrJ mice the turnover of the newly synthesized PMP22 by the proteasome is decreased (Fig. 6 1A ) leading to the formation of cyt osolic protein aggregat es (Fortun et al., 2003; Fortun et al. 2006) In these models impaired PMP22 trafficking has been proposed to play a role in the disease process which eventually leads to demyelination (Fig. 6 1A ) (Sanders et al., 2001) Currently, there is no cure for CMT1A neuropathies and treatment is limited to rehabilitation and corrective surgery (Pareyson and Marchesi, 2009) In this study, t wo pro tein quality contr ol pathways of therapeutic interest were explored for aiding the folding and/or degradation of misfolded proteins and improve myelination, namely chaperones (Chapter 2) and autophagy (Chapter 3) (Fig. 6 1B [1,3]) Our investigations in Chapter 2 and Chapter 3 directly stem from our previous proof of principle studies First, the HS induced chaperones and n utrient deprivationinduced autophagy are able to suppress the formation of PMP22 aggregates in a toxininduced cellular model (Fortun et al., 2003; Fortun et al., 2007) Secondly, intermittent fasting, a dietary approach to i nduce both chaperone and autophagy pathways improve locomotor performance and myelination in TrJ mice (Madorsky et al., 2009) Pharmacological stimulation of the expressi on of protein chaperones by synthetic smallmolecule inhibitors of HSP90 in DRG explants from C22 neuropathic mice lead s to an improvement in myelin formation increase in myelin internodes and correct processing of PMP22 (Chapter 2) (Fig. 6 1B [1,2,5,7]) This treatment was performed
156 during late stage of myelination which is 1014 days after initiating myelination with ascorbate ( Fig. 2 5 ) Preliminary resul t s from our lab show that exposure o f DRGs from C22 neuropathic mice with HSP90 inhibitors at early t imepoints (i.e) 2 days after initiation of myelination also leads to improvement in myelin protein expr ession (data not shown) In addition to the improved myelin in C22 model first round of results show that activating chaperones using HSP90 inhibitor in DRGs from TrJ mice also leads to enhanced myelination (data not shown) Together, t hese results warrant further studies with HSP90 inhibitors as potential therapeutic candidates, in vivo for both overexpressor (C22) and point mutation (TrJ) mouse models of hereditary demyelinating neuropathies. With regards to the autophagy pathway, t he treatment of DRG explant cultures from neuropathic mice with RM (25 nM) to activate autophagy improves the processing of PMP22 through the secretory pathway (Chapter 3) (Fig. 6 1B [3,5]) Furthermore, RM increases the abundance and length of myelin internodes, as well as the expression of myelin proteins (Chapter 3) (Fig. 6 1B ) The improvement in m yelination was observed for early and late treatment paradigms in bot h disease models. The se result s signif y that the beneficial effect of autophagy on myelination, similar to that of the chaperone pathway, overlaps among distinct genetic models of PMP22 neuropathies Abolishment of the improvement in myelination by l entivi rus mediated knockdown of the autophagy related gene 12 ( Atg12 ) in the TrJ model further reinstates the fact that autophag ic activity is critical for the observed benefits. Together, these results support the potential use of RM and other autophagy enhanci ng compounds as therapeutic agents for PMP22 associated demyelinating neuropathies and necessitate the testing of
157 RM in vivo in the mouse models In all of the studies in Chapter 2 and 3, late treatment with chaperoneor autophagy inducing compounds was c onsistently better in improving myelination compared to early treatment, in vitro The fact tha t pharmacological activation of chaperones and autophagy independent ly improves myelination in C22 and TrJ models (Chapters 2 and 3) prompts us to test whether combined treatment of these pathway activators would be additive for improvement in myelination. The commonality between the C22 and TrJ mouse mo dels is the misfolding of PMP22 associated with accumulation of pUb substrates (Fig. 6 1A ) P rotein aggregates could interfere with essential cellular functions, such as myelination in the case of SCs (Fig. 6 1A ) Myelination is a highly energy and quality control demanding process that requires massive synthesis and processing of myelin proteins by SC s (D'Antonio et al., 2009) Hence it is not surprising that a defect in the trafficking of one extremely hydrophobic glyco protein, namely PMP22, would affect the expression and trafficking of other myelin proteins such as P0 and MBP in our mouse models. Furthermore, in addition to endogenous activation of chaperones and autophagy (Fortun et al., 2003; Fortun et al., 2006) the quality control mechanism that is commonly affected in these two models is the proteasomal machinery (Fortun et al., 2005; Fortun et al., 2006) (Fig. 6 1A ) which is the major pathway for turnover of myelin proteins such as PMP22 (Pareek et al., 1997) and MBP (Akaishi et al., 1996) Intriguingly, MBP is one of the unrelated proteasome substrates that is trapped in with the PMP22 inclusions in nerves of neuropathic mice (Fortun et al., 2005) (Fig. 6 1A ) On the otherhand, P0 is thought to be turnedover by the endosomal lysosomal pathway (Yin et al., 2000) There is an emerging body of evidence for the cross talk
158 between these quality control pathways (Ryhanen et al., 2009) W hen inefficiency in one pathway occurs, activation of another pathway has the capacity to protect the overall proteostatic balance of the system (Ryhanen et al., 2009) In our studies activation of chaperones (Chapter 2) and autophagy (Chapter 3) independently show reduction in pUb substrates i n SCs from both neuropathic models (Fig. 6 B ) Due to limited samples, we could not directly measure the proteasomal activitiy in t hese studies (Chapters 2 and 3). However, accumulation of pUb substrates has been previously shown to correlate with impaired proteasome activity (Fortun et al., 2005; Fortun et al., 2006) (Fig. 6 1A ) Hence, t he reduction in the accumulation of pUb substrates achieved by activating chaperones and autophagy in this study (Chapter 2 and 3; also see Chapter 5) (Fig. 6 1B ) likely reflects the allev ia tion of the burden on the proteasomal pathway (Fortun et al., 2005; Fortun et al., 2006) (Fig. 61B ) Such a r estoration of protein homeostasis would possibly provide a more conducive environment for impr ovement in PMP22 processing and myelination by SCs from n europathic mice (Fig. 6 1B [5,7]) In addition to the improved protein expression and trafficking of our protein of interest PMP22, the total levels of other my elin proteins namely P0 and MBP improv ed as well leading to an overall increase in myelination. This result could be explained by taking into account of the fact s that P0 and MBP genes are coexpress ed with PMP22 in myelinating SCs (Kuhn et al., 1993) and at the protein level PMP22 and P0 are coregulated ( Hagedorn et al., 1999; Notterpek et al., 1999a) and they interact (D'Urso et al., 1999; Hasse et al., 2004) Hence, t he improvement in PMP22 processing by chaperones (Chapter 2) and autophagy (Chapter
159 3) most likely contributed to the concomitant increase in the protein expression of its as soci ated partners, P0 and MBP in the both C22 and TrJ neuropathic samples The onset of CMT1A in humans is ~35 years and is progressive with age (Shy et al., 2008; Verhamme et al., 2009a) (Fig. 6 1A) The big question remains, as to why, a person born with a defective PMP22 gene manifests disease symptoms only after ~35 years. To address whether normal aging contributes to the onset and disease progression of CMT1A, we focused on the molecular changes in quality control pathways in the PNS with normal aging (Chapter 4 and 5) The responsiveness of SCs isolated from aged nerves to stress stimuli such as HS and starvation is w eakened (Chapter 4) likely account ing for the observed accrual of damaged molecules including pUb substrates in sciatic nerves with age (Chapter 5) (Fig. 6 1A ) The defective chaperone autophagy (Chapter 4) and proteasomal pathway s (Chapter 5) in the nerves of aged rats, is associated with a loss of myelination, degeneration of axons, and drastic expansion of ion channels at the nodes of Ranvier (Chapter 4) (Fig. 6 1A [4,5,6]) The se results indicate that age is possibly a contributing factor in peripheral neuropathies. We employed a well defined diet restriction method namely lifelong CR, kn own to slow the aging process and activate chaperone and autophagy pathways in many organ systems (Heydari et al., 1993; Bergamini et al., 20 03; Selsby et al., 2005; Wohlgemuth et al., 2007) to determine its influence on the peripheral nerves (Chapter 4) (Fig. 6 1B [1,3]) Age associated reduction in the expressi on of the major myelin proteins, neurofilaments and widening of the nodes of Ranv ier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated SC phenotype (Fig. 6 1B [7,8,9]) Notably, t he improvement in nerve architecture with diet restriction
160 are underlined by sustained expression of pr otein chaperones and markers of the autophagy lysosomal pathway (Fig. 6 1B [1,3,9]) similar to the other studies (Heydari et al., 1996; Bergamini et al., 2003; Selsby et al., 2005; Wohlgemuth et al., 2007) in other organ systems as well In a n independent study from our lab, an intermittent fasting diet is able to activate chaperones and autophagy, decrease p Ub substrates improve myelination and alleviate neuropathic behavioural phenotype (Madorsky et al., 2009) Together, the in vitro and in vivo results in Chapter 4 suggest that there might be an age limit by which dietary intervention or a similarly effec tive pharmacological intervention needs to be initiated to elicit a beneficial response on peripheral nerve health, in the context of aging and disease (Fig. 6 1B) I n addition to the myelin and protein quality control pathway defects, normal a ging is asso ciated with oxidative damage in the PNS (Chapter 5) The age related alterations in cellular protein homeostatic mechanisms (Chapter 4) likely lead to a buildup of oxidatively modified damaged proteins with age, which is associated with the conglomeration of distended lysosomes and lipofuscin adducts These results underscore the close relationship with which these pathways function (Chapter 5) The occurrence of these detrimental structures is notably less frequent within nerves of agematched rodents kept on a life long reduced calorie diet likely by reducing the accumulation of damage buildup or by a sustained activation of protein quality control pathways Myelination is a highly energy demanding process that likely becomes less efficient due to oxidat ive damage. CMT1A is an age related disease and oxidative stress is known to take place with age in a variety of organ systems (Bokov et al., 2004) T here are currently no reports about oxidative damage in neuropathic mouse
161 models and human pati ents. Little is known about the involvement of oxidative stress with d isease progression in CMT1A. Although, the antioxidant ascorbic acid has been tried as a therapeutic intervention for CMT1A neuropathies (Pareyson et al., 2006; Verhamme et al., 2009b) We looked at the oxidative stress pathway with aging in the PNS, since a major effort in the development of therapeutics for CMT1A i n the past 5 years was turning towards administration of ascorbic acid, an antioxidant (Pareyson et al., 2006; Micallef et al., 2009) The results from Chapter 6 show that there is significant oxidative damage to axonal and glial proteins of sciatic nerves with age (Fig. 6 1A) Restricting diet by life long CR is able to alleviate the agerelated oxidative damage. Clinical studies with ascorbic acid that showed promise in rodent models did not yield positive results in human trials (Micallef et al., 2009) It is possible that a combined pharmacological treatment that can activate more than one quality control pathway and simultaneously alleviate oxidative damage to mimick diet restriction will be cumulatively beneficial for myelin i mprovement and associated defects for the disease in humans (Fig. 6 1B) In CMT1A disease, dedifferentiation of SCs, aberrant expression and reorga nization of axonal ion channels, pronounced damage to axonal cytoskeleton and transport as well as recruitment of macrophages have been reported (Kohl et al., ; Kohl et al., ; Maier et al., 2002; Misko et al., 2002; Devaux and Scherer, 2005; Martini et al., 2008) and similar changes have been documented with normal aging in this study (Chapter 4 and 5) (Fig. 6 1A [5,6,7]). Strikingly, food restriction is able to attenuate all of the above mentioned deleterious processes with age (Fig. 6 1B [8,9,10]). In the human disease as well as in CMT1A mouse models, macrophages have been shown to be the
162 primary i mmune cell type exacerbating the disease (Kohl et al., ; Hartung et al., 1992; Misko et al., 2002) They are likely activated and recruited due to the deg enerating myelin fragments. With CR, p revention of the agerelated deg eneration of myelin sheaths due to sustained chaperone autophagy and proteasomal pathways, as well as minimal oxidative damage, likely leads to decline in macrophage recruitment (Fig. 6 1B [1,3,6]) Since CMT1A disease has an immune component, RM which is an FDA approved immunosuppressant is expected to provide additional benefi ts for curtail ing the macrophage cell infiltration. Also, a recent study shows that RM extends lifespan of aged mice and this could offer further advantage in the treatment of neuro pathy without toxic side effects (Harrison et al., 2009) I n CMT1A, the early disease manifestations include muscle loss, weakening of extremities and reduction in nerve conduction velocity (Verhamme et al., 2009a) These disease characteristics are replicated by neuropathic mice as well (Huxley et al., 1996; Huxley et al., 1998; Notterpek and Tolwani, 1999) In order t o analyze the disease progression with aging i n humans a 5 year long study has recently been completed with adult CMT1A patients ( PMP22 gene duplication) and agematched controls (Verhamme et al., 2009a) The results of this study show that there is significant physical disability in CMT1A patients but not in controls over the 5year period, as expected (Fig. 6 1A ) However, decline in nerve conduction velocity and muscle strength is detected even in control subjects with normal aging (mean age, 40 years; median age, 2565 years; n=26) and is at a similar rate compared to patients (mean age, 41 years; median age, 1769 years; n=44) (Verhamme et al., 2009a) Hence, i t is possible that the reduction in nerve conduc tion velocity and muscle strength with normal
163 aging (Verhamme et al., 2009a) combined with the decline in functional glial and axonal proteins and the accumulation of damaged proteins with age seen in our study (Chapter s 4 and 5) together become additive for the progression of the neuropathy. Thus, the physical diability seen in CMT1A patients seems to be a combination of having a defective PMP22 gene and the events triggered by normal aging (Fig. 6 1A ) Studies from our collaborators (Marzetti et al., 2008; Xu et al., 2008) as well as our correlation studies (Chapter 4) indicate a significant decline in forelimb g rip strength with age which is a measure of muscle strength (Xu et al., 2008) This result in our rodent study with normal aging is comparable to the decline in muscle strength observed in the human study (Verhamme et al., 2009a) Significantly a CR diet that is able to minimize aberrant changes in quality control pathways (Chapters 4 and 5) correlates with maintenance of grip streng th (Fig. 6 1B ) in agematched rodents (Chapter 4; also see (Xu et al., 2008) ) Thus, there is evidence from humans as well as from rodent studies that normal aging contributes to the progressive nature of this disease to a considerable extent This could explain why the onset of the disease takes place after the individual s with the disease mutations have lived asymptomatic upto midlife. Together, these studies described in this dissertation support our hypothesis that, activating quality control pathways such as HSR and autophagy by pharmacological or dietary means is indeed beneficial for the processing of PMP22 and associated myelin proteins. Overall activating protein homeostatic pathways has the ability to improve myelination in the context of diseas e and aging (Fig. 6 1B) which shows great potential for therapeutic benefits for a neurodegenerative disease such as CMT1A. Although, it is hard to decipher the specific sequence of events through which CR thwarts agerelated
164 changes in quality control mechanisms, the results in Chapter 4 and 5 show that dietary restriction is an efficient means of defying agerelated oxidative damage and maintaining a younger state in peripheral nerves. As a matter of fact, it becomes a chicken o r the egg question of whether sustained activation of quality control pathways by CR prevents damaged protein accumulation or a decrease in accumulation of undesirably modified proteins with CR leads to better functioning of the proteostatic mechanisms. Either way, the end result of CR is the maintenance of myelinated phenotype and better nerve health. Since adverse dietary means such as intermittent feeding and calorie reduction might not be suitable therapeutic options in humans the pharmacological approaches described in Ch apt er s 2 and 3 are desirable.
165 Figure 61. Working model: Quality control mechanisms with disease and age in the PNS (A) In SCs from animals with disease mutation or normal aging , there is an accumulation of P MP22 aggregates (PMP22 agg) and pUb subst rates (pUb subst) , respectively. This is associated with an impairment of protein degradation by the proteasome  As a result, the trafficking of PMP22 as well as other myelin proteins to the plasma membrane is affected, leading to defects in myelination  With disease and aging myelination decreases and corresponding ly axonal degeneration , widening of nodes of Ranvier  and macrophage infiltration  take place. In normal aging, oxidative damage has a contributing role to these deleterious processes. Together, these events lead to physical disability with disease and age . (B) Exp osure of SCs to HSP90 inhibitor/RM/ CR promotes the sustained activation of the chaperone [1, 2] and autophagy  pathways The induction of these protein homeostatic pathways by pharmacological or dietary means prevent the aggregation of damaged pUb substrates  and subsequently promote the correct processing and trafficking of PMP22 , as well as other myelin proteins  Restoration of subcellular prote o stasis with CR diet improves myelin formation  along with preservation of axonal proteins including neurofil aments and channel proteins  and maintenance of nerve architechture and differentiated SC phenotype  These improvements are are associated with minimum oxidative damage in the normal aging process . These beneficial effects likely lead to a maintained physical strength .
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192 BIOGRAPHICAL SKETCH Sunitha Rangaraju was born i n the year 1984 in the Union Territory of Pondicherry a former French colony in India and was raised in the coastal city of Chennai in the southern part of I ndia. She is the eldest daughter of Mr. Venga Kandar Rangaraju who is a retired government officer and Mrs. Bala Sarassa Manicassamy who is a retired Professor of Botany. She has a younger sister, Vidhya Rangaraju and a younger brother, Vengada Karthik Rangaraju. After completing high school in 2001, she pursued her undergraduate studies at Anna University, Chennai In 2004, during her undergraduate studies, she gained research experience as a scholarship student under the guidance of Dr.K.S.Krishnan at the Tata Institute of Fundamental Research (TIFR), in Mumbai, the financial capital of India. In 2005, she carried out her undergraduate research thesis in the laboratory of Dr.Vinay Kumar Nandicoori at the National Institute of Immunology (NII), in New Delhi, the capital of India. She graduated with a Bachelors degree in Industrial Biotechnology in 2005. She came to the U S to join the Interdisciplinary Program in Biomedical Sciences at University of Florida in 2005 and a year later in 2006, became a member in the laboratory of Dr.Lucia Notterpek.