1 EFFECTS OF INTERMITTENT FASTING ON THE PHENOTYPE OF PERIPHERAL NEUROPATHIC MICE By AMANDA WABER NGUYEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007
2 Copyright 2007 Amanda Waber Nguyen
3 ACKNOWLEDGMENTS I would like to thank my mentor Dr. Notterp ek for assistance a nd guidance throughout my graduate studies. The time, suggestion and a ssistance of my committee members Dr. Dunn, Jr. and Dr. Foster were also great ly appreciated. I also acknow ledge Debbie Akins of the Dunn laboratory for her preparation of the EM thick secti ons of sciatic nerve, Asha Rani of the Foster laboratory for her assistance with initial grip stre ngth acquisition, and Iri na Madorsky of the Notterpek laboratory for her tec hnical expertise and assistance with the sciatic nerve western blots. Dr. Amici helped with g-ratio analys is, and gave immeasurab le amounts of emotional support and advice for which I am grateful. I would also like to thank Priel Schmalbach of the Notterpek lab for assistance with western blots, an e ffort which was very helpful at a crucial time. I would also like to thank all other past and present members of the Notterpek lab for their assistance and support during my time in the lab.
4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF FIGURES................................................................................................................ .........6 LIST OF ABBREVIATIONS..........................................................................................................7 ABSTRACT....................................................................................................................... ..............8 CHAPTER 1 INTRODUCTION................................................................................................................... ...10 Hereditary Peripheral Neuropathies............................................................................................10 CMT1A Treatment Studies........................................................................................................ .11 Genes Linked to CMT............................................................................................................ ....12 Myelination in the Peri pheral Nervous System..........................................................................13 Peripheral Myelin Protein 22................................................................................................... ...15 Trembler-J Mouse Model of CMT1A.........................................................................................17 Ubiquitin-Proteasome System....................................................................................................18 Protein Aggregation............................................................................................................ ........20 Autophagy...................................................................................................................... .............21 Molecular Chaperones........................................................................................................... .....24 Intermittent Fasting Regimen................................................................................................... ..27 2 MATERIALS AND METHODS................................................................................................31 Introduction................................................................................................................... ..............31 Subjects....................................................................................................................... ................31 Intermittent Fasting Regimen................................................................................................... ..31 Accelerated Rota-rod........................................................................................................... .......32 Grip Strength.................................................................................................................. .............32 Statistical Analysis........................................................................................................... ...........33 Morphometry.................................................................................................................... ..........33 Western Blot Analyses.......................................................................................................... ......34 3 RESULTS........................................................................................................................ ...........36 Body Weight Does Not Decrease Signi ficantly with IF Regimen in Mice................................36 Forelimb Grip Strength Is Not Effected by IF Regimen in TrJ or Wt Mice...............................36 IF Regimen Improves Hindlimb Grip Strength in TrJ Mice.......................................................37 Improvement in Complex Motor Function of TrJ Mice with IF Regimen.................................38 Increase in Myelin Thickness with IF Re gimen in Sciatic Nerves of TrJ Mice.........................39 Increase in Myelin Protein Levels with IF Regimen in Mice Sciatic Nerves.............................40 Molecular Chaperones Are Upregulated with IF Regimen in Mice Sciatic Nerves...................41
5 Autophagy Is Induced in TrJ Mice Sciatic Nerve with IF Regimen...........................................42 The Lysosomal Degradative Pathway Is I nduced with IF Regimen in Mice Sciatic Nerve.......................................................................................................................... .............43 4 DISCUSSION..................................................................................................................... ........54 LIST OF REFERENCES............................................................................................................. ..59 BIOGRAPHICAL SKETCH.........................................................................................................59
6 LIST OF FIGURES Figure page 3-1. The body weight of mice on IF regimen is not significantly different that of AL fed mice. ........................................................................................................................ .........45 3-2. Forelimb gripstrength in wt and TrJ mice is consistent throughout the study.....................46 3-3. Mouse hindlimb gripstrength is affected by the IF regimen. ............................................47 3-4. IF regimen improves the performan ce of TrJ mice on the acce lerated Rota-rod. ..............48 3-5. The g-ratio of the sciatic nerves of Tr J mice decreases with 5 months on IF regimen. .....49 3-6. Myelin protein levels are upregulated in the sciatic nerve of mice on IF regimen. .............50 3-7. Some cytosolic molecular chaperones are upregulated in mice sciatic nerve with IF regimen. ..................................................................................................................... ......51 3-8. Autophagy degradative pathway is upregul ated in TrJ mice sciatic nerve with IF regimen. ..................................................................................................................... ......52 3-9. The lysosomal degradative pathway is upreg ulated in the sciatic nerves of mice on the IF regimen, but the UPS degradative pathway is not effected. ........................................53
7 LIST OF ABBREVIATIONS ACTH Adrenocorticotropic hormone AL Ad libitum Atg Autophagy-related proteins BDNF Brain-derived ne ural growth factor C22 CMT1A mouse with 22 copies of human PMP22 gene CR Caloric restriction CMT Charcot-Marie-Tooth disease CNS Central nervous system DPV Diastolic blood pressure variability DSS and CH Dejerine-Sottas Syndrome and chronic hypomyelination ER Endoplasmic reticulum GAPDH Glyceraldehydes-3phosphate dehydrogenase Gas3 Growth arrest specific gene 3 HD Huntingtonâ€™s disease HNPP Hereditary neuropathy [with] pressure palsies HRP Horseradish peroxidase HSF-1 Heat shock factor-1 Hsp Heat shock protein IF Intermittent fasting LAMP Lysosomal associated membrane protein LC3 Microtuble associated protein 1 light chain 3 MAG myelin-associated glycoprotein MBP Myelin basic protein MPZ(P0) Myelin protein zero PMP22 Peripheral myelin protein 22 PNS Peripheral nervous system SC Schwann cell SPF Specific pathogen-free conditions TrJ Trembler-J mouse UPS Ubiquitin-Proteasome system wt Wildtype
8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF INTERMITTENT FASTING ON THE PHENOTYPE OF PERIPHERAL NEUROPATHIC MICE By Amanda Waber Nguyen May 2007 Chair: Lucia Notterpek Major: Medical Sciences--Neuroscience Hereditary neuropathies are a prevalent fam ily of genetic diseas es in the peripheral nervous system, affecting approximately 1:2500 pe ople. The demyelinating neuropathy CharcotMarie-Tooth disease type 1A is the most co mmon form. Currently there are no effective treatments for patients with this disease. In previous studies, the intermittent fasting regimen (IF) (alternating feed/f ast days) has been neuropro tective in the central ne rvous system in animal models of multiple neurodegenerative disorders and neural injury. Levels of chaperones (heat shock response) and neurotrophins increase with IF. Male, 8 week old wildtype mice and heterozygous Trembler J littermates, which are CMT1A models with a point mutation in the peripheral myelin protein 22 gene, were fed either ad libidum (AL) or by IF during the 5 month study. Mice on IF lost less than 10% body weight compared to AL fed. Trembler J mice motor function improved with IF in hindlimb grip strength after 5 months, and complex motor performance on the Rota-rod after 4 months. Myelin thickness of peripheral nerves increased in Trembler J mice on IF. Increased levels of cytosolic chaperones and stimulation of the autophagic and lysosomal degradative pathways observed through wester n blot are possible mechanisms for the partial amelioration of the Trember J neuropathy. The promising results of
9 this study warrant further investigation to even tually pursue pharmacol ogical approaches to increase these pathways as possi ble treatment for CMT1A patients.
10 CHAPTER 1 INTRODUCTION Hereditary Peripheral Neuropathies The most common family of genetic diseases in the peripheral nervous system (PNS) is hereditary neuropathies. A large degree of pheno typic variability is evident, and hereditary peripheral neuropathies are linked to mutations in a variety of genes (Vigo et al., 2004). Currently there are no effective treatments for patients. The two forms are demyelinating, type 1, and a primary axonal degeneration peripheral ne uropathy, type 2. The three main classes of demyelinating hereditary peripheral neuropathy are: Charcot-Marie-Tooth Disease (CMT), Dejerine-Sottas Syndrome with Chronic Hypom yelination (DSS and CH), and Hereditary Neuropathy [with] Pressure Palsie s (HNPP). The disease DSS and CH has onset in infancy or early adolescence with delayed motor milestones and severe progressive loss of neuromuscular function. Furthermore, HNPP has onset in the second or third decade of life, with mild peripheral neuropathy. The most prevalent form of hereditary periphe ral neuropathy, CMT, was first identified in 1886 separately by the Charcot and Marie group and Tooth, and affects approximately 1:2500 people (Skre, 1974; reviewed in Young and Suter, 2003). The CMT type 1 is a demyelinating peripheral neuropathy and CMT type 2 is caused by a loss of myelinated axons ( Dyck et al., 1993; reviewed in Young and Suter, 2003) . The most common form is CMT subtype 1A (CMT1A), afflicting approximately 50% of patie nts. Although a large degree of phenotypic variability is evident even in patients with the same mu tation (Marques et al., 1999), CMT symptoms typically begin in the first or second decade of life. The disease progression begins with distal muscle weakness and atrophy in the legs and feet, and later progresses to the arms and hands causing foot (pes cavus and claw toes ) and hand deformities (Dyck and Lambert, 1968 ;
11 reviewed in Dyck et al 1993; reviewed in Young and Suter, 2003) . These impairments are due in CMT1 to demyelination in the PNS resulting in a reduction of nerve conduction velocity (Dyck and Lambert, 1968, reviewed in Dyck et al., 1993; reviewed in Young and Suter, 2003) . The focus of this study will be CMT1A. CMT1A Treatment Studies The current treatment of patients with CMT1A aims at ameliorating symptoms with physical therapy, surgery, and pain management, and does not slow or stop disease progression. Therapy addressing the root cause of the dis ease, demyelination, needs to be developed. Resistance training exercise improves muscle strength and activity levels of CMT patients (Chetlin et al., 2004). However, recent studies in CMT1A animal models have had promising results. Addition of L-ascorbic acid is necessa ry to promote Schwann cell (SC) differentiation, and basal lamina and myelin forma tion in co-cultures with dorsal root ganglion cells (Eldridge et al., 1987). The C22 mice are models of CMT1 A which trangenically overexpress peripheral myelin protein 22 (PMP22) (Huxley et al., 1996). A study found that administering ascorbic acid to male C22 mice improved motor performan ce and increased the number and thickness of myelinated axons in the periphera l nerves (Passage et al., 2004). This dramatic improvement is a possible result of ascorbic ac id inhibiting the stimulation of cAMP for PMP22 expression, reducing the amount of PMP22 expressed. Ascorb ic acid controls the expression of certain genes directly, in addition to having general antioxidant proper ties, the former eliciting the improved phenotype of the C22 (Passage et al., 2 004). Currently a phase III clinical trial is underway to determine the effects of long-term treatment of ascorbic acid in CMT1A patients (Pareyson et al., 2006). In another study, male , transgenic PMP22 overexpression rats were given a progesterone antagonist. Treatment pa rtially corrected the neuropathic phenotype, decreased PMP22 overexpression, and increased the number of myelinated axons in the
12 peripheral nerves (Serada et al., 2003). Progesterone is an epigenetic regulator of the expression of the myelin genes PMP22 and myelin protein zer o (MPZ) in the SC (Melcangi et al., 1999). In TrJ mice and nude mice xenographed with nerves from CMT1A patients, motor performance improved after treatment with neurotrophin-3 by improving axonal regeneration (Sahenk et al., 2005). Neurotrophin-3 is an important component of the SC autocrine survival. The need for effective treatment for CMT1A is still evident. Genes Linked to CMT Mutations in myelin proteins, such as MPZ or PMP22 are linked to CMT1 (Reilly, 1998; Marques et al., 1999; Young and Sute r, 2003), (for a complete updated list of proteins related to CMT see http://molgen-www.uia.acbe/CMTMutations/ ). Mutations in the MPZ gene have been associated with CMT1B, as well as DSS and CH (reviewed in Shy, 2006). The most common gene linked to CMT1A is the myelin protein PM P22 (reviewed in Young and Suter, 2003). The missexpression of the PMP22 protein, through point muta tion or duplication, is associated to demyelinating hereditary peripheral neuropathy of all three types (Naef and Suter, 1998; Suter and Patel, 1994). Duplications in a 1.5 mega base section of human chromosome 17 region p11.2-p12 that includes the locus for the PMP22 gene are most prevalent in CMT1A (Lupski et al., 1991; Roa et al., 1993). However, point mutati ons in the PMP22 gene are also linked to the disease (Lupski et al., 1991; Ro a et al., 1993). The deleterious effects of missexpression of PMP22 are due to a toxic gain of function (Tobler et al., 1999). Proper myelination is necessary for the conduction of nerve signal along a xons, promoting efficient saltatory impulse propagation. These mutations impair myelin formation or result in demyelination of PNS nerves. In CMT1A myelin does not form properly around large caliber motor axons, or is degraded overtime in a process of demyelinati on. Some hypermyelination is also a feature of
13 CMT1A, which is caused by the SCs over wra pping around axons (onion-bulb formations) and excessively proliferating (Thomas et al., 1996; reviewed in Young and Suter, 2003). Myelination in the Peripheral Nervous System Myelin is essential for efficient signal trans duction along the axon. The lipid-rich myelin sheath insulates the axon with intermittent secti ons of unmyelinated axon denoted as nodes of Ranvier. This allows the depolarization to seem ingly leap from node to node, promoting faster signal transmission: â€œsaltato ry conductionâ€ (reviewed in Garbay et al., 2000). During development a SC initially comes in contact with a bundle of unmyelinated PNS axons, and this contact triggers the cell to proliferate (Bunge and Wood, 1975 ; reviewed in Garbay et al., 2000). The SC then undergoes lateral elongation, and esta blishes a 1:1 ratio with a PNS axon (reviewed in Garbay et al., 2000). The SCs depend on a xonal signals for differentiation and survival (Lobsiger et al., 2002; reviewed in Maier et al., 2002). The wrappi ng of the basal lamina of a SC around the peripheral axons is the first stage of myelin formation (reviewed in Bunge et al., 1993). This process is closely regulated by axon signals, and then the SC through neurotrophic factors . Levels of the neurotrophic factors neurotr ophin-3 and brain-derive d neurotrophic factor (BDNF) are high as the SCs proliferate. Then neurotrophin-3 levels decrease, since this neurotrophic factor inhibits ac tive myelination (Chan et al., 2001). The neurotrophin BDNF is needed for active myelination by th e SCs (Chan et al., 2001). Als o, BNDF inhibits the migration of SCs (Yamauchi et al., 2004). Then TrkC inhi bits SC gene activation. The decrease in neurotrophin-3 and the binding of BDNF to p75NTR receptor prompts the SC to elongate and wrap around the axons. The p75NTR receptor on SCs binds BNDF a nd enhances the myelination process (Chan et al., 2001; Cosgaya et al., 2002), while inhibiting the SCs ability to migrate (Yamauchi et al., 2004).
14 The p75NTR receptors are therefore n ecessary for proper myelin formation. Increase in p75NTR receptors is evident in development, and nerve injury, during demyelination and remyelination (Cosgaya et al., 2002 ). Myelinating SCs affect th e arrangement and clustering of sodium channels in the axon, forming nodes of Ra nvier (reviewed in Martini, 2001; reviewed in Maier et al., 2002). After formation of the matu re myelin internodes, all of the neurotrophic factors are down regulated, with assistance of TrkB-T1 which removes excess BDNF (Cosgaya et al., 2002). Compact myelin forms when the cytoplasm is excluded from the SC forming the layers around the axon, until only 2nm of space remains (Bunge et al., 1989; reviewed in Garbay et al., 2000). Myelin, compact and uncompact, is approxi mately 70% lipids, with high concentrations of galactosphingolipids and saturated fatty acids (reviewed in Garbay et al., 2000). The protein composition of compact myelin of the PNS incl udes MPZ, Myelin Basic Protein (MBP) as well as PMP22. All are integral membrane proteins except MBP (reviewed in Garbay et al., 2000). MPZ is a member of the immunoglobulin superf amily, and the primary protein in peripheral myelin, composing 50% of all prot ein. This targets to regions of compact myelin with PMP22 and MBP, and is needed for proper structure a nd function. Axonal signal s induce the expression of MPZ, and all myelin proteins, in myelina ting SCs after the developing SC achieves a 1:1 relationship with the PNS nerve axon. The protei n MBP is essential in the adhesion of central nervous system (CNS) myelin, bu t not in the PNS (Sm ith-Slatas and Barbarese, 2000). In both the CNS and PNS, the protein is found in compact myelin (Omlin et al., 1982; reviewed in Smith-Slatas and Barbarese, 2000). In the PNS, MBP has four prominent peptide isoforms evident on western blot-14kDa, 17kDa, 18.5kDa, and 21.5kDa, and can be phosphorylated and methylated (reviewed in Garbay et al., 2000). Furthermore, MB P regulates posttranscriptionally
15 the number of Schmidt-Lanterman Incisures (SL I) in the PNS (Smith-Slatas and Barbarese, 2000). The SLI are narrow channels which connect the layers of compact myelin to the SC body, and have gap junctions and al so adherens juctions (reviewed in Garbay et al., 2000). The uncompact myelin of the PNS is contai ns E-cadherin, myelin-associated glycoprotein (MAG), and the gap junction protein connexin32 (Sch erer et al., 1996; review ed in Garbay et al., 2000). The internodal SLI incisures and paranodal regions have uncompacted myelin (Scherer et al., 1996). The SLI contain some organelles and gap junctions, which are composed of connexin32 proteins, to link the layers of myelin ( Balice-Gordon et al., 1998; reviewed in Paul, 1995 ; reviewed in Smith-Slatas and Barbarese, 2000). E-cadherin is the major adhesive glycoprotein of the SC (Fannon et al., 1995). Th e adhesive transmembrane glycoprotein MAG is concentrated to regions of uncompact myelin at paranodes in SLI (Tra pp et al., 1984, 1989). Ion channels including the delayed rectifying K(+) channels Kv1 .1 and Kv1.2 and Caspr are the juxtaparanodal region, and this positioning is determined by the myelinating SC. Thus the myelin sheath organizes the underlying axona l membrane (Arroyo et al., 1999). The thick, highly lipid-rich layers of mye lin sheath surrounding the axon act as an electrical insulator, assisting the rapid conduction of the signal along the axon (rev iewed in Garbay et al., 2000). Peripheral Myelin Protein 22 The 22 kDa PMP22 is a tetraspan, intermem braneous (Manfioletti et al., 1990), highly hydrophobic glycoprotein ( Pareek et al., 1993). PNGaseF re moves all the car bohydrate residues, leaving the core PMP22 peptide of 18 kDa indica ting it is a glycoprotein (Snipes et al., 1993). On the structural level, the first transmem brane domain of PMP22 was found to be an helix (Deber et al., 1992). A point mutation of the leuc ine 16 residue to a proline in PMP22 results in a dramatic shift to a -sheet conformation in the first transmembrane domain, due to the
16 destabilization effects of the proline on the helix (Yamada et al., 2003). This L16P point mutation was seen first in the CMT1A mouse mode l Trembler-J (TrJ) (Suter et al., 1992) and then in patients with CMT1A (Valentijn et al., 1993; Ionasescu et al., 1997). The PMP22 protein is involved in forming and maintaining peripheral myelin and is expressed in high levels in compact myelin (Sni pes et al., 1992; Adlkofer et al., 1995; Robertson et al., 1997). Since PMP22 is localized at cell-cell junc tions, the protein may also be involved in process of myelin wrapping of the axons, and ad hesion (Notterpek et al ., 2001; Roux et al., 2004, 2005). The PMP22 protein binds the 6 4 integrin complex, which is important in the SC and basal lamina interactions, and lack of PMP22 re sults in peripheral neuropathy (Amici et al., 2006). PMP22, additionally identifi ed as Growth Arrest Specific Gene 3 (Gas3) (Manfioletti et al., 1990), additionally modulates th e ability of cells to spread. The N-glycosylation of PMP22 is necessary to modify cell spreading, and the cell surface expression of the protein is essential for proper function in signaling activi ties (Brancolini et al., 2000). Point mutations in the transmembrane domain of PMP22 result in intracellular retention of the protein (Brancolini et al., 2000). PMP22 has a high turnover ra te in the cell, with the degradation of approximately 80 % of the nascent protein by th e Ubiquitin-Proteasome system (UPS), possibly resulting from the misfolding of the highly hy drophobic protein. While the other 20% of PMP22 folds successfully and translocates to the cell membrane (Pareek et al., 1997). The UPS is the primary method of degradation of proteins, primarily cytosolic proteins, with a high turnover rate in the cell (Groll and Clausen, 2003). The m echanism of targeting PMP22 to the proteasome is yet to be ascertained; however there are three possible ubiquitination sites.
17 Trembler-J Mouse Model of CMT1A There are a number of mous e models of CMT1A, including models with overexpression of the PMP22 protein and point mutations in PMP22. The Trembler-J (TrJ) mouse was discovered as a spontaneously occurring mutati on in the Jackson Laboratories (Henry and Sidman, 1983). The peripheral neur opathic phenotype of TrJ mice is linked to a point mutation in the PMP22 gene (Suter et al., 1992). Th e heterozygous TrJ/+ have a trembling gait and progressive hindlimb muscle weakness and at rophy. Homozygous TrJ have a very severe phenotype modeling DSS and CH. Upon examination of the peripheral ne rves of the TrJ mice severe demyelination and reduced axon diamet er was evident. Additionally, TrJ have hyperproliferating SCs with excessive basal lamina , due to the alterations in myelination, and extracellular matrix and impr oper myelination including onionâ€“bul b formations (Henry et al., 1983). Abnormal interactions of the SC with the nerve axon are also seen in TrJ/+ (Robertson et al., 1997). The nerve conduction veloci ties in TrJ/+ peripheral nerves are reduced in a similar in magnitude to patients with the same point mutati on (Meekins et al., 2004). Characteristics of the TrJ/+ (hereto referred to as Tr J) closely model the human progression of CMT1A. In the TrJ mice, PNS myelin formation is seen during deve lopment, although not as prolifically as in wildtype. Then the peripheral myelin is progressi vely lost over time in TrJ (Henry et al., 1983; Notterpek et al., 1997). The reduction in myelin in TrJ sciatic nerves correl ates to reduced levels of the myelin proteins MBP, MPZ, a nd PMP22 (Notterpek et al., 1997). The peripheral nerves of TrJ mice have an upregulation of the lysosomal degradative pathway (Notterpek et al., 1997). There is an increase in the chap erone levels of PNS nerves in TrJ, including hsc70 (Fortun et al., 2003). Molecu lar chaperones assist in proper protein folding, and increase when the cell is under stress (M osser and Morimoto, 2004). Unlike wt-PMP22 in the SC membrane, TrJ-PMP22 is endo-H sensitive, indicating it accumulates in the intermediate
18 compartment before entering the trans-Golgi network (Tobler et al., 1999). Endo-H sensitivity indicates that the protein has ye t to reach the medialand tran sGolgi. Endo-H cleaves only high mannose sugar moieties which have yet to be cleaved by mannosidase II (Snipes et al., 1993). The TrJ-PMP22 forms heterodimers w ith wt-PMP22, impeding some wt-PMP22 from reaching the SC membrane as well (Tobler et al., 1999). Normally PMP22 has a short half life in the cell of approximately 45 min (Pareek et al., 1997), while TrJ-PMP22 has a half life of approximately 4 hrs (Fortun et al., 2003). A simila r increase in PMP22 half life occurs in SCs of C22 mice (Fortun et al., 2006). The 8-fold slower turnover rate of TrJ-PMP22 correlates with reduction of proteasomal activity (Fortun et al., 2005). In CMT1A a portion of PMP22 is insoluble, endo-H sensitive, and has a high mol ecular weight, so potentially is contained in aggregates (Notterpek et al., 1999). The Tr J mice peripheral neur opathic phenotype and underlying subcellular al terations result in its effectiv eness as a model for CMT1A. Ubiquitin-Proteasome System The ubiquitin-proteasome system (UPS) is a hi ghly regulated proce ss for degradation of cellular proteins. Degradation mediated by U PS commonly begins with the tagging of the protein with a chain of covalently attached ubi quitin molecules. This signals the 26S proteasome to degrade the protein. The ubiquitin molecules are released and r ecycled in the cell. The 26S proteasome complex is composed of a barrelsh aped 20S catalytic core and capped by two 19S regulatory particles reviewed in Ciechanover and Brundin, 2003). Ubiquitin-independent targeting for UPS is also possibl e by the 26S proteasome complex in special cases, as with the rapidly turned over mammalian enzyme ornithine decarboxylase (Murakami et al., 1992). Proteasome enzyme activity generally declines with age, so cells are less able to clear mutated proteins (Zhou et al., 2003).
19 In neurodegenerative diseases proteasomal inhibition may result in the accumulation of inclusion bodies in the cell. The overproduction of misfolded proteins overwhelms the UPS, and in some cases can possibly bind and inhibit no rmal proteasomal function as in Huntingtonâ€™s Disease Sakata et al., 2003; reviewed in Ciechanove r and Brundin, 2003). There are potentially more complex interrelationships between concen trations of proteins in aggresomes, the equilibrium between the synthesis, folding and degr adation of proteins, and the efficiency of the UPS. It has been shown that pharmacological in hibition of the proteasome leads to an increase in the formation of inclusion bodies in different cellular models. The long-term inhibition of the UPS in neurons is detrimental to normal cellular functioning reviewed in Ciechanover and Brundin, 2003). The misfunction of this system is implicated as a direct or secondary pathomechanism in a number of neurodegenera tive diseases. A rela tionship between UPS dysfunction and the formation of inclusion bodi es that include ubiqui tinated proteins and proteolytic components has been seen in a variet y of neurodegenerative disorders, but only as a secondary mechanism. Inclusion bodies are cytoso lic aggregated proteins within the cell. The chymotrypsin-like activity of the proteasome d ecreases by approximately 60% in TrJ sciatic nerves correlating to an increase in ubiquitin-positive prot ein aggregates (Fort un et al., 2005). In C22 sciatic nerves impairment of proteasomal degradative function and an upregulation of the lysosomal degradative pathway, with recruitmen t of proteasomal subunits to aggregates, are observed (Fortun et al., 2006). The extended pr esence of the highly hyd rophobic protein in the cytosol of the SCs in TrJ mice may contribute partly to th e formation of PMP22-positive cytosolic aggregates in TrJ nerves, as well as the impairment of the proteasomal function.
20 Protein Aggregation Formation of aggresomes, which are membra ne-free structures, occurs when small aggregates of proteins travel by retrograde transport along micr otubules to a perinuclear region (Kopito et al., 2000). This process necessitates the redistribution of the cytoskeletal protein vimentin, and intact microtubles (Johnston et al., 1998; Notterpek et al., 1999; Kopito et al., 2000). The presence of protein aggregates is not unique to CMT1A, aggregates are also observed in a number of other neurodegenerative diseases including Parkinsonâ€™s disease (Sakata et al., 2003), Alzheimerâ€™s disease, Amyo-Latera l Sclerosis, Prion disease, and Huntingtonâ€™s disease (reviewed in Sherman and Goldberg, 2001; reviewed in Ci echanover and Brundin, 2003). Aggresomes form of the PMP22 protein in CMT1A (Notterpek et al., 1999; Ryan et al 2002), the huntingtin protein in Huntingtonâ€™s disease (Waelter et al., 2001), and parkin and synuclein proteins in Alzheimerâ€™s disease (Junn et al., 2002). Initially small accumulations of electron dens e, non-laminar material were observed in some of the TrJ SCs (Henry et al., 1983). The significance of these findings was later determined as the transmembrane protein PMP22 accumulated in ubiquitinpositive, cytosolic, perinuclear aggregates in TrJ (R yan et al., 2002) and C22 sciatic nerves (Fortun et al., 2005). Also, in nerve biopsies of patients with CM T1A an accumulation of cytosolic PMP22 was observed (Nishimura et al., 1996) . The missexpression of PMP22 through overexpression or mutation results in the formation of PMP22 positi ve aggregates and aggresomes (Notterpek et al., 1999). Additional proteins re cruit to these ubiquitin-PMP22 positive aggresomes including MBP, proteasomal subunits, and ch aperones (Fortun et al., 2006). Pharmacological inhibition of the proteasome also causes aggregate formation, increasing the percentage of insoluble endo-H sensitive PMP22. . A higher number of perinuclear aggregates (aggresomes) form with mutated
21 PMP22 than wt-PMP22 (Ryan et al., 2002). Therefore a relationship between proteasome function and aggregate formation is evident Autophagy Autophagy is one mechanism for the degradatio n of aggregated and missfolded proteins. The word â€œautophagyâ€ derives from the Greek to eat (â€œphagyâ€) oneself (â€œautoâ€), describing the role of the pathway for differentiation and pr otein degradation. There are two types of autophagy in eukaryotic cells: microautopha gy and macroautophagy. Mi croautophagy occurs when the lysosome directly engulfs a component of the cytoplasm through the invagination of the lysosomal membrane (reviewed in Yang et al., 2005). Macroautophagy is more relevant for aggregate clearance and protein removal. Mo lecular characterizati on of autophagy was done primarily in nutrient-deprived yeast models, with proteins being identified as Autophagy-related proteins (Atg) which have high levels of e volutionary conservation in higher eukaryotes (reviewed in Yang et al., 2005). Autophagy is th e main pathway for targeting proteins for lysosomal degradation (Dunn et al., 1990). In the induction of macroautophagy (which w ill be furthermore be referred to as autophagy) an isolation membrane forms that surrounds the portion of pr otein aggregate, or region of cytosol, with Atg1 and Atg13 being part of the initiation complex. It is postulated that initiation of this isolation me mbrane, or preautophagosomal structure, is budded from the endoplasmic reticulum (ER), although the mechanism is not well understood. Next, a double membrane vesicular structure called the autoph agosome forms with the sequestering of the protein/cytosolic component by th e isolation membrane, in a pro cess called vesicular nucleation. This double-membrane structure is a morphological hallmark of autophagy activity (Dunn et al., 1990). Phosphatidylinositol (PtdIn s) 3-kinase complex I is im portant in the beginning of vesicular nucleation, and the pr otein Atg8, which has higher euka ryotic orthologs, undergoes an
22 enzyme modification before modifying PtdIns-k inase. The E1-like activating enzyme Atg7 (autolog of gsa7) conjugates to Atg8 to form the autophagosomal membrane. Another component of this complex necessary for autopha gy is the protein beclin -1 (Atg6 in yeast) (Kihara et al., 2001) which also has interactions with Bcl-2 family antiapoptotic proteins in higher eukaryotes ( Aita et al., 1999 ; Liang et al., 1999; Liang et al., 2001). Cells with lower beclin-1 expression have a reduction in autophagy (Qu et al., 2003). The actual engulfment of the cytosolic compone nt by the isolation membrane necessitates that the membrane be deformed, and a transi ent coat by Atg12-Atg5 complex formed. The enzyme activity of Atg7 is needed for the formation of the Atg12-Atg5 complex. The Atg5dependent conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II (LC3phosphatidylethanolamine (LC3-PE) conjugate), is currently the major marker for autophagy activation (Ohsumi et al., 2001). This autophagosome then fuses with a lysosome, forming the autophagolysosome. The lysosomal-associated membrane protein (LAMP) is a marker for lysosomes, and composes approximately 50% of all lysosomal membrane proteins (Kornfield and Mellman, 1989; reviewed in Eskelinen, 2006). If both LAMP-1 and LAMP-2 glycoproteins are knocked out, lysosomal function is disrupted (reviewed in Eskelinen, 2006). The lysosomal proteolytic enzymes within the lumen of th e lysosome can degrade the prot ein components for recycling in the cell, and also ridding the ce ll of the misfolded or aggregated protein, or other cytosolic inclusion. Extracellular stresses, such as st arvation and high temperat ure, and intracellular stresses, like the accumulation of misfolded protei ns, can activate autophagy (reviewed in Levine and Klionsky, 2004). Autophagy and chaperone -mediated autophagy, in which molecular
23 chaperones target proteins for degradation, decline with aging, seen in rat liver cells (Cuervo and Dice, 2000; Ward et al., 2002). Transgenic mouse models have further elucid ated the importance of autophagy, especially in the nervous system. When the Atg7 gene is knocked out in mice, inhibiting autophagy, starvation-induced protein degrad ation is suppressed, and the tu rnover of organelles and longlived proteins decreases, in turn accumulating in cells of these mice (Komastu et al., 2005). Atg7 knock out mice have ubiquitin-positive protein a ggregates even without proteasomal inhibition, indicating the importance of aut ophagy as well as the UPS for ba sal level protein degradation (Komastu et al., 2005). A conditional knock out m ouse with deficiency of Atg7 activity only in the CNS, was developed by crossing with a Cre mous e with a nestin promoter resulted in severe neurodegeneration (Komastu et al., 2006). The la ck of CNS autophagy activity results in marked motor and behavioral impairments, and neuronal ce ll death, markedly in the cerebral cortex and amygdala. Also, ubiquitin-positive aggregates in the neurons of the amygdala and hippocampus increase without any proteasomal impairment. Th ese neurons are consider ed sensitive to such aggregates, seen in multiple neurodegenerative disorders such as Alzheimerâ€™s disease (Komastu et al., 2006). Ubiquitin-positive aggregates are al so noted in myelinated axons, but the effect on myelin thickness or composition is not investigat ed. Therefore, autophagy deficiency alone can result in neurodegeneration (Komatsu et al., 2006) . Inhibition of basal level autophagy through the conditional knock out of the Atg5 gene in the CNS resulted in formation of inclusion bodies in the cytoplasm of neurons which increas ed in number as the mice aged. The neuropathogenesis in the At g5 CNS knock-out mice may also be due to the presence of additional, diffuse mutated proteins in the cyto sol (Hara et al., 2006). The presence of the current primary marker for autophagy, the enhanced conversion of LC3 I to LC3II, is not usually
24 evident even with nutrient starva tion in the CNS (Wang et al., 2006). It is possible that the CNS has different autophagy regulation due to MAP1B inhibition of LC3-positive autophagosomes. In mice with CNS axonal dystrophy and degeneration the reduction of the mTor kinase pathway phosphorylation indicates activ ation of autophagy (Wang et al., 2006). Recently it was determined that the inhibiti on of autophagy increased levels of p62/SQSTM1 protein, which binds ubiquitin and LC3, and was recruited to autophagosomes selectively for degradation (Bjorkoy et al., 2005). Based on this finding, the lack of detectable levels of p62/SQSTM1 in the degenerating axon terminals of the Purkinje ne urons of the mouse model further indicated activation of autophagy (Wang et al., 2006). Proteasomal inhibition transien tly induces aggresome formati on in SCs in culture. These aggresomes can be subsequently cleared by th e autophagic-lysosomal degradative pathway. Formation of ubiquitin-positive aggregates in the cell induces the autophagic-lysosomal degradative pathway (Fortun et al ., 2006). There is an upregulation of autophagic markers which colocalize with lysosomal markers. In the sc iatic nerves of one year old TrJ mice active autophagy is evident (Fortun et al., 2003). In C22 SCs activation of autophagy is also seen, marked by colocalization of Atg7 with PMP22-pos itive aggregates (Fortun et al., 2006). Treatment with rapamycin induced autophagy and thereby enhanced the clearance of insoluble polyglutamine inclusion bodies and aggregates in a Huntingtonâ€™s disease model (Webb et al., 2003; Qin et al., 2003). Autophagy is crucial in degrading aggregat ed proteins, and controlled upregulation of this degradative pathway can have therapeutic applications for diseases which have aggregated proteins. Molecular Chaperones Molecular chaperones assist in the proper fold ing and translocation of nascent proteins, as well as binding and targeting of irretrievably misfolded prot eins for degradation in the
25 proteasome. The chaperone response is also pa ramount in the response of the cell to stress (reviewed in Mosser and Morimoto, 2004). H eat Shock Proteins (Hsps) are molecular chaperones that bind to partially unfolded or highly hydrophobic stretches of amino acids in proteins preventing aggregation and guiding correct folding. Nas cent proteins proceed from the ribosome and initially interact with Hsp70 (revi ewed in Sherman and Goldberg, 2001). The activity of the inducible Hsp70 is assisted by co-chaperones, such as Hsp40. Co-chaperones bind Hsp70 at its c-terminus activating the ATPase act ivity, increasing the rate of protein-chaperone interactions. The chaperone Hsp90 binds specia lized client proteins, recognizing non-native state proteins, and holds these prot eins in a â€œfolding-competentâ€ st ate, even at high temperatures up to 42oC. The Hsp70 chaperones then bind and refold the proteins in the cytosol in correct conformation (reviewed in Freeman and Morimot o, 1996). The activity of small Hsps, such as Hsp27, minimizes damage resulting in misfolded pr oteins due to cellular stresses such as an increase in heat (reviewed in Mosser and Mori moto, 2004). A secondary function of Hsp70 is rescuing and refolding proteins al ready misfolded in th e cytosol, if possible, or targeting the proteins for degradation. Since cellular stress increases the number of cytosolic chaperones present in the cell, there is some cytoprotective function in these cells to new stressors (reviewed in Mosser and Morimoto, 2004). The importance of Hsps for proper PNS myelin ation is evident since mutations in some small molecular chaperones, such as Hsp27 and Hs p22, are linked to patients with CMT2 disease (Evgrafov et al., 2004; Tang et al., 2005; Kijima et al., 2005) . Normally Hsp27 is involved in the organization of the neurofilament network. Ther efore, mutations in Hsp27 cause dysfunction of the cytoskeleton of the a xon and transport along the axon (Evgrafov et al., 2004). The small chaperones Hsp27 and Bcrystallin inhibit caspase activity, which is associated with cellular
26 apoptosis and cell death (Kamradt et al., 2002). Increasing leve ls of Hsp27 increased sensory and motor neuron survival after injury (reviewed in Mosser a nd Morimoto, 2004). The lectin molecular chaperone calnexin binds to a single oligosaccharide residu e N42 on PMP22 directly and is important for quality control of the hi ghly hydrophobic protein. It is the only known ER molecular chaperone to direct bind PMP22. The association of TrJ-PMP22 with the ER chaperone calnexin is extended compar ed to wt-PMP22 (Dickson et al., 2002). Some molecular chaperones ar e recruited to aggresomes (Garcia-Mata et al., 1999). Proteasomal inhibition results in a large increase in th e amount of soluble and insoluble forms of the molecular chaperones Hsp40 and Hsp70, with Hsp70 localized around aggregates (Ryan et al., 2002). The chaperone Hsc70 is recruited to PMP22-postive a ggregates (Fortun et al., 2003). The cytosolic molecular chaperone Bcrystallin is upregulated and localized to aggresomes of PMP22 (Ryan et al., 2002). The presence of aggr esomes in the cell initiates an increase in chaperones, which then bind to th e aggregated proteins to attemp t to refold the proteins if possible, or target the prot eins for degradation. Induction of chaperones through pharmacological or other mechanisms is important for a variety of disease, including those with muta ted proteins which form aggregates. Inducing hyperthermia causes a general increase of Hsps. Adrenergic agents like amphetamine increase Hsps through inducing hyperthermia (re viewed in Tolson a nd Roberts, 2005). Bimoclomol increases Hsps by enhancing the activation of he at shock factor-1 (hsf -1) (reviewed in Tolson and Roberts, 2005). Other pharmacological treatments, like anti-inflammatory agents and salicylates increase the DNA binding properties of hsf-1 (reviewed in Tolson and Roberts, 2005). Inhibition of Hsp90 activity enhances Hsp expres sion since Hsp/hsf-1 complexes decrease and hsf-1 has a decreased rate of bi nding. Examples of Hsp90 inhibito rs are geldamycin, and the less
27 toxic derivative 17-AAG (17-ally lamino-17-demethoxygeldamycin) (Z ou et al., 1998; Ali et al., 1998). Besides pharmacological chaper one induction, chronic exercise can increase chaperone levels in some tissues (reviewed in Tolson and Roberts, 2005). Cold stress increased Hsp70 in brown adipose tissue, and transiently in the he art of mice (reviewed in Tolson and Roberts, 2005). Psychological stress increased Hsp27 and Hs p70 mRNA levels in the cerebral cortex and stomach of mice (Fukudo et al., 1997; reviewed in Tolson and Roberts, 2005). In another psychological stress paradigm, mi ce had increases in Hsp70 in adrenal and vascular tissues (Blake et al., 1995; reviewed in Tolson and R oberts, 2005). Internal and external stresses increase chaperone levels. Additionally, dietary modification through caloric restriction (CR) or intermittent fasting (IF) has shown to increase chap erone levels in some tissues (Yu and Mattson, 1999; Colotti et al., 2005; review ed in Mattson and Wan, 2005). Intermittent Fasting Regimen The CR and IF regimens have cytoprotective effects in a number of tissues, including the CNS. Both regimens increase rodent lifespa n (reviewed in Mattson and Wan, 2005) and the CR regimen increased the lifespan of non-human prim ates in a 17 years study (Mattison et al., 2003; reviewed in Mattson and Wan, 2005). The CR re gimen also stimulates the turnover of membranes, organelles, and proteins in the cell, important in aging cells (reviewed in Bergamini et al., 2004; Hagopian et al., 2003; Sp indler et al., 2001). The IF re gimen entails that the animals are fed one day, and starved the next at the sa me time each day. The CR regimen is a reduction in daily caloric intake, resulting in significant weight loss, a 3040% reduction in food intake is common. In some rodent models the IF regimen results in a significant we ight loss as well, but the C57/BL/6 mouse strain sustains less than 10% reduction in weight. The C57/BL/6 mice
28 gorge on fed days, consuming approximately th e same amount of food as would have been consumed over two days (Anson et al., 2003). The effects of dietary restri ction through IF and CR are not completely understood, but an increase in some chaperone levels, such as Hs p70 and glucose related protein 78 are seen in rat CNS (Yu and Mattson, 1999). The expression of Hs p70 is down regulated in the hearts of aged rats, in the heart tissue of aged rats on IF and CR regimens Hsp70 increases significantly. The hearts of aged rats on the IF regimen had the gr eatest retention of heat shock response induction of chaperones (Colotti et al., 2005 ). Furthermore, BDNF levels are augmented in the CNS of mice on IF regimen (Duan et al., 2003). When mice that are heterozygous for BDNF knock out are placed on the IF regimen, insulin sensitivity improves, which is important for proper glucose processing. The increase in BDNF levels resu lts in behavioral and metabolic improvement (Duan et al., 2006). Rats on the IF regimen have improve d cardiac functioning, including lower blood pressure (Ahmet et al., 2005). The number of d ead myocytes after corona ry artery ligation and the overall region of myocardial infarction damage in the heart decreases with IF regimen. A decrease in the number of neutrophils infiltrating the injury site is al so seen in the hearts of rats on IF regimen (Ahmet et al., 2005). Heart rate variability is a sign of cardiac health, and it increases in rats on the IF regimen. High diasto lic blood pressure variab ility (DPV) is a risk factor for heart disease, and leve ls of DPV are decreased with IF regimen in rats. There is an overall improvement in the cardiovascular stress re sponse with IF regimen in rats (Mager et al., 2006). The IF regimen is neuroprotective (reviewe d in Mattson and Wan, 2005). When insulted with an immobilization stress ta sk, rats on IF regimen had incr eased activation of hypothalamic-
29 pituitary-adrenal neuroendocrine system, ev ident with increased plasma levels of adrenocorticotropic hormone (ACTH) and cor ticosterone (reviewed in Mattson and Wan, 2005) . The CR regimen protected hippocampal and cort ical neurons in presenilin-1 mice, an Alzheimerâ€™s disease model, from excitotoxic ity and apoptosis (Zho et al., 1999). Also CR regimen protected hippocampal neurons from ch emical hypoxia. In rats undergoing a central artery inclusion induced stroke, preconditioning with CR had improved behavioral outcome and reduced brain damage, with fewer striatal neur ons lost (Yu and Mattson, 1999). In another study mice were treated with kainic acid in the hippocampus, which causes neuronal loss and induces seizures which damage pyramidal neurons, afte r 20 weeks on the IF regimen (Anson et al., 2003). Compared to mice fed ad libitum, a significant increase in the number of CA3 and CA1 region neurons in the hippocampus survived in IF mice after the neurotoxic insult of kainic acid. However, CR protected only CA1 region neurons. An increase in serum beta hydroxybutyrate levels is seen in mice on the IF diet, a marker of ketone body formation. The adipose reserve of mice on the IF regimen is also increased, i ndicating ketogenesis may contribute to the neuroprotective effects. The neuroprotective eff ects may also be due to an upregulation of molecular chaperones due to the intermittent stress of the IF regimen (Anson et al., 2003). Thus, the mechanism of neuroprotection of the CNS with IF has multiple possibilities, including being correlated to increased chaperone levels. The IF regimen increases molecular chaperone levels, such as Hsp70, in rodent models in tissues including the CNS, but effects by the IF regimen on PNS chaperone levels have yet to be determined. An increase in the levels of mo lecular chaperones in the PNS could help to correctly fold proteins and target missfolded proteins for degradati on. Although other methods can induce the chaperone response, initially inve stigating the effects of the non-pharmacological
30 method of the IF regimen eliminates potential side effects of drug treatmen ts. The effects of IF on autophagy activation have yet to be studied. However, sin ce starvation induces autophagy and clears aggregates in ce ll culture, and chaperone respons e can mediate autophagy, the IF regimen may likewis e induce autophagy in vivo. Autophagy induction woul d be beneficial to clear aggregated proteins from the cells. In the TrJ sciatic nerves increasing the chaperone response and autophagy induction may clear aggr egated PMP22, thereby improving myelination and consequently improving phenotypic sympto ms. This study will explore the possible beneficial effects of the IF regimen on the phenotype of the CMT1A mouse model TrJâ€”motor performance, myelination and possible molecu lar changes in levels of chaperones and autophagic reponse. Our hypothesis is that th e IF regimen will im prove the neuropathic phenotype of TrJ mice through the induction of ch aperones and autophagy. After determining if the IF regimen improves the TrJ phenotype conc omitantly with increasing the levels of chaperones and autophagy, pharmacological approaches aimed to increase these two pathways as an eventual therapy for CMT1A patients can be further pursued. If Tr J mice exhibit improved motor performance and PNS myelination that is no t correlated with increases in chaperones and autophagy, other aspects of the IF re gimen must be investigated. It is possible that the increase in neurotrophins such as BDNF, seen previously in the CNS, may account for the amelioration of the neuropathic symptoms.
31 CHAPTER 2 MATERIALS AND METHODS Introduction In efforts to delineate the effects of the IF regimen on the phenotype of TrJ/+ peripheral neuropathic mice, a three tiered approach was used. The first aspect was to test the motor function of the mice, looking at muscle strength, testing hindlimb and forelimb grip strength, and at complex motor function and balance with the accelerated Rota-rod. Secondly, the gross morphometry of the peripheral sciatic nerve, to de termine if the thickness of myelin is changed with treatment through use of g-ratio calculations was investigated. Then biochemical analysis was conducted of sciatic nerve lysates using west ern blot to determine relative molecular level changes in chaperones, myelin proteins, and degradative pathways-autophagy, lysosomal, and UPS. Subjects Mice used for this study were +/+ and TrJ/+ ma le littermates, 8 weeks old at start of the study. Genotyping was done by PCR from genomi c DNA isolated from tail biopsies of mice younger than 10 days old (Notterpek et al., 1997 ). The TrJ (The Jackson Laboratory, Bar Harbor, ME, USA) mouse breeding colony is housed under specific pathogen-free conditions (SPF) at the University of Florida McKnight Brai n Institute animal facility. The use of animals for these studies was approved by the Institutiona l Animal Care and Use Committee. Mice were kept in plastic cages with wire mesh lids in a 12:12-h light-dark cycle and control mice were fed ad libitum (AL) ( Fortun et al., 2003) . Intermittent Fasting Regimen Mice on IF diet had food added on fed days, and all food removed on subsequent starve days , between 12 and 1 pm each day, with alternating feed/fast days . All mice were given access
32 to water at all times and were weighed weekly during the study to monitor weight (Anson et al., 2003). Accelerated Rota-rod The accelerated Rota-rod is a complex balance and motor task. The mice were placed on a textured rotating rod. The rod is pliable enough for ease of walking, while not so malleable that the mice can simply grip with there claws and ro tate with the rod. Ther e are dividers between each mouse, and 5 mice can be tested simultaneous ly. The mice are placed individually on the Rota-rod by holding the base of th e tail and then gently â€œflicki ngâ€ the mouse onto the rod, in a â€œpaintbrush strokeâ€-like motion, careful to not allow the mouse to grip the sides of the Rota-rod section with its hindlimbs. A ll tests were conducted monthly dur ing the study on an accelerated Rota-rod (Ugo Basile, Camerio VA, Italy), by th e same observer in a SPF behavioral testing room on the same day for all mice during th e light period. Method is modified from Chapillon et al., 1998 and McIIwain et al., 2001, w ith both the AL and IF mice trained for 2 subsequent days with 3 trials per day of 60 sec at a fixed 5 rpm, with a 30 min rest between each trial. On the subsequent 2 days the mice were tested individually with 3 trials per day on the Rota-rod which accelerates at a steady pace from 4-36 rpm, over a period of 300 sec, with a 60min rest period between each trial. Both the acceleration a nd time on the Rota-rod contribute to the motor difficulty of the task. Therefore, maximum time each mouse spent on the accelerating Rota-rod for each trial was recorded. Grip Strength All hindlimb and forelimb grip strength te sts were conducted monthly during study on the Chatillon DFE series Digital Force Gauge appara tus (Chatillon Systems, AMETEK Inc., Florida, USA) adapted for mice in a SPF behavioral testi ng room, on the same day during the light period for all mice. Hindlimb grip strength was done by initially grasping the mouse by nape of the
33 neck and base of the tail. Then after allowing mous e to grip inclined metal grid attached to force gauge, compression force necessary to pull mouse from grid is measured. To measure forelimb grip strength the mouse is first allowed to grip a horizontal metal grid which is attached to the force gauge. Forelimb grip strength is measured as the amount of force necessary to pull mouse by base of tail away from the metal grid ( modified from Meyer et al., 1979). The mice are allowed 3 attempts to grip the bar, and the gr ip strength used is the average of the grips by individual mice in each trial. Statistical Analysis Repeated measures ANOVA were used to de termine genotype and condition differences, as well as changes over the course of the st udy. The repeated measure ANOVA with between subjects factors is used when analysis includes separate groups , between subjects factor, and within subjects factor. When the F value (facto r) is high, the p value is low thereby indicating significant differences. The F va lue is denoted with [F(degre es of freedom of the groups, degrees of freedom of the number of individu al measurements [trials])=value, p=value] (Statview manual, SAS Institute Inc., Cary, NC, US A). Analysis of behavior data was done used StatView (SAS Institute Inc., Cary, NC, USA). Additional anal ysis conducted using Excel 2000 software (Microsoft, Redmond, CA). Morphometry All reagents used for these studies were obtai ned from Electron Micros copy Sciences (Fort Washington, PA). Sciatic nerves were collected from mice at end of the 20 week study, and the mice were ~28 weeks old. Samples were fixe d by immersion in 1% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffe r, pH 7.4, 1 h at room temperature, followed by osmication in 2% OsO4 in 0.1 M sodium cac odylate buffer for 1 h at room temperature, dehydrated in an ascending ethanol and acetone se ries, and embedded in Spurrâ€™s medium. Thick
34 sections of the samples were stained with tolu idine blue and surveyed by light microscopy using a SPOT camera (Diagnostic Instrume nts, Sterling Heights, MI) a ttached to a Nikon (Melville, NY) Eclipse E800 microscope. Measurements of axon and fiber (axon with myelin) diameters on light level images were analyzed on a PC using the public domain NIH Image J program (developed at Research Servi ces Branch, National Institute of Health, Bethesda, Maryland, USA, and available on the internet at http://rsb.info.nih.gov/nigh-image/ ) . G-ratios were determined by dividing the axon diameter by the fi ber diameter. To quantify potential differences in g-ratios ~100 individual fibers per animal were analyzed, using four mice per genotype and condition. Statistical analysis was performed using Excel 2000 software (Microsoft, Redmond, CA). Images were formatted for printing by using Photoshop 5.5. ~400 axonâ€“SC profiles per genotype/treatment were measure d. Axon fibers have without di scernable myelin at the light microscope level (previously estimated as fewer than five lamellae) were not measured. These fibers are demyelinated, with axons are of suffi cient diameter that one would normally expect them to be myelinated. Also axon fibers that were misshapen, or had a g-ratio of less than .55( m axon diameter/ m fiber diameter), indicating hyperm yelination, were not included in average g-ratio for each nerve (modified from Amici et al., 2006) . Western Blot Analyses Sciatic nerves collected from genotyped mice we re frozen immediately in liquid nitrogen. For total protein analyses, nerves from three mice were crushed under liquid nitrogen and then solubilized in SDS gel sample buffer (62.5mM Tris, pH 6.8, 10% glycerol, and 3% SDS). Protein concentrations were determined using BCA reagents (Pierce, Rockford, IL). Samples (27 g/lane) were separated on 7.5, 12.5 or 15% acry lamide gels under reducing conditions and transferred to nitrocellulose or PVDF membranes (Bio-Rad, Hercules, CA). Blots were blocked
35 in 5% nonfat milk in PBS, and then incubate d with primary antibodies . Polyclonal rabbit antiPMP22 (1:2000) developed against a pep tide corresponding to the second extracellular loop of the human or the rat PMP22 (Pareek et al., 1997). The other an tibodies used were: monoclonal rat anti-MBP antibody (Chemicon, Temencula, CA), hsp27 (Cell Signaling Technology, Inc, Beverly, MA), hsp40 (Stressgen, Victoria, Br itish Columbia, Canada ), polyclonal hsp70 (Stressgen), alpha B crystallin (Stressgen), m onoclonal mouse anti-actin (Sigma), and polyclonal beclin (Cell Signaling Technology Inc, Danvers, MA ). Polyclonal rabbit gsa7 (glucose induced autophagy protein 7, Atg7 autolog) and polyclo nal LC3 were a kind gi ft form Dr. Dunn, Jr., University of Florida, Gainesville, FL. Additi onal antibodies were used including: polyclonal poly-ubiquitin (Dako Denmark A/S, Colorado, US A affiliate), polyclonal cathepsin D (Cortex Biochem Inc, Leandro, CA), polyclonal LAMP 1 (hybridoma 1D4B in lab), and monoclonal mouse anti-glyceraldehyde-3-phos phate dehydrogenase (GAPDH) (a kind gift from Dr. G. Shaw, University of Florida, Gainesville, FL). Then blots were incubated with the corresponding secondary antibody conj ugated to horseradish peroxida se (HRP) (Sigma, St Louis, MO). Bound antibodies were visualized using an enhanced chemiluminescence substrate kit (PerkinElmer Life Sciences, Boston, MA). F ilms were digitally imaged using a GS-710 densitometer (BioRad Laboratories) and were formatted for printing by using Photoshop 5.5 (Adobe Systems Inc, San Jose, CA). Relative quantifications done w ith Photoshop CS (Adobe Systems Inc), and graphs prepared with Excel 2000 (Microsoft). (modified from Fortun et al., 2003).
36 CHAPTER 3 RESULTS Body Weight Does Not Decrease Significantly with IF Regimen in Mice Previously it was seen that C57/B6 mice had a less that 10% reducti on in body weight with IF regimen (Anson et al., 2003). A large degree of individual variability is evident within each group, seen in Figure 3-1a. The general trend is a decrease in body weight of mice on the IF regimen compared to AL fed mice, though not more than 10% across the 20 week study, seen in Figure 3-1b. However at the 18 week time point the wt IF group deviated by approximately 20% from the AL fed. The differences between the wt IF and wt AL were more evident than the TrJ IF and TrJ AL groups. Since no significant weight loss was experienced by mice on the IF regimen, all subsequent differences seen with the IF regimen are li kely the result of the dietary regimen and not weight reduction. Forelimb Grip Strength Is Not Effected by IF Regimen in TrJ or Wt Mice The forelimb grip strength of TrJ mice is not decreased compared to wt mice at baseline, when mice are 8 weeks old, (p>0.05), seen in Figure 3-2a. The distribution of wt measurements tends to be higher than the median at baseli ne, and TrJ measurement distribution pools lower than the median also in Figure 3-2a. In Figure 3-2b, the IF regimen does not affect wt littermates forelimb grip strength, (p>0.05). The IF regi men significantly decreases the forelimb grip strength of TrJ mice at the 2 and 4 month time points on the regime n, [F(1,25)=19.42, p<0.005], [F(1,25)=7.633,p<0.05], in Figure 3-2b. The apparent d ecrease in forelimb grip strength in TrJ mice may result from a period of adjustment to the regimen. This phenomenon was seen in previous treatment studies involvi ng CMT1A mice (Passage et al., 2004 ). No general trend of IF regimen affect on forelimb grip strength of TrJ mice is seen ove r the course of the 5 month study, (p>0.05), seen in Figure 3-2b. The Tr J mice fed AL did not experience decreased
37 forelimb grip strength either. A significant im provement with IF regimen in TrJ mice would not be expected since there is no no ticeable impairment in this motor functional aspect during this study. IF Regimen Improves Hindlimb Gr ip Strength in TrJ Mice Muscle atrophy in the hindlimbs, as well as the decreased peripheral motor control, are evident in CMT1A mouse models such as TrJ (H enry et al., 1983). In a previous study a CMT1A mouse model had significantly reduced hind limb grip strength at 8 weeks (Norreel et al., 2001). TrJ mice hindlimb grip strength was al so significantly reduced at the baseline time point, when the mice were 8 weeks old, (p< 0.005), in Figure 3-3a. The wt individual measurements were evenly distri buted above and below the media n. The grip strength of wt mice increases with time as the mice mature fr om 8 weeks to adulthood (Miller et al., 2005). Our data also found a general trend of increasing hindlimb grip strength as the wt mice matured, then reaches a plateau [F(1,5)=6.103,p<0.005)], Figur e 3-3b. The hindlimb grip strength of the two groups of wt mice in the study deviated fr om the baseline time point, indicating significant individual variability [F (5, 30) =2.903, p<0.05)], in Figure 3-3b. The wt IF group had significantly lower hindlimb grip strength at the 1 and 3 month timepoints (p<0.05). There is not impairment in hindlimb grip strength of the wt mice due to peripheral neuropathy, so this propensity for significant differences between wt AL and IF groups needs further investigation. Technical difficulties in the hindlimb grip strength task may contribute to the variability in measurements observed. TrJ mice had significantly reduced hindlimb gr ip strength compared to the wt mice over the course of the study. Although ha ving individual variability in hindlimb grip strength, the AL fed and IF groups of TrJ mice did not deviate significantly at ba seline, (p>0.05). At the 5 month timepoint in the study, the TrJ mice on the IF regi men had a significant in crease in hindlimb grip
38 strength compared to AL fed [F (1, 25) =9.612, p<0.005] in Figure 3-3b. However, there was no general trend of IF regimen eff ect seen across the course of th e study (p>0.05) two-way repeated measures ANOVA, also in Figure 3-3b. An additi onal factor possibly affec ting the variability of results is the spastic hindimb griping in TrJ mice, which is observed as coordinated hindlimb movements are decreasing. This factor may impede the hindlimb grip strength assessment in the TrJ mice, resulting in the lack general trend for improvement with the IF regimen. Previously, there was not a hindlimb grip st rength assessment on the TrJ m ouse. C22 mice, for which the grip strength test was previously conducted, have only a decrease in grip strength, with no spastic griping is seen, thereby resulting in a possibly more accurate assessment of muscle strength (Norreel et al ., 2001). As the TrJ mice on the IF re gimen showed a re lative increase in hindlimb grip strength at the 5 month time point, fu rther assessment is necessary to determine if the IF regimen has an actual effect. Improvement in Complex Motor Functi on of TrJ Mice with IF Regimen Previous studies indicated transgenic rode nt CMT1A models with PMP22 overexpression have severe impairment performing the complex motor and balance task of the Rota-rod (Serada et al., 1996; Passage et al., 2004). As demyelination of the peri pheral nerves increases as the mice age, coordinated motor function decreases. The TrJ mice performance is impaired at 8 weeks, compared to wt littermates, Figure 3-4a, (ANOVA, p<0.0001). Results in this study indicate that there is a main effect of genotype, with TrJ being severely impaired in motor performance compared to wt [F(1,65)=146, p<0.0001]. A repeated measures ANOVA was conducted across the months of testing comparing genotype as well as treatment effects in the mice. As seen in Figure 4b and previously, wt mice are capable of remaining on an accelerated Rota-rod for ~300sec (McIlwain et al., 2001). After 1 month on the IF regimen wt mice accelerated Rota-rod performance decreases (p<0.05) , but then recover by 2 months, with no
39 residual impairment over the course of the 5 month study. Subsequent ANOVAs examining the treatment effects within each genotype indica ted Rota-rod performance of wt mice was not effected by the IF regimen over the course of the study (p>0.05, ANOVA). This temporary lag of performance of wt mice may result from a peri od of adjustment to the new regimen, seen in previous studies (Passage et al ., 2004), as well as with forelimb grip strength in TrJ mice. It would be expected to see a similar performance la g effect due to adjustment to the regimen in both wt and TrJ mice across all three motor behavi oral assessments, however differences in test sensitivities, and high individua l variability due to small numb ers of individuals per condition may account for these seemingly conflicting result s. Also, the general adjustment period may result in a lag in performance in all groups, t hough not significantly. In teractions between the effects of the treatment on TrJ mice compared to wt mice is significant [F(1,65)=5.8, p<0.05], as is the effect of treatment on performan ce across the months on the IF regimen [F(5,65)=3.0,p<0.05]. A sustained impairment of TrJ mice performan ce across the 5 month study as compared to wt mice was observed [F(5,65)=3.1,p<0.05]. A si gnificant improvement in motor performance on the accelerated Rota-rod in TrJ mice on the IF regimen was observed compared to AL fed TrJ mice across the 5 months of testing [F(5,20)= 4.3,p<0.01]. The AL fed TrJ mice had a general trend of decreased motor performance during th e study, though not significant. The TrJ on IF experienced a significant improvement in acceler ated Rota-rod performance, which was a trend across the 5 month study compared to AL fed. Increase in Myelin Thickness with IF Regimen in Sciatic Nerves of TrJ Mice G-ratios are the ratio of axon to nerve fiber diameter , and indicate the thickness of the myelin sheath. A lower g-ratio of a nerve indica tes a thicker myelin shea th, and the g-ratios of TrJ sciatic nerve are lower than wt sciatic nerve . This decrease in gratio, indicating thinner
40 myelination of peripheral axons, is evident in other CMT1A animal models and in patients with CMT1A as well (reviewed in Young and Suter, 2003). In the sciatic nerve cross sections in Figure 3-5a-b, large numbers of improperly or th inly myelinated axons are evident. Also, a number of demyelinated axons ar e evident in Figure 3-5a-b. Th e sciatic nerve section of a TrJ AL mouse in Figure 3-5a has seemingly thinner myelin thickness than TrJ IF in Figure 3-5b. The sciatic nerves of AL and IF fed TrJ mice ha ve significantly reduced myelin thickness when compared to previously observed g-ratios of wt sciatic nerves. In a pr evious study the sciatic nerves of 3 month old wt mice had a g-ratio of .59 m axon diameter/ m fiber diameter (Passage et al., 2004). It is important to note that the exact g-ratio is dependent on the preparation of the nerve for EM, and fixation and staining of the sample , but the trend for g-ratios is still evident. The sciatic nerves of TrJ on the IF regimen ha ve significantly higher gratios than AL fed, observed in Figure 3-5c (p<0.005, St udentâ€™s t-test). The increase in g-ratio observed in sciatic nerves of TrJ mice on the IF regimen in Figure 3-5c correlates to an increase in myelin protein levels seen in lysates of the TrJ sciatic nerves in Figure 3-6b-d. Increase in Myelin Protein Levels with IF Regimen in Mice Sciatic Nerves The TrJ mice have an increased number of unmyelinated and thinly myelinated peripheral axons (Henry and Si dman, 1983). Therefore relative steady state myelin protein levels were investigated, to dete rmine the effects of the IF regime n. All subsequent proteins are observed at steady-state levels within pooled, whole sciatic nerv e lysates by western blot. In Figure 3-6a the level of the primary myelin pr otein in peripheral myelin, MBP, is markedly reduced in TrJ sciatic nerve markedly compared to wt, as seen previously (Fortun et al., 2006). Additionally, in the same figure in wt mice scia tic nerve, the 14kDa is oform of MBP increases by an approximately 8-fold magnitude. The 17kDa, 18.5kDa, and 21.5kDa MBP isoforms
41 slightly increase with the IF regimen, Figure 3-6a. With th e IF regimen, MBP levels are increased in TrJ sciatic nerve as well with the 14kDa and 17kDa isoforms have a 10-fold and 2fold increase respectively. The 18.5kDa and 21.5kDa is oforms are also slightly increased in TrJ sciatic nerve observed in Figure 3-6a. The protein levels of PMP22 and P0 are drasti cally reduced in TrJ sciatic nerve compared to wt sciatic nerve (N otterpek et al., 1997) . In response to IF regimen, a slight augmentation in PMP22 levels, and a 1.5-fold increas e in P0 (MPZ) levels was obser ved in wt sciatic nerve, in Figure 3-6b-c. In the same Figure, PMP22 and P0 levels increase with IF regimen in TrJ mice with approximately 2-fold and 1.5-fold, respectively. Regarding the relative fold increases it is important to note that the quantifications of all proteins were based on one wester n blot with a pooled sample of 3 animals per condition. This is true for all subsequent western blot quantif ications in this paper. The large genotypic differences between TrJ and wt sc iatic nerves with myelin protei n levels may also affect the relative precision of the densitometry due to the consequential high degree of intensity difference. Nonetheless, the compact myelin proteins MBP, PMP22, and P0 levels increase slightly in the sciatic nerve of wt mice, and ma rkedly in TrJ with IF regimen compared to AL fed. Molecular Chaperones Are Upregulated with IF Regimen in Mice Sciatic Nerves An increase in the cytosolic chaperone Hsp70 was observed in the hearts (Colotti et al., 2005), and the CNS of rats on the IF regi men (Yu and Mattson, 1999). Therefore we investigated if a similar increas e in levels of Hsp70 was evident in the PNS due to IF. In the sciatic nerve of both wt and TrJ mice on the IF regimen, Figure 3-7a, levels of increased compared to AL fed. As Hsp70 is the primary ch aperone in the heat sh ock cellular response to stress, increase in these proteins levels can be beneficial for the cell (reviewed in Freeman and
42 Morimoto, 1996). The small heat shock proteins hsp40 and hsp27 are also increased with the IF regimen in wt and TrJ sciatic nerv es in Figure 3-7b-c. Levels of Bcrystallin are increased in wt and TrJ sciatic nerves Figure 3-7d. An upre gulation of the molecular chaperones Hsp70, Hsp40, Hsp27 and Bcrystallin in TrJ sciatic nerve compared to wt sciatic nerve is observed in Figure 37a-d, and seen previously, due to the stress of a ggregates in the SCs (Fortun et al., 2003). The relative increase in chaperone leve ls due to IF was similar for a ll of the heat shock protein and small cytosolic chaperones investigated, with approximately a 2 to 3-fold increase in both genotypes. The Hsps and small molecular chaperone Bcrystallin investigated were induced in the sciatic nerve of both wt and TrJ mice with IF. This indicates chaperone induction is potentially a very important aspect of the neuroprotective effects of the IF regimen. Autophagy Is Induced in TrJ Mice Sciatic Nerve with IF Regimen Atg7, the mammal autolog of gsa7, is a ke y regulator of autophagy (Komatsu et al., 2006). Levels of atg7 (Gsa7 antibody) (Dorn et al., 2001) are increased in TrJ sciatic nerve, compared to wt sciatic nerve seen in Figure 3-8a. This increase in atg7 was seen in TrJ sciatic nerves previously, markedly in the insoluble fr action of nerve lysate, when compared to wt (Fortun et al., 2003). Atg7 increases very slightly in wt sciatic ne rve, but increases by 1.5-fold in TrJ sciatic nerves in response to the IF regimen in Figure 3-8a. Beclin is an essential protein in the initial phase of autophagy (Qu et al., 2003). In Figure 3-8 b, Beclin levels are initially elevated in TrJ sciatic nerves compared to wt scia tic nerves. The level of Beclin is increased in both wt and TrJ sciatic nerve with IF regimen, by 1-fold and 3-fold respectively. Observation of the increased conversion of LC3I to LC3II is the standard marker for autophagy induction ( reviewed in Levine and Klionsky, 2004; Wang et al., 2006). In wt sciatic nerve LC3I is present, but there is no perceivable LC3II observed in Figure 3-8c. LC3I levels increase by
43 approximately 2-fold in wt mice sciatic nerve and 3-fold in the sciatic nerve of TrJ littermates. TrJ exhibit levels of LC3II, indicating aut ophagy induction. Inducti on of autophagy in TrJ sciatic nerves was seen previ ously through morphometr ic studies and by an increased level of Atg7 (Gsa7 antibody) (Fortun et al., 2003). LC3II le vels increase four-fold in response to IF in TrJ mice sciatic nerve, and the ratio of LC3II/ LCI is elevated, indica ting increased autophagy seen in Figure 3-8c. Thus, autophagy is not active in wt sciatic nerve, bu t is active in TrJ sciatic nerve, and this activity is increased with IF regimen. Autophagy is therefore a potential mechanism by which the phenotypic improvement is observed in TrJ mice with the IF regimen. The Lysosomal Degradative Pathway Is Induced with IF Regimen in Mice Sciatic Nerve The lysosomal degradative pathway activity is increased in the TrJ sciatic nerve compared to wt (Notterpek et al., 1997) and in Figure 3-9a, possibly in a compensatory mechanism for the decrease in UPS function (Fort un et al., 2003). Increase in levels of LAMP-1, a lysosome associated membrane glycoprotei n, in Figure 3-9a, and the lysosomal enzyme cathepsin D, in Figure 3-9b, are observed in wt and TrJ sciati c nerves on IF. A two-fold induction of both isoforms of cathepsin D was seen in wt sciatic nerve with IF re gimen, while a four-fold increase was evident in TrJ sciatic nerves. The most co mmon degradative pathway for proteins which are short-lived in the cell, including PMP22, is the UPS reviewed in Ciechanover and Brundin, 2003). The amount of poly-ubiquitinated proteins was higher in TrJ sciatic nerves than in wt sciatic nerves, seen previously and in figure 9c suggesting UP S impairment (Ryan et al., 2002; Fortun et al., 2003). Also in Fi gure 3-9c, the IF regimen did not increase UPS activity in wt or TrJ sciatic nerve, which would be indicated by a decrease in poly-ubiquitinated proteins. Observation of proteasomal activity would be a mo re direct method to determine activity of the UPS. However, since poly-ubiqu itin levels did not change, the IF regimen has no evident effect
44 on the UPS activity. Proteasomal activity is decreas ed in TrJ sciatic nerves, possibly due to the overwhelming of the system due to mutated PMP22. The lysosomal pathway may also be assisting to remove missfolded and aggregated pr oteins in SCs of TrJ mice on the IF regimen, to improve myelination of peripheral nerves. Together these data suggest that the IF re gimen improves the motor function of TrJ mice, and increases myelination. Levels of molecular chaperones increased in both wt and TrJ mice on the IF regimen, and autophagy and lysosomal marker levels also increase in TrJ mice, indicating possible mechanisms for the phenotypic improve ments observed in the neuropathic TrJ mice.
45 Figure 3-1. The body weight of mice on IF regimen is not significantly diffe rent that of AL fed mice. A large degree of individual variabil ity in weight is evident within each group (a). Wildtype mice on IF regimen (n=5) have a decrease in body weight over the course of the 20 week study compared to wt AL fed mice (n=3), but the decrease is not significant (p>0.05, Studentâ€™s t-test). Similarly, the decrease in body weight of TrJ mice on IF regimen (n=5) is not signifi cantly reduced compared to AL fed (n=4) (p>0.05, Studentâ€™s t-test).
46 Figure 3-2. Forelimb gripstrength in wt and TrJ mice is consis tent throughout the study. TrJ mice forelimb gripstrength is not impaired at baseline, mice are 8 weeks old (TrJ mice n=9, wt mice n=8), (p>0.05, ANOVA), (T ukey-whisker box plots, error bars represent quartile distributi on of values from median li ne) (a). No trend of IF regimen effect in wt mice (p>0.05, two-wa y repeated measures ANOVA), or in TrJ mice (p>0.05, two-way repeated measures ANOVA) (b). Significant decrease in forelimb gripstrength in TrJ mice on IF regimen at 2 months and 4 months on regimen compared to AL fed TrJ mice (*p<0.05, two-way repeated measures ANOVA), then recovery (b).
47 Figure 3-3. Mouse hindlimb grip strength is affected by the IF regimen. At baseline, the hindlimb gripstrength of TrJ mice (n=9) is significantly less than wt mice (n=8) (**p<0.005, ANOVA), (Tukey-whisker box plots, error bars repr esent quartile distribution of values from me dian line) (a). The AL (n=3) and IF fed (n=5) wt mice groups have significantly different hindlimb gripstrength at base line, at 1 month on regimen, and at 3 months on regime n (*p<0.05 ANOVA). After 5 months on regimen, TrJ IF mice (n=5) have significantl y greater hindlimb gripstrength than AL fed TrJ mice (n=4) (**p<0.005, two-way re peated measures ANOVA). TrJ mice have significantly lower hindlimb gripstre ngth than wt mice over the 5 months on the regimen (**p<0.005, two-way repeated measures ANOVA) (error bars SEM) (b).
48 Figure 3-4. IF regimen improves the performanc e of TrJ mice on the accelerated Rota-rod. At baseline, TrJ mice (n=9) have significan tly impaired motor performance on the accelerated Rota-rod compared to wt mice (n=8) (**p<0.005 ANOVA) (error bars SEM) (a). The performance of wt mice on the accelerated Rota-rod is consistent (p>0.05, two-way repeated measures ANOVA) . The performance of TrJ mice (n=5) on IF regimen significantly improves with 4 months on regimen compared to AL fed TrJ mice (n=4) (*p<0.05, two-way repeated measures ANOVA) (b). There is a general trend of mo tor performance improvement in TrJ mice on the IF regimen compared to AL fed over the course of the 5 month study (*p<0.05, two-way repeated measures ANOVA)(error bars SE M) (error bars SEM) (b).
49 Figure 3-5. The g-ratio of the sc iatic nerves of TrJ mice decreases with 5 months on IF regimen. TrJ AL mice sciatic nerves have a large number of thinly myelinated or dysmyelinated axons (a). The nerves of th e TrJ IF mice also have a large number of thinly myelinated and dysmyelinated axons , but an increase in myelin thickness compared to AL fed TrJ are seen (b). Scale bar is 10 m. The increase in myelin thickness is evident as the average g-ratio of the sciatic nerve TrJ mice on IF regimen (n=4) is lower than that of the sciatic nerve of AL fed TrJ (**p<0.005, Studentâ€™s ttest), (error bars SEM) (c).
50 Figure 3-6. Myelin protein levels are upregulated in the sciatic nerve of mice on IF regimen. In the MBP graph, isoform I is medium grey, II da rk grey, III white, IV light grey (b). The MBP 14kDa isoform increases in wt scia tic nerve in response to the IF regimen, as does the 17kDa isoform. However the 18.5kDa and 21.5kDa MBP isoforms do not seemingly change in wt sciatic nerve (a). MBP levels are lower in TrJ than wt sciatic nerve (a). The 14 and 17 kDa MBP isoforms increase in TrJ sciatic nerve with IF regimen, and the 18.5 and 21.5 isoforms increase slightly (a). Levels of MPZ (P0) increase slightly with IF regimen in wt sc iatic nerve (b). The myelin protein P0 is lower in TrJ sciatic nerve than in the wt littermates (b). An increase in levels of MPZ (P0) is also seen in TrJ sciatic nerve with the IF regimen (b). PMP22 levels increase slightly in the sciatic nerve of wt and Tr J mice on the IF regimen (c). Levels of PMP22 are reduced in the sciatic nerve of TrJ mice compared to the sciatic nerve of wt littermates (c). The graphs represen t the ratio of protei n concentration after correction for GAPDH, as determined by wester n blot and WT AL control is set at 1 (100%). GAPDH is a constitutive marker for loading control. (n=3 animals per condition).
51 Figure 3-7. Some cytosolic molecular chaperones are upregulated in mice sciatic nerve with IF regimen. Hsp70, Hsp40, Hsp27, and Bcrystalin levels are higher in TrJ mice sciatic nerve than wt mice sciatic nerve (a-d). Hsp70, Hsp40, Hsp27, and Bcrystallin levels are upregulated with IF regimen in wt mi ce sciatic nerve compared to AL fed mice (a-d). Similarly chaperone Hsp70, Hsp40, Hsp27, and Bcrystallin levels are increased in TrJ mice in response to If regime n (a-d). The graphs represent the ratio of protein concentration af ter correction for GAPDH, as determined by western blot and WT AL control is set at 1 (100%). GAPDH is a constitutive marker for loading control. (n=3 animals per condition).
52 Figure 3-8. Autophagy degradative pathway is upregulated in TrJ mice sciatic nerve with IF regimen. Levels of atg7 are in creased in TrJ compared to wt mice sciatic nerve (a). With IF regimen, levels of atg7 does not change in wt sciatic nerve and beclin increases (a-b). In the LC3 graph grey bars represent isoform I and black bars represent isoform II (c). Th e conversion of LC3I to LC 3 II is evident in TrJ mice sciatic nerve, and not wt mice sciatic nerve (c ). LC3I levels slightly increase in both wt and TrJ sciatic nerve with IF regimen (c ). LC3 II levels increase with IF regimen in TrJ mice sciatic nerve, and the ratio of LC 3I/II decreases (c). The graphs represent the ratio of protein concen tration after correction for GAPDH, as determined by western blot and WT AL contro l is set at 1 (100%). GAPDH is a constitutive marker for loading control. (n=3 animals per condition).
53 Figure 3-9. The lysosomal degradative pathway is upregulated in the sciatic nerves of mice on the IF regimen, but the UPS degradative pathwa y is not effected. Levels of LAMP 1, cathepsin D, and poly-ubiquitin are higher in the sciatic nerves of TrJ mice than in wt sciatic nerve (a-c). LAMP 1 levels are incr eased in wt and TrJ sciatic nerve with IF regimen (a). In the cathepsin D graph, white bars represent isoform I and grey bars represent isoform II (c). In wt sciatic nerv e cathepsin D levels are slightly increased, in TrJ sciatic nerve the levels are increased (b-c). Levels of poly-ubiquitin are not effected by IF regimen in wt or TrJ sciatic nerve (d). The graphs represent the ratio of protein concentration af ter correction for GAPDH, as determined by western blot and WT AL control is set at 1 (100%). GAPDH is a constitutive marker for loading control. (n=3 animals per condition).
54 CHAPTER 4 DISCUSSION The TrJ mice on the IF regimen have improved motor performance and thicker myelination of peripheral nerves . Possible factors which yiel d this phenotypic improvement with IF regimen treatment are an increase in the steady-state levels of my elin proteins, cytosolic chaperones, and induction of autophagy and the ly sosomal pathway; all observed in the sciatic nerves of TrJ mice on the IF regimen in this stud y. Further investigation to support these results, and delineate the mechanisms of th e improvement seen is necessary. Although previous benefits of the IF regimen have been observed in the CNS of rodents, including induction of chaperone s and neuroprotection (Yu and Ma ttson, 1999; Zho et al., 1999; Anson et al., 2003; Duan et al., 2003), the effect s of the regimen on the PNS have not yet been investigated. Cytosolic stress induces the chaper one response, and dietar y restriction through the IF regimen appears to be a general stress that also stimulates chaperone production. Chaperone levels are increased in both wt and TrJ sciati c nerves with IF regimen, seen in Figure 3-8. Increasing chaperones helps to correctly fold proteins, and target mi ssfolded proteins for degradation, thereby decreasing th e number of aggregated proteins . This induction of the chaperone response can be very beneficial, since increase of a single chap erone through transgenic studies has showed promising results in protecting the heart from damage due to myocardial infarction. Mice with increased levels of Hsp70 or Bcrystallin have increased resistance to heart damage due to myocardial in farct and reduced myocyte death (Plumier et al., 1995; Mestril, 2005). An increase in chaperone leve ls with the IF regimen in aged rat hearts was also seen to improve outcome after myocardial infarct (Colotti et al., 2005) Importantly, an increase in the chaperones Hsp70 was seen in the CNS of mice and ra ts on the IF regimen correlated to neuroprotection be nefits (Yu and Mattson, 1999). Pharmacological interventions
55 aimed at increasing chaperone response have been attempted for a variety of neurological diseases with aggregate pathology. It was ascertained that incr easing the levels of Hsp70 in a Huntingtonâ€™s disease mouse model transiently de creased huntingtin protein aggregates, and use of geldamycin decreased aggregate formation ove r a longer period in cell culture (Hay et al., 2004). Autophagy and lysosomal pathways are induced in TrJ sciatic nerves in response to IF regimen. The lysosomal pathway is upregulated and autophagy induced in TrJ sciatic nerve compared to wt (Notterpek et al., 1997; Ryan et al., 2001; Fortun et al ., 2003. Aggregates and aggresomes of PMP22, which recruit other protei ns, form in TrJ mice SCs progressively. This occurs concurrently with an age related genera l decline in autophagy and chaperone levels seen in other tissues (Cuervo and Dice, 2000 ; Jin et al., 2004). The IF regimen enhances these pathways, and may assist in clearance of the a ggregated and missfolded proteins. Autophagy is activated by starvation, and the starve-fast diet of the IF re gimen further induces autophagy in TrJ sciatic nerve. This pathway is also induced in other tissues of mi ce on IF regimen, including the heart and muscle determined by measuring relati ve LC3I/II levels with western blot (data not shown). This increase in chaper one levels can also result in the increased autophagy activity, as chaperones mediate autophagy. Interestingly, autopha gy is not induced in the sciatic nerve of wt mice on the IF regimen since the wt were not under additional PNS stress. The TrJ peripheral nerves are under additional stress due to the accumulation of the mutated PMP22 protein, which aggregate with other proteins in the cytosol, possibly leading to the induction of autophagy in the PNS of TrJ but not wt mice. Myelin protein levels increased in TrJ sciatic ne rves in response to the IF regimen as seen in Figures 6. The increase in myelin proteins in the sciatic nerves of TrJ mice on the IF regimen
56 is very promising, as the phenotypic motor functi on impairments stem from this demyelination of the peripheral nerves. The myelin protein MB P is increased in both wt and TrJ sciatic nerve in response to IF regimen. It was previously determined that the constitutive chaperone Hsp70 (Hsc70) is necessary for the optimal synthesis of MBP (Aquino et al., 1998). As myelin protein levels also increase in wt sciatic nerves, other po ssible additional factors include an increase in neurotrophin levels, such as BDNF. BDNF e nhances myelination of PNS axons by binding the receptor P75NTR on SCs (Chan et al., 2001; Cosgaya et al., 2002; Yamauchi et al., 2004). Increasing BDNF levels increases myelin protein levels in the PNS (Chan et al., 2001), and this thicker myelin is maintained throughout adulthood (Tolwani et al., 2004). In previous studies IF increased BDNF levels in the CNS of mice (Duan et al., 2003). Since neur otrophin increases are a possible mechanism for neuroprotec tion in the PNS, future studies will aim at also determining if neurotrophins are increased in the PNS in response to IF. Phenotypic improvement of the TrJ mice on the IF regimen is noted by significant increases in the thickness of my elin around large caliber motor axons in the sciatic nerve, evident with a decrease in g-ratios when compared to AL fed in Figure 3-5a-c. A hallmark of TrJ sciatic nerves is thinly myelinated axons. Increased myelin thickness of peripheral nerves, noted by the decreased g-ratios could improve motor pe rformance through enhancing nerve conduction velocity. Results support the hypothesis th at the TrJ mice phenotype woul d be improved with the IF regimen, possibly due to induction of chaperones and autophagy. Notably, an improvement in the complex motor performance of TrJ mice on the IF regimen as compared to the TrJ AL fed littermates was seen in Figure 3-4b. Also, alth ough a general trend of increased hindlimb grip strength was not seen in IF TrJ mice, a significant increase in hi ndlimb grip strength was seen by
57 5 months on the regimen in Figure 3-3b. The IF regimen results in general improvement in cardiovascular function in rodent models (Ahm et et al., 2005; review ed in Mattson and Wan, 2005). The observed increase in PNS myelin thic kness seen in TrJ mice with IF would account for the improved motor performance. Mechanisms for the increased myelination are possibly the increased chaperone levels and induction of the autophagic pathway. These findings suggest the IF regimen has be nefits in the PNS as well as the general improvements already seen in rodents in cardiovascular functi on, lifespan, and CNS neuroprotection (reviewed in Matt son and Wan, 2005). Previously the IF regimen increased the levels chaperones (Colotti et al., 2005; Yu a nd Mattson, 1999) a possible mechanism for the PNS myelin protection seen in TrJ mice. In this study, increases in chaperone levels and the autophagic degradative pathway were most evident in the PNS. The BDNF levels were not yet investigated. Currently there are no effective treatments for patients with CMT1A. Treatment studies in CMT1A rodent models have had pr omising results however, improving phenotype and increasing the number of myelinated axons in the sciatic nerves. Th is study is the first to use a natural treatment paradigm, modifying diet through IF, to improve the phenotype and myelination in a CMT1A mouse model. It is also the first study to see improvement in a mouse with a point mutation in PMP22, ra ther than a transgenic overexpre ssor, and to aim at alleviating phenotypic symptoms by induci ng chaperones and autophagy. Although all results are based on an initi al study with 3-4 mice per condition, and necessitates further studies to support these findings, the promis ing results of this first study warrant further investigation. Recently our lab conducted a 20 we ek IF regimen treatments with additional TrJ animals, to verify that results are consistent. Also, the affect of the IF regimen on C22 mice, since overexpression of PMP22 is the most common cause of CMT1A, was conducted
58 in our laboratory and the results are currently be ing analyzed. Determining if BDNF levels are increased in response to IF regimen in the PNS would better elucidate another possible mechanism for phenotypic improvements observed in TrJ mice. Immunohistochemistry of teased nerve fibers can investigate if the number of ubiquitin-positive protein aggregates decreases with the IF regimen in TrJ peripheral nerves. Muscle weight of hindlimb muscles can be ascertained to denote if IF has any effect s on muscle atrophy in CMT1A. Since voluntary movements are effected in CMT1A, further testing to determine mu scle tone effects, including sensory testing, to determine if CMT1A has more focal impairments. Delineating which aspects of the IF regimen are most beneficial for the improvement in TrJ mice phenotype is also important, as further pharmacologi cal interventions can be aimed to increase these pathways, and eventually be a possible effective therapy to a lleviate peripheral neurop athy of patients with CMT1A.
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70 BIOGRAPHICAL SKETCH Amanda Jeanne Waber Nguyen was previously from Jacksonville, FL, where she graduated from Sandalwood High School after moving from Corpus Christi, TX. She attended University of Florida and Univ ersity of North Florida to ach ieve her undergraduate degree in Biology. During her final undergraduate year she was UNF-Mayo Scholar of the Year, and conducted research at Mayo Clinic Jacksonville in molecular neuroscience. Currently Mrs. Nguyen, her husband and son are living in Folsom, CA.