MECHANISMS OF NEURODEGENERATION AND STEM CELL MIGRATION: A STUDY OF MOLECULAR SIGNALS AFTER PERIPHERAL NERVE INJURIES JI JUN FENG MBBS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2004 i ACKNOWLEDGMENTS I would like to express my deepest appreciation to my supervisor, Associate Professor Samuel Sam Wah Tay, Department of Anatomy, National University of Singapore, for his innovative ideas, invaluable guidance, constant encouragement, infinite patience, and friendly critics throughout this study. I am greatly indebted to Assistant Professors S. Thameem Dheen and He Bei Ping, Department of Anatomy, National University of Singapore, for their valuable suggestions and friendly help during this study. I am very grateful to Professor Ling Eng Ang, Head of Anatomy Department, National University of Singapore, for his constant support and encouragement to me, and also for his full support in using the excellent research facilities. I must also acknowledge my gratitude to Mrs Ng Geok Lan, Mrs Yong Eng Siang and the late Miss Margaret Sim for their excellent technical assistance; Mr Yick Tuck Yong for his constant assistance in computer work; Mr P. Gobalakrishnan for his constant guidance in photomicrography; Mr Tajuddin B. Marican Ali for his help in animal operation; Mr Lim Beng Hock for looking after the experimental animals; Miss Teu Cheng Hong Kate for her assistance in cell culture work; and Mdm Ang Lye Gek Carolyne, Mdm Diljit Kaur, Mdm Teo Li Ching Violet for their secretarial assistance. I would like to thank all other staff members and my fellow postgraduate students at Department of Anatomy, National University of Singapore for their help and support. I would like to express my special thanks to my friends, Drs.Ouyang Hongwei and Wang Zhuo at Department of Orthopaedic Surgery, National University Hospital for their friendly help and advice. ii Certainly, without the financial support of the National University of Singapore, which offered a research grant (R181-000-059-213), this work would not have been brought to a reality. I would like to take this opportunity to express my heartfelt thanks to my parents and sister for their full and endless support for my study. Finally, I am greatly indebted to my wife, Mdm Yu Xiao Liang for her understanding and encouragement during this study. iii This thesis is dedicated to my beloved family iv PUBLICATIONS Various portions of the present study have been published or accepted for publication. International Jounals: 1. Ji J, Dheen ST, Tay SSW (2002). Molecular analysis of vagal motoneuronal degeneration after right vagotomy. J Neurosci Res 69 (3): 406-17. 2. Ji JF, He BP, Dheen ST, Tay SSW (2004). Expression of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 in the neural stem cells isolated from the subventricular zone of the adult rat brain. Neurosci Lett 355 (3): 236-40. 3. Ji JF, He BP, Dheen ST, Tay SSW (2004). Interactions of chemokines and chemokine receptors mediate the migration of bone marrow stromal cells to the impaired sites in the brain after hypoglossal nerve avulsion. Stem Cells 22 (3): 41527. 4. Ji JF, Dheen ST, Tay SSW. Expressions of cytokines and chemokines in the dorsal motor nucleus of the vagus of the rat after right vagotomy. (Submitted). Conference papers: 1. Ji JF, Dheen ST, Tay SSW (2002). Upregulation of inducible nitric oxide synthase and its mRNA expression in vagal motor nuclei following right vagotomy in rat. Australia Neuroscience Society Meeting, Sydney, Australia. 2. Ji JF, Dheen ST, Tay SSW (2002). Activation of apoptotic and N-methyl-Daspartate (NMDA) receptor-calcium-neuronal nitric oxide synthase (nNOS) pathways in the vagal motor nuclei of rats after right vagotomy. Annual Meetings of Experimental Biology, New Orleans, LA, USA. 3. Ji JF, Dheen ST, Tay SSW (2002). Site-specific migration of transplanted mesenchymal stem cells into the hypoglossal nucleus after unilateral avulsion of hypoglossal nerve. Society for Neuroscience 32nd Annual Meeting, Orlando, Florida, USA. v 4. Ji JF, He BP, Dheen ST, Tay SSW (2003). Expressions of chemokine receptors on neural stem cells from adult rat brains. Society for Neuroscience 33rd Annual Meeting, New Orleans, LA, USA. 5. Ji JF, He BP, Dheen ST, Tay SSW (2004). Expression of cytokines in the dorsal motor nucleus of the vagus nerve after vagotomy. 4th ASEAN Microscopy Conference, Hanoi, Vietnam. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………… .i DEDICATIONS…………………………………………………………………………iii PUBLICATIONS……………………………………………………………………… iv TABLE OF CONTENTS……………………………………………………………….vi ABBREVIATIONS…………………………………………………………………….xvi SUMMARY…………………………………………………………………………… xx CHAPTER 1: INTRODUCTION……………………………………………………….1 1. General introduction: Animal model of axotomy to study neurodegeneration .2 2. Neuronal and glial responses to axotomy…………………………………………… 2.1. Axonal reaction………………………………………………………………… .2 2.1.1. Morphological changes in nerve fibre………………………………………2 2.1.2. Changes in axonal transport…………………………………………………3 2.2. Perikaryal alteration……………………………………………………………… 2.2.1. Morphological changes…………………………………………………… .4 2.2.2. Metabolic changes………………………………………………………… 2.3. Glial cell reaction………………………………………………………………… 2.4. Fate of axotomized neurons 3. Neuronal death after axotomy………………………………………………………… 3.1. Apoptosis 3.1.1. Discovery of apoptosis…………………………………………………… 3.1.2. Morphological characteristics of apoptotic cell death………………………8 vii 3.1.3. Apoptosis in the model of axotomy……………………………………… .9 3.2. Necrosis……………………………………………………………………………9 4. Mechanisms of neurodegeneration……………………………………………………10 4.1. Involvement of Cytokines .10 4.1.1. Pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) ………………………………………… 11 4.1.2. Interleukin-6 (IL-6)……………………………………………………… .13 4.1.3. Transforming growth factor-beta (TGF-β1)…………………………… 14 4.2. Role of Nitric oxide (NO) in neurodegeneration.……………………………… .14 4.2.1. Historical perspective of NO.…………………………………………… .14 4.2.2. Isoforms of nitric oxide synthase (NOS).………………………………….15 4.2.3. Biological functions of NO.……………………………………………… 16 4.2.4. Roles of NO in the nervous system.……………………………………… 16 4.2.5. Roles of NO in the model of axotomy.…………………………………….19 4.3. Involvement of apoptosis associated molecules.…………………………………19 4.3.1. Bcl-2 and Bax.…………………………………………………………… 19 4.3.1.1. Discovery of Bcl-2 and Bax 19 4.3.1.2. Functions of Bcl-2 and Bax in apoptosis.…………………………20 4.3.1.3. Bcl-2 and Bax in the model of axotomy.………………………….21 4.3.2. Caspase-3.………………………………………………………………….22 4.3.2.1. Discovery of caspases.…………………………………………….22 4.3.2.2. Functions of caspase-3 in apoptosis.………………………………23 4.3.2.3. Caspase-3 in the model of axotomy.………………………………23 viii 4.4. N-methyl-D-aspartate receptor (NMDAR)-calcium-neuronal nitric oxide synthase (nNOS) pathway in neurodegeneration…………………………24 4.4.1. Functions of NMDAR ………………………………………………… 24 4.4.2. Calbindin D28 K (CB) 25 4.4.3. nNOS ……………………………………………………………………26 4.5. Chemokines/chemokine receptors……………………………………………….27 4.5.1. Historical discovery of chemokines/chemokine receptors……………… 27 4.5.2. Chemokines/chemokine receptors in the normal central nervous system (CNS) .28 4.5.2.1. Chemokines/chemokine receptors and brain development……….28 4.5.2.2. Chemokines/chemokine receptors in the normal adult brain…… 29 4.5.3. Roles of chemokines/chemokine receptors in neurodegeneration……… .30 4.5.3.1. Stromal Cell-Derived Factor (SDF-1)………………………….30 4.5.3.2. Fractalkine……………………………………………………… .31 4.5.3.3. Monocyte Chemoattractant Protein (MCP-1)………………… 32 5. Animal model of the present study for molecular analysis of neurodegeneration: vagotomy…………………………………………………………34 5.1. Structure and function of the vagus nerve system.……………………………….34 5.2. Degeneration of the vagal motoneurons in the model of vagotomy…………… .37 6. Cell replacement in the CNS: potential neuroregeneration by stem cells…………….38 6.1. Mesenchymal Stem Cells (MSCs)……………………………………………… 38 6.1.1. Historical discovery of MSCs…………………………………………… .38 6.1.2. Biological characteristics of MSCs……………………………………… .39 ix 6.1.2.1. Heterogeneity of MSCs……………………………………………39 6.1.2.2. Expression of cytokines by MSCs……………………………… .40 6.1.2.3. Proliferation and multipotent differentiation of MSCs……………40 6.1.3. Transdifferentiation of MSCs into neurons and glia……………………….41 6.1.4. Therapeutic potential of MSCs to treat CNS diseases and injuries……… 42 6.1.5. Directed migration of transplanted MSCs…………………………………43 6.1.6. Mechanisms of cell migration: role of chemokines/chemokine receptors 43 6.1.6.1. SDF-1/CXCR4…………………………………………………….44 6.1.6.2. Fractalkine/CX3CR1………………………………………………45 6.1.6.3. MCP-1/CCR2…………………………………………………… 45 6.1.7. Animal model of the present study of MSCs migration: unilateral avulsion of the hypoglossal nerve….…………………………………… .46 6.1.7.1. Structure of the hypoglossal nerve system……………………… 46 6.1.7.2. Unilateral avulsion of the hypoglossal nerve…………………… 47 6.2. Adult neural stem or progenitor cells……………………………………………48 6.2.1. Historical discovery of neural stem or progenitor cells in the adult brain .48 6.2.2. Neural stem or progenitor cells in the subventricular zone (SVZ)……… 49 6.2.2.1. Origin of the postnatal SVZ………………………………………49 6.2.2.2. Architecture of the SVZ………………………………………… 49 6.2.2.3. Identity of neural stem or progenitor cells in the SVZ………… .50 6.2.3. Migration of endogenous neural stem or progenitor cells……………… .51 6.2.3.1. Migration of endogenous neural stem or progenitor cells in the SVZ……… ……………………………………………….51 A CCR2 B CCR5 C CXCR4 D CX3CR1 Fig.48. rMSCs express CCR2, CCR5, CXCR4 and CX3CR1 at the protein level. rMSCs are stained with anti-CCR2 (A), CCR5 (B), CXCR4 (C) or CX3CR1 (D) antibodies and are then analyzed by flow cytometry. The shaded regions represent isotype control staining and the open regions represent the chemokine receptors staining. A CCR2 B CCR5 C CXCR4 D CX3CR1 E Fig. 49. Localization of CCR2, CCR5, CXCR4 and CX3CR1 in rMSCs. Immunohistochemistry reveals the localization of CCR2 (A), CCR5 (B), CXCR4 (C) and CX3CR1 (D) in the membrane and cytoplasm of rMSCs. Note that the immunostaining of CXCR4 is more intense in RS cells (arrow heads) than in mMSCs (arrows). (E): Immunostaining of CCR5 on parietal cortex as a typical negative control for localization of CCR2, CCR5, CXCR4 and CX3CR1 in rMSCs. No CCR5 immunopositive cells are observed in the parietal cortex. Scale bar = 25µm Number of migrating cells/HPF Effect of Fractalkine on Migration of rMSCs in vitro PTx untreated 60 PTx treated ** 40 ** 20 Medium 50 250 500 Fractalkine Concentration [ng/ml] Fig. 50. Effect of rrfractalkine on migration of rMSCs. A significant increase in the number of migrated rMSCs is found at the concentrations of and 50 ng/ml fractalkine (p[...]... causally related to the degree of brain injury, includes: (1) the capacity of pro-inflammatory cytokines to exacerbate brain damage; (2) the capacity of proinflammatory cytokine blockade to reduce ischemic brain damage; and (3) depletion of circulating neutrophils reduces ischemic brain injury (Feuerstein et al., 1998) On the other hand, anti-inflammatory cytokines in the CNS maintain homeostasis and. .. that re-establishes a resting immunological status However, cytokines are involved not only in the immune response but also in a variety of physiological and pathological processes In general, they are crucial participants in receptor-mediated intercellular signaling that regulate cells engaged in innate and adaptive immunity, inflammation, cell growth and differentiation, cell death, angiogenesis and. .. (frequently in a INTRODUCTION 9 subplasmalemmal distribution), a decrease in cell volume, and alterations to the plasma membrane resulting in the recognition and phagocytosis of apoptotic cells, thereby preventing an inflammatory response 3.1.3 Apoptosis in the model of axotomy Inappropriate apoptosis is implicated in many human diseases, including neurodegenerative diseases such as Alzheimer’s disease and. .. in the content of intracellular fluid and subsequent swelling The organelles of the necrotic cells also become swollen and finally disintegrated The DNA becomes randomly fragmented and the entire cell undergoes lysis with the resultant spillage of intracellular contents to the surrounding tissues The spillage of lytic enzymes can cause damage to the surrounding tissues and may lead to inflammation INTRODUCTION... defined by the movement of macromolecules and organelles through an axon Most materials like tubulin, actin and neurofilamentous proteins are transported anterogradely from the perikaryon to the axon terminal at a fast and a slow rate In mammals, fast transport advances at 200-400 mm a day while slow transport at 0.2-6 mm a day (Lasek et al., 1984; McQuarrie et al., 1986) Some materials such as neurotrophic... (Grafstein and Murray, 1969; Grafstein, 1971) After 1-2 days, there may be a decrease in the amount of transported transmitterassociated materials (Boyle and Gillespie, 1970; Kirk, 1974), which may reflect their decreased content in the neuron Still later, after axonal outgrowth has been initiated, there may be an increase in the overall amount of proteins that are axonally transported or in their rate... and Dubois-Dauphin, 1996) in neonatal and adult rodents after axotomy of the optic, sciatic and facial nerves, respectively 3.2 Necrosis In contrast to the active form of apoptotic cell death, necrosis is a passive form of cell death caused by gross physical or chemical insults Necrotic cell death is characterized by the early loss of plasma membrane integrity The leaky membrane leads to increase in. .. The expression of iNOS protein and mRNA was induced in the DMV of vagotomized rats as shown by immunohistochemistry, in situ hybridization and real-time PCR analysis The enhanced bcl-2 and reduced bax mRNA levels and subsequent upregulation of both bcl-2 and bax mRNA as well as protein level in the DMV of vagotomized rat were observed In addition, the increase of caspase-3 mRNA level was detected in. .. protein levels Recombinant human SDF-1α (rhSDF-1α), the ligand for CXCR4 and recombinant rat fractalkine (rrfractalkine), the ligand for CX3CR1 induce the migration of rMSCs in a heterotrimeric G protein-dependent manner in vitro Furthermore, rhSDF-1α injected intracerebrally acts as a potent stimulus for the homing of transplanted rMSCs to the site of injection in the brain rMSCs, transplanted into the. .. ischemia, brain trauma, and neurodegenerative diseases have been developed to examine the mechanisms underlying neurodegeneration Animal models of axotomy in different systems have been studied for many years to gain insight into the mechanisms of progressive neuronal injury and degeneration (Lieberman, 1971; Torvik and Skjorten, 1971; Matthews, 1973; Decker, 1978; Al Abdulla et al., 1998; Ginsberg and Martin, . Associate Professor Samuel Sam Wah Tay, Department of Anatomy, National University of Singapore, for his innovative ideas, invaluable guidance, constant encouragement, infinite patience, and. ABC avidin-biotin complex α-MEM alpha minimal essential medium AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate AP alkaline phosphate BBB blood-brain-barrier CaBPs Ca 2+ binding. Roles of chemokines in the migration of neural stem or progenitor cells 54 7. Aims of the present study ………………………………………………………… 55 7.1. Molecular analysis of the degeneration of the vagal motoneurons