Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease, characterised by demyelination in the central nervous system (CNS). The exact pathophysiology of MS is still unknown but it is believed to be associated with infiltration of T cells and activation of microglia that result in myelin damage leading to neurological deficits including neuropathic pain. Current treatment strategies such as glatiramer acetate have recognized the importance of BDNF in myelin repair. In addition, the proposed role of the chemokine CX3CL1 and its receptor CX3CR1 in the control of microglia activation and leukocyte infiltration place this chemokine in an important position in regulation of MS-induced neuropathic pain. In this research study, the experimental autoimmune encephalomyelitis (EAE) rat model of MS was used to examine the role of BDNF in myelin repair as well as CX3CL1’s role in neuropathic pain. Methods: A total of 66 adult female Lewis rats are divided into 3 experimental groups: naïve control, active control and active EAE. Naïve control animals do not receive any injections. Active control animals receive 2 intraperitoneal injections of pertussis toxin and injections of Freund’s adjuvant and Mycobacterium Tuberculosis. Active EAE animals receive the same regimen administered to active controls plus full inoculation with fatty acid and Guinea pig myelin basic protein. Expressions of BDNF, CX3CL1 and CX3CR1 in a time dependent mansion (day 0, 3, 6, 9, 12 &15) were examined using immunohistochemistry (IHC), ELISA, Western blot, RT-PCR and real time-PCR. Results: There was a significant increase in BDNF, CX3XL1 and CX3CR1 expression of protein and mRNA in DRG at day 12 after induction of MS. The neurons and glial cells were identified to express BNDF, CX3XL1 and CX3CR1 in the spinal cord of EAE animal. Conclusion: The antigenic-induced expression of BDNF within the DRG may represent a key element involved in facilitating central myelin repair. In addition, the chemokine CX3CL1 and its receptor CX3CR1 represent key mediators involved in the development of MS-induced pain. Keywords: Multiple sclerosis, MS, experimental autoimmune encephalomyelitis, EAE, CX3CL1, CX3CR1, neuropathic pain, myelin repair

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease, characterised by demyelination in the central nervous system (CNS). The exact pathophysiology of MS is still unknown but it is believed to be associated with infiltration of T cells and activation of microglia that result in myelin damage leading to neurological deficits including neuropathic pain. Current treatment strategies such as glatiramer acetate have recognized the importance of BDNF in myelin repair. In addition, the proposed role of the chemokine CX3CL1 and its receptor CX3CR1 in the control of microglia activation and leukocyte infiltration place this chemokine in an important position in regulation of MS-induced neuropathic pain. In this research study, the experimental autoimmune encephalomyelitis (EAE) rat model of MS was used to examine the role of BDNF in myelin repair as well as CX3CL1’s role in neuropathic pain. Methods: A total of 66 adult female Lewis rats are divided into 3 experimental groups: naïve control, active control and active EAE. Naïve control animals do not receive any injections. Active control animals receive 2 intraperitoneal injections of pertussis toxin and injections of Freund’s adjuvant and Mycobacterium Tuberculosis. Active EAE animals receive the same regimen administered to active controls plus full inoculation with fatty acid and Guinea pig myelin basic protein. Expressions of BDNF, CX3CL1 and CX3CR1 in a time dependent mansion (day 0, 3, 6, 9, 12 &15) were examined using immunohistochemistry (IHC), ELISA, Western blot, RT-PCR and real time-PCR. Results: There was a significant increase in BDNF, CX3XL1 and CX3CR1 expression of protein and mRNA in DRG at day 12 after induction of MS. The neurons and glial cells were identified to express BNDF, CX3XL1 and CX3CR1 in the spinal cord of EAE animal. Conclusion: The antigenic-induced expression of BDNF within the DRG may represent a key element involved in facilitating central myelin repair. In addition, the chemokine CX3CL1 and its receptor CX3CR1 represent key mediators involved in the development of MS-induced pain. Keywords: Multiple sclerosis, MS, experimental autoimmune encephalomyelitis, EAE, CX3CL1, CX3CR1, neuropathic pain, myelin repair

Bien que les troubles anxio-dépressifs représentent une des principales causes d’invalidité et un des plus sérieux problèmes de santé dans le monde, les mécanismes neurobiologiques à l’origine de ces affections demeurent méconnus. Les processus mis en jeu semblent multiples et complexes : passant par des déséquilibres au niveau des neurotransmetteurs jusqu’à des modifications de la plasticité neurale et du remodelage cellulaire. Les neurotrophines étant considérées comme les principaux régulateurs de la plasticité, l’hypothèse d’un lien causal entre le niveau de neurotrophine, principalement de l’une d’entre elles, le Brain-Derived Neurotrophic Factor (BDNF) et l’apparition de troubles anxio-dépressifs a ainsi été proposée. Notre travail a donc eu pour objectif l’étude de l’implication du BDNF dans l’étiopathogenèse et le traitement des troubles anxio- dépressifs à travers l’utilisation de modèles murins. Etant donné que plusieurs études cliniques et précliniques ont montré une implication du BDNF dans plusieurs traits psychologiques et comportementaux, nous avons cherché à déterminer si des différences dans le gène BDNF pouvaient être à l’origine de la grande hétérogénéité comportementale des différentes souches de souris. Nous rapportons dans cette étude l’existence d’un polymorphisme sur un seul nucléotide (SNP) à l’origine d’un changement d’acide aminé (une leucine est remplacé par une méthionine) en position 32 dans la séquence du prodomaine du BDNF. Nous démontrons ensuite que, bien que ce SNP ne modifie par l’expression basale de BDNF dans le cerveau, ce polymorphisme est associé au phénotype « anxieux » des souris. Par contre, il n’est pas impliqué dans le comportement alimentaire, le toilettage, l’activité, l’apprentissage et la mémoire. / Although anxio-depressive disorders are a major cause of disability with important health consequences, the underlying neurobiological mechanisms remain largely unknown. Neurotransmitter imbalances can change numerous intracellular signaling pathways that ultimately result in lasting modifications in neural plasticity and cellular remodeling. Since neurotrophins, and particularly brain-derived neurotrophic factor (BDNF), are considered main regulators of neural plasticity, changes in the expression of these genes are an attractive mechanism by which normally differentiated brain cells may transform into a pathological anxio-depressive phenotype. A first study seven strains of mice with large behavioral differences were used to investigate whether differences in the expression and sequence BNDF may be associated with anxiety and depressive traits. A change of a single nucleotide in position 32 of prodomain sequence of BNDF, which resulted in a leucine being replaced by methionine, was associated with mice exhibiting the anxious phenotype. Mice carrying the Met allele had greater propensity to neophobic reactions and behaviors related to anxiety. However, this polymorphism did not alter the basal expression of the gene and other behavioral activities including appetite, thirst, grooming, learning and memory. Furthemore, when chronic stress slightly unpredictable (UCMS) was used in seven mouse lines, no association was observed between Leu 32 Met and depression-like effectors due to antidepressant treatments. When heterozygous (+/-) BNDF mice were used in the UCMS model, no changes were noted in physical observations (i.e, hair coat, weight), behavioral responses (to mimic anxiety, aggressiveness and resignation) and in the stress hormones; by contrast, some effects to the anti-depressants were different from the ones seen in homozygous (+/+) mice. In conclusion, BDNF polymorphisms can contribute to the various behavioral profiles exhibited by different strains of mice in test of anxiety. However, it does not appear to be involved in the etiopathogenesis of depression.

Polymerase gamma POL G1 mutator mice (POL G) are deficient in the mitochondrial DNA proof-reading capacity leading to an accumulation of mtDNA point mutations, resulting in accelerated aging phenotype and brain atrophy. Endurance exercise training reverses the phenotypic manifestations and rescues much of the progeroid aging phenotype, including brain atrophy. Neurogenesis is mediated by neurotrophins that stimulate cell growth and survival. One of the main neurotrophins is brain-derived neurotrophic factor (BDNF), which is secreted by muscle cells and has been shown to increase with acute exercise in brain and serum. Therefore, we investigated whether continuous delivery of BDNF by in-vivo gene therapy would improve the neurogenesis on the dentate gyrus in POL G mutator mice. Wild-type controls and POL G mutator mice were given intra-peritoneal injections of capsules containing recombinant G8 myoblasts that secreted BDNF, or vehicle (veh), over five months. Cell survival analysis at the level of the dentate gyrus in the brain was measured by BrdU analysis. By nine months of age, BDNF-injected POL G mutator mice did not exhibit improvements in neurogenesis in comparison with POL G controls. Motor assessment through rotarod performance showed no differences between wild type and POL G. CLAMS assessment demonstrated impairment of locomotor activity in POL G mice as expected; and no improvement in the POL G group treated with BDNF. Unexpectedly, wild type animals treated with BDNF exhibited decreased levels of locomotor activity similar to the POL G mutator mice. In conclusion, continuous BDNF administration did not improve neurogenesis at the level of the dentate gyrus in the POL G animal model. It is likely that the prevention of brain atrophy seen with endurance exercise is mediated by additional molecular factors, including BDNF. / Thesis / Master of Health Sciences (MSc) / Polymerase gamma POL G1 mutator mice (POL G) are deficient in the mitochondrial DNA proof-reading capacity leading to an accumulation of mtDNA point mutations, resulting in accelerated aging phenotype and brain atrophy. Endurance exercise training reverses the phenotypic manifestations and rescues much of the progeroid aging phenotype, including brain atrophy. Neurogenesis is mediated by neurotrophins that stimulate cell growth and survival. One of the main neurotrophins is brain-derived neurotrophic factor (BDNF), which is secreted by muscle cells and has been shown to increase with acute exercise in brain and serum. Therefore, we investigated whether continuous delivery of BDNF by in-vivo gene therapy would improve the neurogenesis on the dentate gyrus in POL G mutator mice. Wild-type controls and POL G mutator mice were given intra-peritoneal injections of capsules containing recombinant G8 myoblasts that secreted BDNF, or vehicle (veh), over five months. Cell survival analysis at the level of the dentate gyrus in the brain was measured by BrdU analysis. By nine months of age, BDNF-injected POL G mutator mice did not exhibit improvements in neurogenesis in comparison with POL G controls. Motor assessment through rotarod performance showed no differences between wild type and POL G. CLAMS assessment demonstrated impairment of locomotor activity in POL G mice as expected; and no improvement in the POL G group treated with BDNF. Unexpectedly, wild type animals treated with BDNF exhibited decreased levels of locomotor activity similar to the POL G mutator mice. In conclusion, continuous BDNF administration did not improve neurogenesis at the level of the dentate gyrus in the POL G animal model. It is likely that the prevention of brain atrophy seen with endurance exercise is mediated by additional molecular factors, including BDNF.

Neurotrophins are important signaling molecules in neuronal survival and differentiation. The precursor forms of neurotrophins (proneurotrophins) are the dominant form of gene products in animals, which are cleaved to generate prodomain and mature neurotrophins, and are sorted to constitutive or regulated secretory pathway and released. Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the brain development and in the pathogenesis of neurological diseases. In Huntington’s disease, the defective transport of BDNF in cortical and striatal neurons and the highly expressed polyQ mutant huntingtin (Htt) result in the degeneration of striatal neurons. The underlying mechanism of BDNF transport and release is remains to be investigated. Current studies were conducted to identify the mechanisms of how BDNF is transported in axons post Golgi trafficking. By using affinity purification and 2D-DIGE assay, we show Huntingtin-associated protein 1 (HAP1) interacts with the prodomain and mature BDNF. The GST pull-down assays have addressed that HAP1 directly binds to the prodomain but not to mature BDNF and this binding is decreased by PolyQ Htt. HAP1 immunoprecipitation shows that less proBDNF is associated with HAP1 in the brain homogenate of Huntington’s disease compared to the control. Co-transfections of HAP1 and BDNF plasmids in PC12 cells show HAP1 is colocalized with proBDNF and the prodomain, but not mature BDNF. ProBDNF was accumulated in the proximal and distal segments of crushed sciatic nerve in wild type mice but not in HAP1-/- mice. The activity-dependent release of the prodomain of BDNF is abolished in HAP1-/- mice. We conclude that HAP1 is the cargo-carrying molecule for proBDNF-containing vesicles and plays an essential role in the transport and release of BDNF in neuronal cells. 20-30% of people have a valine to methionine mutation at codon 66 (Val66Met) in the prodomain BDNF, which results in the retardation of transport and release of BDNF, but the mechanism is not known. Here, GST-pull down assays demonstrate that HAP1 binds Val66Met prodomain with less efficiency than the wild type and PolyQ Htt further reduced the binding, but the PC12 cells colocalization rate is almost the same between wt prodomain/HAP1 and Val66Met prodomain/HAP1, suggesting that the mutation in the prodomain may reduce the release by impairing the cargo-carrying efficiency of HAP1, but the mutation does not disrupt the sorting process. Recent studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro. Here, we show that proBDNF plays a role in the death of axotomized sensory neurons. ProBDNF, p75NTR and sortilin are highly expressed in DRG neurons. The recombinant proBDNF induces the dose-dependent death of PC12 cells and the death activity is completely abolished in the presence of antibodies against the prodomain of BDNF. The exogenous proBDNF enhances the death of axotomized sensory neurons and the antibodies to the prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduces the death of axotomized sensory neurons. We conclude that proBDNF induces the death of sensory neurons in neonatal rats and the suppression of endogenous proBDNF rescued the death of axotomized sensory neurons.

<p><p>Heterotrimeric G proteins play invaluable roles in cellular processes involving transmembrane signaling, particularly at sites of neuronal connectivity within the central nervous system (CNS). G&alpha;z is a member of the G&alpha;i subfamily of heterotrimeric G proteins that displays unique biochemical characteristics and is primarily expressed in neuronal and neuroendocrine cells. Studies in Gz&ndash;null mice over the past decade reveal that Gz significantly impacts responses to psychoactive drugs, and is capable of coupling to D2 dopamine, 5&ndash;HT1A serotonin, &mu;&ndash;opioid, and &alpha;2A&ndash;adrenergic receptors. These studies have suggested that Gz may play a critical role in diseases and disorders involving disruptions of monoamine neurotransmitter signaling in the brain such as depression, anxiety, drug abuse, ADHD, schizophrenia, drug addiction, and pain sensitivity. Much is still unknown about the roles and mechanisms of action of Gz in biology. </p></p><p><p>In this thesis, I have built on what is known regarding G&#945;z biochemistry by conducting a series of studies that provide further understanding of its role in the CNS, particularly in neuronal development and seizure susceptibility. Gz interacts with several proteins that act as regulators and effectors: RGSZ, adenylyl cyclase, EYA2, and Rap1GAP being the best characterized. A finding regarding its impact of Gz on neurotrophin signaling through RAP1GAP in particular has led to much of the work described here. The studies presented in this thesis indicate that G&alpha;z inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for G&alpha;z in regulation of neurotrophin signaling in the CNS that may have important implications for development and plasticity. Furthermore, G&alpha;z was shown to be uniquely distributed to synaptic vesicles suggesting that one mechanism underlying Gz biology may be the regulation of vesicle loading, docking, or release. Finally, I demonstrate that Gz-null mice are hypersusceptible to pilocarpine&ndash;induced seizures, and provide histology data indicating increased levels of zinc in the hippocampus. Taken together, these findings suggest that Gz plays a regulatory role at the intersection of neurotrophin and GPCR signaling in the CNS. </p></p> / Dissertation

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

Previous research in our laboratory has shown that the spinal cord is capable of a simple form of instrumental learning. Spinally transected rats that receive controllable shock to an extended hindlimb exhibit a progressive increase in flexion duration that reduces net shock exposure. Subjects that receive uncontrollable shock, on the other hand, do not exhibit an increase in flexion duration, and are unable to produce this instrumental response even when they are later tested with controllable shock. This behavioral deficit can also be elicited by intermittent shock to the tail, and as little as 6 minutes of this shock is sufficient to produce a deficit that can last up to 48 hours as shown by Crown, Ferguson, Joynes, and Grau in 2002. Instrumental training has been shown to provide a number of beneficial effects. The instrumental training regimen produces a lasting effect that enables learning when subjects are later tested with a more difficult response criterion. Similarly, instrumental training can provide protection against the deleterious effects of uncontrollable shock as shown by Crown and Grau in 2001. The present study aims to determine the role of brain-derived neurotrophin factor (BDNF) in the beneficial effects of instrumental training. Experiments 1 and 2 examined the role of BDNF in the facilitory effect of instrumental training. Through the inhibition of endogenous BDNF, Experiment 1 showed that BDNF is necessary for the facilitation effect. Experiment 2 demonstrated that exogenous BDNF can produce the facilitation effect in dose-dependent fashion. Experiment 3 showed that the inhibition of BDNF attenuates the protective effect of instrumental training. Likewise, Experiment 4 showed that exogenous BDNF can substitute for instrumental training, and produce this protective effect. Experiment 5 showed that exogenous BDNF can block the development of the deficit when given immediately after uncontrollable shock. Experiment 6 showed that exogenous BDNF can block the expression of the deficit. Taken together, these experiments outline a major role for BDNF in mediating the beneficial effects of instrumental learning in the rat spinal cord.

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