Parkinson’s disease (PD) is the second most common neurodegenerative disorder primarily affecting the aging population over the age of sixty. Characterized by the significant degeneration of dopaminergic (DAergic) neurons of the substantia nigra causing severe motor dysfunction. Although the exact pathogenesis of this disease is still unknown endoplasmic reticulum stress and mitochondrial dysfunction are believed to play a role. PD is diagnosed after severe DAergic neuron degeneration, and yet is still often misdiagnosed. There is a need for a definitive diagnostic test for the early detection of PD. Current therapies only relieve symptoms and do not stop disease progression. Neurotrophic factors (NTF) are naturally occurring proteins that promote the survival, differentiation and maintenance of neurons and present a promising candidate for the treatment of PD. Cerebral dopamine neurotrophic factor (CDNF) is a novel NTF that protects and rescues DAergic neurons. The present study investigated the role of DAergic activity and CDNF mRNA expression in C. elegans, as well as understanding how does PD affect the endogenous levels of CDNF protein and mRNA expression. We demonstrated that of the various dopamine (DA) synthesis and transport mutants tested, the impaired synthesis of DA from levodopa is linked to the up regulation of CDNF. Also, following unilateral 6-hydroxydopamine (6-OHDA) lesioning protein and mRNA expression of CDNF was not affected implicating ER stress as inducing a possible compensatory up regulation of CDNF, thus returning levels to normal. CDNF mRNA expression was determined to decline with age and possibly increase ones vulnerability to developing a neurodegenerative disorder. An increase mRNA expression of CDNF in the PD patient population was found to be specific to platelets. Stroke patients showed an increase in CDNF expression in whole blood. In conclusion, these findings highlight the importance of the relationship between CDNF and ER stress and warrants further investigation. / Thesis / Master of Science (MSc)

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No description available.

Alzheimer's disease is a progressive neurodegenerative disorder of the central nervous system. One pathological characteristic is excessive neuronal loss in specific regions of the brain. Among the areas most severely affected are the basal forebrain cholinergic neurons and their projection regions, the hippocampus and cortex. Neurotrophic factors, particularly the neurotrophins nerve growth factor and brain-derived neurotrophic factor, play an important role in the development, regulation and survival of basal forebrain cholinergic neurons. Furthermore, brain-derived neurotrophic factor regulates the function of hippocampal and cortical neurons. Neurotrophins are synthesized in hippocampus and cortex and retrogradely transported to the basal forebrain. Decreased levels of neurotrophic factors are suspected to be involved in the neurodegenerative changes observed in Alzheimer's disease. We examined autopsied parietal cortex, hippocampus and nucleus basalis of Meynert samples from age- and gender-matched Alzheimer's diseased and neurologically non-impaired individuals using the quantitative technique of competitive RT-PCR. We also examined parietal cortex samples by Western blotting. We demonstrate a 3.4-fold decrease in brain-derived neurotrophic factor mRNA levels in the parietal cortex of patients with Alzheimer's disease compared to controls (p < 0.004) but fail to observe changes in BDNF protein levels in that brain region. We also demonstrate, for the first time, BDNF mRNA in the nucleus basalis of Meynert and report an age-related decline in the levels of BDNF mRNA in both control and AD samples. Using the competitive RT-PCR technique we fail to observe differences in BDNF mRNA levels in the hippocampus between AD and control subjects, conflicting with previous in situ hybridization studies and RNase protection assays. A decrease in brain-derived neurotrophic factor synthesis could have detrimental effects on hippocampal, cortical and basal forebrain cholinergic neurons and may account for their selective vulnerability in Alzheimer's disease. / Thesis / Master of Science (MS)

Dans le système nerveux central (SNC), l'hippocampe est une structure majeure pour les fonctions cognitives et comportementales. Le Brain-Derived Neurotrophic Factor (BDNF), un acteur clé dans ces fonctions neuronales, est fortement exprimé dans l'hippocampe. La structure du gène Bdnf murin est complexe, comportant 8 exons non codants (I à VIII), chacun avec un promoteur spécifique (1 à 8) et un exon IX codant commun. Les glucocorticoïdes (GC) exercent des actions pleiotropes sur ces processus neuronaux en se liant et en activant le récepteur des glucocorticoïdes (GR), et le récepteur des minéralocorticoïdes (MR). Le GR est un facteur de transcription, modulant la transcription de ses gènes cibles, en se liant directement aux éléments de réponse des glucocorticoïdes ou en interagissant indirectement sur d’autres facteurs de transcription. Il a été suggéré que l'expression de Bdnf est régulée par le stress et les concentrations élevées de GC. Cependant, il reste à définir si BDNF est un gène cible du GR et quels sont les mécanismes moléculaires impliqués. Dans ce travail, nous avons démontré que les fortes concentrations de GC diminuent l'expression de l'ARNm de Bdnf via le GR dans divers modèles cellulaires neuronaux. Dans des cultures primaires de neurones hippocampiques de souris et dans les cellules BZ, les transcrits de BDNF contenant l’exon IV et VI sont reprimés par le GR. Par ailleurs les transfections transitoires démontrent que l’activité du promoteur 4 est diminuée par GR. Les expériences de mutagenèse et de ChIP ont révélé que la répression induite par le GR sur l'expression et l’activité transcriptionnelle de Bdnf implique un petit fragment de 74 bp situé dans le promoteur en amont de l'exon IV. La localisation précise de l’interaction génomique du GR et les facteurs de transcription potentiels mis en jeu restent à identifier. Ce travail a contribué à une meilleure compréhension des mécanismes impliqués dans la régulation de l’expression de Bdnf par GR. Il apporte de nouveaux éléments sur les interactions moléculaires et fonctionnelles entre la signalisation GC et celle de BDNF dans les neurones, d’importance majeure dans la physiopathologie du SNC. / In the central nervous system (CNS), the hippocampus is a structure of major importance for cognitive and behavioral functions. The brain-derived neurotrophic factor (BDNF), a key player in such neuronal functions is highly expressed in the hippocampus. Rodent Bdnf gene structure is relatively complex, composed of 8 noncoding exons (I to VIII), each one with a specific promoter (1 to 8), and one common coding exon IX. Glucocorticoids (GC) exert pleiotropic actions on neuronal processes by binding to and activating the glucocorticoid receptor (GR), as well as the mineralocorticoid receptor (MR). GR functions as a transcription factor, directly by interacting to glucocorticoid response elements or indirectly by interacting with other transcription factors, leading to the regulation of target gene transcription. It has been suggested that Bdnf expression is regulated by stress and high GC concentrations. However, it remains to define whether Bdnf is a GR target gene and what are the underlying molecular mechanisms. Herein, we demonstrate that high GC levels downregulate total Bdnf mRNA expression via GR in various in vitro neuron-like cellular models. In primary cultures of mouse hippocampal neurons and BZ cells, BDNF IV- and VI-containing transcripts are involved in this regulatory mechanism. Moreover, in transient transfections, promoter 4 activity was reduced by activated GR. Furthermore, ChIP analysis and mutagenesis experiments demonstrate that the GR-induced repression on Bdnf expression and transcriptional activities occurs through GR binding to a small 74 bp promoter sequence upstream of exon IV. The exact GR binding site on DNA and its putative transcription factor partners are currently under investigation. Altogether, these findings contribute to a better understanding of the mechanisms by which GR represses BDNF expression. Our study brings new insights into the molecular interactions between GC signaling and BDNF signaling in neurons, both important pathways in the pathophysiology of the CNS.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 86-111).

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy