No description available.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.

No description available.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non-specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock-out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain-specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

Rotenone is a commercially available pesticide with a variety of industrial applications. However, occupational exposure to rotenone has been implicated in the development of Parkinson’s disease. To explore the mechanism of dopamine neuron death secondary to rotenone exposure, the zebrafish was used as a live animal screening tool for environmentally-induced Parkinson’s disease. After testing a variety of small molecule compounds on embryonic zebrafish for their potential to cause dopamine neuron loss, we identified that rotenone exposure induces a bradykinetic dopamine neuron loss phenotype. This phenotype was characterized by decreased locomotion, sensory insensitivity, and a transient but marked decrease in the number of dopamine neurons in embryos exposed to 100nM rotenone, with a concomitant decrease in dopamine transporter mRNA levels. The dopamine neuron deficits were observed in the subpallium, pretectum, olfactory bulb, but these losses were most pronounced in the ventral diencephalon after rotenone exposure. Rotenone damages the mitochondria, generating reactive oxygen species (ROS), and subsequently induces ROS-mediated apoptosis in these dopamine neurons. The rotenone-induced dopamine neuron loss and locomotion phenotypes could be partially rescued in zebrafish larvae with ascorbic acid co-treatment during rotenone exposure. Adults raised from zebrafish embryos exposed to rotenone did not show any deficits to their dopamine neuron distribution, but did show anxiety-like behaviors and upregulation of dopamine receptor D1 mRNA levels. These results suggest that rotenone exposure can cause dopamine neuron death through ROS- mediated apoptosis, and supports an environmental cause of Parkinson’s disease.
 La roténone est un pesticide disponible dans le commerce et utilisé pour une variété d’usages industriels. Cependant, l’exposition à la roténone a été impliquée dans le développement de la maladie de Parkinson. Afin d’explorer le mécanisme menant à la perte de neurones dopaminergiques suite à une exposition à la roténone, le poisson-zèbre (Danio rerio) a été utilisée comme modèle animal du développement de la maladie de Parkinson attribuable à des causes environnementales. Après avoir testé une série de petites molécules sur des embryons de poisson-zèbre pour déterminer leur capacité à causer une perte de neurones dopaminergiques, nous avons identifié la roténone comme causant un phénotype de perte de neurones associée à une bradykinésie. Ce phénotype était caractérisé par une perte de la locomotion, une insensibilité sensorielle, et une diminution transitoire mais marquée du nombre de neurones dopaminergiques chez des embryons exposés à une concentration de roténone de 100nM. Ceci coïncidait avec une réduction des niveaux d’expression du gène du transporteur de la dopamine (dat). Des pertes de neurones dopaminergiques furent observés dans le sub-pallium, le pré-tectum et le bulbe olfactif mais étaient plus prononcées dans le diencéphale ventral. La roténone cause des dommages aux mitochondries en générant des dérivés réactifs de l’oxygène (ROS) et, subséquemment, induit une apoptose attribuable aux ROS dans les neurones dopaminergiques. La perte de neurones dopaminergiques due à la roténone ainsi que les déficits locomoteurs ont pu être partiellement empêchés par un co-traitement à l’acide ascorbique. Les poissons adultes ayant été exposés à la roténone au stade embryonnaire ne montrèrent pas de déficits quant à la distribution des neurones dopaminergiques mais présentaient des comportements indiquant un plus haut niveau d’anxiété ainsi qu’une augmentation des ARNm du récepteur D1 de la dopamine. Ces résultats suggèrent que l’exposition à la roténone peut causer la mort des neurones dopaminergiques via une apoptose médiée par les ROS et pourrait constituer une cause environnemental de la maladie de Parkinson.

Parkinson’s disease is a movement disorder characterized by nigrostriatal dopamine pathway degeneration and neuronal α-synuclein accumulation. Pathogenesis is associated with mutations in α-synuclein and Gba1 encoding alleles. Animal models created to date do not recapitulate the spectrum of clinical disease features. This thesis characterizes the bi-genic Synergy mouse, hypothesized to demonstrate motor behavioural and histological abnormalities downstream of α-synuclein overexpression and mutated Gba1. Synergy and SNCA mice (overexpressed α-synuclein with wild-type Gba1) have early onset deficits in motor coordination, muscle strength and nest building. Both exhibit increased α-synuclein concentration in the brain and cerebellar inclusions positive for two markers of pathological α-synuclein processing. Overall mutant Gba1 expression within Synergy mice does not worsen the behaviour or the histopathological findings associated with overexpression of human α-synuclein in SNCA mice. Future studies will determine whether mutant Gba1 expression alters cognitive behaviour and/or lipid homeostasis in this new bi-genic model of Parkinson’s disease.

Previous controlled studies have shown that severe daytime restriction of dietary protein improves the efficacy of L-dopa and reduces response fluctuations in some Parkinson's disease in-patients. The main purpose of the present study was to investigate the nutritional adequacy of the restricted protein diet. Other objectives were to assess the patients' acceptance of the diet and to identify the practical difficulties encountered in following the diet at home. The effect of the restricted protein diet on the subjects' response to levodopa was also examined. Subjects were 11 free-living, otherwise healthy Parkinson's disease patients who suffered from unpredictable response fluctuations to Sinemet (L-dopa containing medicine). This condition is also described as the "on-off" phenomenon. They were counselled to consume a daytime restricted protein diet for 6 weeks (mean ± s.d. protein intake before evening meal 12 ± 2 g for females and 14 ± 3 g for males). Food intake was unrestricted from dinner until bedtime, and subjects were encouraged to consume nutrient-dense foods during this period. The subjects were required to complete a series of three 6-day food records and "on-off" charts (pre-diet, diet week 2, diet week 6). The "on-off" charts indicated the daily number of hours spent in the "on" state (when medication is effective and parkinsonian symptoms are controlled) and in the "off" state (when medication is not effective and symptoms are not well controlled). Hemoglobin, plasma albumin, prealbumin and ferritin were measured before and after the 6-week diet period. Subjective evaluation questionnaires were completed by all participants and their spouses or caregivers. Results from dietary record analysis showed that the restricted protein diet was associated with significant decreases in total intakes of protein, calcium, iron, magnesium, phosphorus, niacin, riboflavin, vitamin B6 and pantothenate, in comparison to "usual" intakes. Intake of energy, carbohydrates, lipids, potassium, thiamin, folacin, and vitamins A, C and B12 did not change significantly. While on the restricted protein diet, only calcium intake was substantially less than the RNI. Biochemical measures of nutritional status were not significantly reduced. Mean body weight tended to decrease (p=.054) over the first 2 weeks and then stabilized until the end of the study period. The results of the "on-off" charts showed that three of the eleven subjects significantly increased their daily time "on" while on the restricted protein diet. Subjectively, six individuals noted an improvement in daytime mobility and indicated that they would maintain the diet for an indefinite period of time. Problems identified by the subjects included hunger prior-'to the evening meal and a lack of variety in food choices. These results show that otherwise healthy and motivated patients with Parkinson's disease can maintain an adequate intake of energy, protein, and most nutrients while on the daytime restricted protein diet. The diet appeared to be relatively well tolerated by patients who obtained a subjective benefit. It is suggested that in patients whose regular diets are marginally adequate, the restricted protein diet might compromise nutrient intakes. Counselling by a registered dietitian is recommended for all patients who undertake this type of diet. / Land and Food Systems, Faculty of / Graduate

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.