No description available.

La maladie de Parkinson (MP) se caractérise par des troubles moteurs d'évolution progressive, conséquence de la dégénérescence des neurones dopaminergiques de la substance noire. Dans cette maladie, le vieillissement est un facteur de prédisposition majeur. Au cours de ma thèse, j'ai examiné dans un premier temps, les modèles in vivo utilisés au laboratoire pour étudier la MP chez la drosophile, à savoir l'expression de l'α-synucléine et l'intoxication au paraquat. J'ai ainsi contribué à l'identification d'une sous-population de neurones dopaminergiques impliquée dans les effets locomoteurs de l'α-synucléine et à la mise en évidence du rôle d'un récepteur dopaminergique de type D1 dans la neurotoxicité du paraquat. J'ai ensuite montré que l'activité des neurones dopaminergiques accélère la sénescence et diminue la durée de vie des drosophiles en contribuant de manière significative à la production des DRO dans le cerveau. Enfin, nous avons identifié et caractérisé chez la drosophile une protéine homologue de LAMP-2A, le récepteur lysosomal de l'autophagie médiée par les chaperonnes (AMC), et démontré que l'augmentation de la clairance neuronale des protéines cytosoliques par l'expression du LAMP-2A humain ou de son homologue de drosophile a des effets positifs sur le déclin locomoteur lié à l'âge et la résistance aux facteurs de la MP, mais qu'elle n'augmente pas la longévité. Ces résultats suggèrent que l'AMC est un mécanisme conservé chez la drosophile et que son activation protège contre le stress oxydatif et le vieillissement neuronal. / Parkinson's disease (PD) is characterized by progressive motor disorders resulting in dopaminergic neurons degeneration in the substantia nigra. In this pathology, aging is a major predisposing factor. During my thesis, I examined initially, in vivo models used in the laboratory to study the PD in Drosophila, namely the expression of α-synuclein and paraquat. I have contributed to the identification of dopaminergic neurons subpopulation involved in locomotor effects of α-synuclein and highlighting the role of a dopamine D1-like receptor in neurotoxicity of paraquat. I then shown that the activity of dopaminergic neurons accelerated senescence and decreases Drosophila lifespan by contributing significantly to the production of ROS in the brain. Finally, we identified and characterized Drosophila homologous of LAMP-2A protein, involves in autophagy-mediated chaperone (AMC), and demonstrated that the increase in neuronal clearance of cytosolic proteins by the expression human LAMP-2A or its Drosophila homologue has positive effects on the locomotor decline associated with aging and resistance against PD factors, but it does not increase longevity. These results suggest that the CMA is a conserved mechanism in Drosophila and its activation protects against oxidative stress and neuronal aging.

No description available.

The aim of this thesis was the functional characterization of the zebrafish parl (Presenilin-Associated Rhomboid-Like) genes which code for mitochondrial proteins involved in cell survival. A mutation in PARL has been described in Parkinson’s disease patients. I investigated the role of mitochondrial PD-related proteins using a zebrafish parla and parlb deficiency model. I found that the knockdown of both parl genes is lethal. Parla plays a larger role in patterning of the DA neurons in the ventral diencephalon than Parlb. The human PARL rescued the double morphant phenotype, suggesting function conservation between zebrafish and humans. I was able to rescue the mortality and DA neuron mispatterning observed in double morphants with synthetic pink1 mRNA. This suggests that parl genes are epistatic to pink1 in zebrafish. To visualize mitochondria specifically in dopaminergic neurons of live zebrafish, I established a transgenic line Tg(dat:tom20 MLS-mCherry) where regulatory elements of the dopamine transporter (dat) were used to drive expression of a Tom20-mCherry fusion protein that is targeted to the mitochondria. I characterised the expression of Tom20-mCherry to the mitochondria of the majority of DA neuron groups. In addition, I observed a decrease in mCherry fluorescence following MPTP exposure of live fish. The PD-related mutation in PARL is located in a cleavage site of the mammalian protein, which is necessary for the production of the beta peptide; however, this site is predicted to be absent in the zebrafish Parls. To establish the cleavage patterns of the zebrafish Parls and compare them to those of human PARL, I examined the cleavage of Parl-Flag constructs in cultured cells. I detected one band for Parla-Flag and two bands representing Parlb-Flag. The parla and parlb deficiency model along with the characterization of the cleavage patterns of Parl and the Tg(dat:tom20 MLS-mCherry) transgenic line are tools which will help elucidate the role of mitochondrial proteins in PD research.

Thesis (M.Sc. (Pharmacy)) - Rhodes University, 2008.

Thesis (M.S.)--Mississippi State University. Department of Basic Sciences. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, February 2010.

Midbrain dopamine (DA) neurons play a key role in a wide range of behaviours, from motor control, motivation, reward and reinforcement learning. Disorders of midbrain dopaminergic signaling is involved in a variety of nervous system disorders including Parkinson’s disease, schizophrenia and drug addiction. Understanding the basis of how dopaminergic neuronal activity in the substantia nigra pars compacta (SNc) governs movements, requires a deep appreciation of how afferent inputs of various neurotransmitter systems create a neuronal circuit that precisely modulates DA neuronal excitability. Two brainstem cholinergic neuclei, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT), have major cholinergic projections to the SNc, despite the fact that the precise mechanisms of cholinergic modulation of DA neuronal activity mediated by nAChRs remain unclear. To dissect out the modulatory roles of the cholinergic system in regulating DAergic neuronal activity in the SNc and locomotion, we employed optogenetics along with electrophysiological and behavioural approaches. My results from whole-cell recordings from lateral and medial SNc DA neurons revealed that lateral DA neurons received predominantly excitatory nAChR mediated cholinergic neurotransmission (monosynaptic nicotinic or disynaptic glutamatergic responses) resulting in greater excitability of DA neurons both at 5 and 15 Hz blue LED light stimulation of cholinergic terminals. However, medial SNc DA neurons received predominantly biphasic current responses that were both inhibitory GABAergic and excitatory nAChR mediated cholinergic neurotransmission. This led to a net inhibition of action potential firing of DA neurons at 5 Hz blue LED light stimulation of cholinergic terminals, while at 15 Hz stimulation there was an initial inhibition followed by a significant increase of the baseline action potential firing frequency. Furthermore, in vivo optogenetic experiments showed that activation of the cholinergic system in the medial SNc resulted in decreased locomotion, while for the lateral SNc led to increased locomotion. Together our findings provide new insights into the role of the cholinergic system in modulating DA neurons in the SNc. The cholinergic inputs to different subregions of the SNc may regulate the excitability of the DA neurons differentially within a tight range from excitation to inhibition which may translate into different kinds of locomotor behaviour. / Graduate

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.