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.

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.

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

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.

No description available.

Orientador: Antonio Francisco Godinho / Resumo: O herbicida atrazina (ATR) é um praguicida usado intensivamente na agricultura, que persiste no meio ambiente e causa toxicidade em espécies não-alvo, inclusive seres humanos. Considerando o potencial da ATR para produzir efeitos no sistema nervoso central (SNC) e a falta de informações na literatura corrente sobre esse tipo de toxicidade, o objetivo deste projeto de pesquisa foi avaliar os efeitos da exposição subcrônica ao herbicida ATR sobre parâmetros neurocomportamentais específicos e estabelecer sua correlação com o estresse oxidativo (EO) em tecido cerebral de ratos e com o sistema dopaminérgico em regiões específicas do cérebro, nominalmente córtex pré-frontal (CPF), hipocampo (HIP) e corpo estriado. Ratos Wistar machos receberam solução salina (0,3 ml; controle - Ct), atrazina (ATR; 100 mg/kg), atrazina mais vitamin E (ATR; 100 mg/Kg + VE; 200 mg/kg), apenas vitamina E (VE; 200 mg/kg), atrazina mais levodopa (ATR; 100 mg/Kg + LEV; 20 mg/kg) e apenas levodopa (LEV; 20 mg/kg), por gavage, durante 28 dias. O comportamento foi avaliado utilizando arena de campo aberto (ACA), labirinto em cruz elevado (LCE) e hole-board (HB). O teste de estresse oxidativo no tecido cerebral avaliou os níveis de malondialdeído (MDA), capacidade antioxidante hidrofílica (CAH) e atividade das enzimas superóxido dismutase (SOD) e catalase (CAT). A dopamina (DA) foi quantificada por HPLC, em áreas cerebrais específicas (CPF, HIP e estriado). Exposição à ATR provocou ansiedade, diminuiu a coord... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Atrazine (ATR) herbicide is a pesticide used intensively in agriculture that persists in environment and causes toxicity on non-target species, include humans. Considering the potential of ATR to produce effects on the central nervous system (CNS) and the lack of information in the current literature about this type of toxicity, the aim of this research project was to evaluate the effects of subchronic exposure to ATR herbicide on specific neurobehavioral parameters and to establish its correlation with oxidative stress (OE) in rat brain tissue and with the dopaminergic system in specific brain regions, nominally prefrontal cortex (PFC), hippocampus (HIP) and striatum. Male Wistar rats received saline (0.3 ml; control - Ct), atrazine (ATR; 100 mg/kg), atrazine plus vitamin E (ATR; 100 mg/Kg + VE; 200 mg/kg) only vitamin E (VE; 200 mg/kg), atrazine plus levodopa (ATR; 100 mg/Kg + LEV; 20 mg/kg) and only levodopa (LEV; 20 mg/kg), via gavage, during 28 days. Behavior was assessed using open field arena (OF), elevated plus-maze (EPM) and hole-board (HB) apparatus. Oxidative stress assay in brain tissue evaluated malondialdehyde (MDA) levels, hydrophilic antioxidant capacity (HAC), and superoxide dismutase (SOD) and catalase (CAT) enzymes activity. Dopamine (DA) was quantified using HPLC, in specific brain areas (PFC, HIP and striatum). ATR exposure caused anxiety-like behavior, decreased motor coordination and did not alter the locomotor activity of the animals. ATR did not chang... (Complete abstract click electronic access below) / Doutor

Male and Female newborn rats were primed with either quinpirole 0.05 mg/kg IP or SKF-38393 0.1 mg/kg IP on days 1-11, 12-22 and 23-33 of their lives. When the rats reached the age of 13 weeks, they were placed on metal rods in an activity avoidance chamber, and light and electric current of 30V/0.8 mA were used on them as conditioning stimuli. Avoidance of the electric shock was considered a positive conditioned reaction. Training and memorizing the conditioned avoidance reflex consisted of a series of ten trials, 60 seconds apart, once a week for ten weeks. The mean number of positive responses after quinpirole was more profound in all priming intervals tested, as compared to SKF-38393, and was higher as the priming started later in life. Significantly higher scores were obtained by the female rats primed with quinpirole, as compared to the male rats primed with the same drug. These differences were much weaker with SKF-38393. These findings confirm that the central D2 receptor system is involved in learn ing and memorizing of Conditioned Avoidance Reflex much more than the D1 receptors do, and that female rats are more sensitive and retentive to this reflex.

Rats lesioned shortly after birth with 6-OHDA have been proposed to be a near-ideal model of severe Parkinson's disease, because of non-lethality of the procedure, near-total destruction of nigrostriatal dopaminergic fibers, and near-total dopamine (DA) denervation of striatum. There are scarce data that in Parkinson's disease, activity of the central histaminergic system is increased. Therefore, the aim of this study was to determine histamine content in the brain and the effect of histamine receptor antagonists on behavior of adult rats. At 3 days after birth, Wistar rats were pretreated with desipramine (20.0 mg/kg ip) 1 h before bilateral icv administration of the catecholaminergic neurotoxin 6-OHDA (67 μg base, on each side) or saline-ascorbic acid (0.1%) vehicle (control). At 8 weeks levels of DA and its metabolites l-3,4- dihydroxyphenylalanine (DOPAC) and homovanillic acid (HVA) were estimated in the striatum and frontal cortex by HPCL/ED technique. In the hypothalamus, hippocampus, frontal cortex, and medulla oblongata, the level of histamine was analyzed by immunoenzymatic method. Behavioral observations (locomotion, exploratory-, oral-, and stereotyped-activity) were additionally made on control and 6-OHDA neonatally lesioned rats. Effects of DA receptor agonists (SKF 38393, apomorphine) and histamine receptor antagonists (e.g., S(+)chlorpheniramine, H 1 ; cimetidine, H 2 ; thioperamide, H 3 agonist) were determined. We confirmed that 6-OHDA significantly reduced contents of DA and its metabolites in the brain in adulthood. Histamine content was significantly increased in the hypothalamus, hipocampus, and medulla oblongata. Moreover, in 6-OHDA-lesioned rats behavioral response was altered mainly by thioperamide (H 3 antagonist). These findings indicate that histamine and the central histaminergic system are altered in the brain of rats lesioned to model Parkinson's disease, and that histaminergic neurons exert a modulating role in Parkinsonian 6-OHDA-lesioned rats.

To assess the possible modulatory effects of noradrenergic and serotoninergic neurons on dopaminergic neuronal activity, the noradrenergic and serotoninergic neurotoxins DSP-4 N-(2-chlorethyl)-N-ethyl-2-bromobenzylamine (50.0 mg/kg, sc) and 5,7- dihydroxytryptamine (5,7-DHT) (37.5 μg icv, half in each lateral ventricle), respectively, were administered to Wistar rats on the first and third days of postnatal ontogeny, and dopamine (DA) agonist-induced behaviors were assessed in adulthood. At eight weeks, using an HPLC/ED technique, DSP-4 treatment was associated with a reduction in NE content of the corpus striatum (> 60%), hippocampus (95%), and frontal cortex (> 85%), while 5,7-DHT was associated with an 80-90% serotonin reduction in the same brain regions. DA content was unaltered in the striatum and the cortex. In the group lesioned with both DSP-4 and 5,7-DHT, quinpirole-induced (DA D2-agonist-agonist) yawning, 7-hydroxy-DPAT-induced (DA D3 agonist) yawning, and apomorphine-induced (non-selective DA agonist) stereotypies were enhanced. However, SKF 38393-induced (DA D1 agonist) oral activity was reduced in the DSP-4 + 5,7-DHT group. These findings demonstrate that DA D2- and D3-agonist-induced behaviors are enhanced while DA D1-agonist-induced behaviors are suppressed in adult rats in which brain noradrenergic and serotoninergic innervation of the brain has largely been destroyed. This study indicates that noradrenergic and serotoninergic neurons have a great impact on the development of DA receptor reactivity (sensitivity).

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation.