Tesis sobre el tema "Dopamine neurons, parkinson's disease, neuroscience"

The midbrain dopamine system is involved in many aspects of purposeful behaviour and, when compromised, can have devastating effects on movement and cognition as seen in diseases like Parkinson's. In the healthy brain, dopamine neurons are thought to play particularly important roles in learning by signalling errors in reward prediction. The objective of this thesis was to investigate the diversity in the functional properties of the midbrain dopamine system, and how this is altered through genetic variation of relevance to Parkinson's and development of cell phenotype. This objective was addressed with a combination of behavioural experiments, in vivo single-cell recording and labelling (both in anaesthetised as well as awake rodents), immunofluorescence labelling, retrograde tracing and stereology. In a first set of experiments, it was demonstrated that chronic as well as acute genetic challenges can alter the firing patterns of midbrain dopamine neurons. Using a novel bacterial artificial chromosome-transgenic rat model, it was shown that the R1441C mutation in human leucine-rich repeat kinase 2, which is linked to Parkinson's, leads to motor deficits and an age-dependent reduction in the in vivo firing variability and burst firing of substantia nigra pars compacta (SNc) dopamine neurons. These findings help reveal processes of early, pre-degenerative dysfunction in dopamine neurons in Parkinson's. Similar effects on firing variability and burst firing of SNc dopamine neurons were found in a mouse model with conditional knock- out of the transcription factors Forkhead box A1 and A2 (FoxA1/2) in midbrain dopamine neurons. These findings indicate that FoxA1/2 are not only crucial for the early development of dopamine neurons, but also their function in the mature brain. In a second set of experiments in wildtype mice, it was demonstrated that midbrain dopamine neurons (located in SNc and ventral tegmental area) show diverse expression of the molecular markers Calbindin, Calretinin, Aldh1a1, Sox6, Girk2, SatB1 and Otx2. It was found that selective expression of these markers is of use for discriminating between midbrain dopamine neurons that project to dorsal striatum or nucleus accumbens. To elucidate whether the diverse molecular marker expression would map onto dopamine neurons whose firing correlates with distinct behavioural events, midbrain dopamine neurons were recorded and labelled in head-fixed awake mice either exposed to neutral sensory stimuli or performing a classical conditioning paradigm. The population activity of midbrain dopamine neurons was not modulated by neutral sensory stimuli. Interestingly, fewer than 50% of identified dopamine neurons showed phasic firing increases following reward- predicting cue and/or reward delivery, despite the common assumption that most (if not all) midbrain dopamine neurons signal reward prediction errors. Instead, firing was modulated by other explanatory factors, such as licking, or showed no modulation during the task. Response types of midbrain dopamine neurons were not correlated with their anatomical location nor the selective or combinatorial expression of the markers Aldh1a1, Calbindin and Sox6. In conclusion, the first set of experiments identified how different genetic burdens can alter the in vivo firing of midbrain dopamine neurons, and provide new insights into how circuits can change in pathological or compensatory ways at early disease stages in Parkinson's. The second set of experiments revealed striking heterogeneity of midbrain dopamine neurons in the intact system, and established further a functional diversity in the response types of identified midbrain dopamine neurons that is only partially consistent with canonical reward prediction error signalling.

The striatum is the major input structure of the basal ganglia, and is composed of two major populations of spiny projection neurons (MSNs), which give rise to the socalled direct and indirect pathways, and several types of interneuron. Dopaminergic inputs to striatum are critical for its proper function. Indeed, loss of dopaminergic neurons in Parkinsonism leads to motor disturbances, grossly disturbs striatal activity, and is associated with the emergence of excessively-synchronized network oscillations at beta frequencies (15-30 Hz) throughout the basal ganglia. How the distinct structural, neurochemical and other properties of striatal neurons are reflected in their firing rates and patterns in vivo is poorly defined, as are their possible cell-type-selective contributions to the aberrant oscillations arising in the Parkinsonian brain. To address these issues, I first used multi-electrode arrays to record the spontaneous firing of ensembles of neurons in dorsal striatum in both anaesthetised dopamine-intact and Parkinsonian (6-hydroxydopamine-lesioned) rats during two well-defined brain states, slow-wave activity (SWA) and spontaneous activation. The chronic loss of dopamine led to an overall increase in the average firing rates of striatal neurons, irrespective of brain state. However, many neurons in the Parkinsonian striatum still exhibited the low firing rates and irregular firing patterns typical of neurons in the dopamine-intact striatum. During SWA in Parkinsonian rats, the firing of striatal neurons was more strongly synchronized at low frequencies, in time with cortical slow (~1 Hz) oscillations. During spontaneous cortical activation in Parkinsonian rats, more striatal neurons engaged in synchronized firing in time with cortical beta oscillations. Under the same experimental conditions, I then recorded the spontaneous firing of individual striatal neurons and juxtacellularly labelled the same neurons to verify their cell types, and locations; indirect pathway and direct pathway MSNs were distinguished by the expression (and lack of expression respectively), of the neuropeptide precursor preproenkephalin (PPE). After chronic dopamine loss, and on average, only indirect pathway (PPE+) MSNs significantly increased their firing rates during both brain states, and engaged in widespread, synchronized firing in the beta-frequency range. This did not hold true for all PPE+ MSNs; the Parkinsonian striatum contained many MSNs that were virtually quiescent, which were just as likely to belong to the indirect pathway as the direct pathway. Direct pathway (PPE-) MSNs increased their firing only during SWA after chronic dopamine loss and rarely engaged in aberrant beta oscillations. Taken together, these data suggest that (1) the firing patterns, as well as the firing rates of many striatal neurons are grossly disturbed by chronic loss of dopamine and (2) that the pathological synchronization of the rhythmic firing of a subpopulation of indirect pathway MSNs could contribute to the propagation of aberrant beta-frequency oscillations to downstream basal ganglia nuclei in Parkinsonism.

In Lesch-Nyhan disease, concentrations of hypoxanthine are elevated especially in the brain and cerebrospinal fluid; dopamine and its metabolites are reduced in the caudate and putamen. Hence we investigated the possibility that hypoxanthine has direct effects on dopamine neurons.
Hypoxanthine, adenine or allopurinol was delivered unilaterally into the rat brain. Behavioural effects were monitored by apomorphine-induced rotation; ipsilateral turning was time and dose-dependent. Turning was competitively blocked by a non-specific DA antagonist, suggesting that dopamine neurons were altered. In hypoxanthine treated animals, a D1 antagonist specifically blocked rotation; catalepsy occurred after caffeine administration.
After two or three weeks treatment all groups had elevated purine levels in the caudate nuclei, while catecholamine levels were variably altered.

One of the main characteristics of Parkinson's disease (PD) is an exaggerated oscillatory activity in the beta band (12-30 Hz). This activity has been linked to the rise of symptoms such as bradykinesia and akinesia. Even if dopamine replacement therapy (oral intake of dopamine pro-drug levodopa) reverses these symptoms, the effect of the treatment on the beta band activity has still not been completely understood. Therefore, here the temporal dynamics of beta band activity in human patients affected by PD were characterized with and without levodopa treatment. Local-field-potential (LFP) recordings from five patients undergoing dopamine replacement therapy were used. From the LFPs, the extracted beta epochs with significantly higher power than expected from a comparable noisy signal were analyzed. This analysis showed that beta band activity occurred in bursts meaning that high amplitude oscillation alternated with silenced periods. The pathological state also distinguished itself for longer epochs and with power that increased with the length of the epoch. The administration of levodopa reduced the duration of bursts and decreased the overall mean power of the beta band activity. Finally, epochs with the same number of cycles were compared. The Coefficient of Variation prior such epochs suggested that the ongoing activity might lock into a synchronization process prior the burst. These results provide important information to better understand how levodopa alleviates some of the symptoms of PD and pave the way to develop better computational models for the emergence of beta oscillations.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder for which the greatest risk factor is age. Four to five percent of 85-year-olds suffer from this debilitating disease, which is characterized by the selective loss of dopaminergic neurons within the substantia nigra and the presence of protein aggregates known as Lewy bodies. While the etiology of this disease is still unknown, recent research implicates oxidative stress, activated microglia, and reactive dopamine (DA) metabolites to play a role in the initiation or progression of the disease. Activated microglia cause injury to dopaminergic neurons via a host of mechanisms, including reactive oxygen species production, release of cytokines, and phagocytic activity. Microglial activation has been detected in the brains of PD patients, but the source of this activation has not been elucidated. Previous research has shown electrophiles and endogenous neurotoxins to play a role in this microglial activation. The interaction between the neurotoxic metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and microglia has not been explored. DOPAL is a highly reactive, bifunctional electrophile produced by oxidative deamination of DA by monoamine oxidase (MAO). DOPAL is oxidized in the major metabolism pathway to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH). DOPAL has previously been shown to be 100-fold more toxic than DA in vitro and in vivo. Potent inhibition of the rate-limiting enzyme in DA biosynthesis, tyrosine hydroxylase, by DOPAL has been well-established. DOPAL-mediated aggregation of Α-synuclein, the primary component of PD-hallmark Lewy bodies, has been suggested but was further explored in this work. Results presented in this body of work include further determination of the aggregation of Α-synuclein by DOPAL, including evidence of covalent modification. The interaction of DOPAL with BV-2 microglia, an immortalized cell line, was addressed in depth through exploration of DOPAL catabolism, toxicity, and generation of an activational response. Metabolism of DOPAL to DOPAC was altered in activated microglia, with the production of DOPAC reduced by ~40%. Metabolism of DOPAL to DOPAC was also inhibited by both 4-hydroxynonenal and malondialdehyde, gold standards of lipid peroxidation. Both of these compounds were found to be significantly toxic to BV-2 cells at concentrations well below those considered toxic to dopaminergic cells. Alternatively, DOPAL and DA were found to be non-toxic to this cell line, while DOPAL was shown to be significantly toxic to dopaminergic cells at concentrations as low as 10 ΜM. Significant activation of BV-2 microglia by DOPAL was observed at 10 ΜM and above by release of TNF-Α. Morphological changes, release of IL-6, and changes in expression of COX-2 also indicated activation by DOPAL but not DA or DOPAC. BV-2-conditioned media, generated by incubation with DA, DOPAL, or DOPAC, was added to MN9D cells, and toxicity was measured by the MTT assay. BV-2 conditioned media generated by DOPAL incubation produced the greatest toxicity for MN9D cells. These results implicate DOPAL in dopaminergic cell death through microglial activation.

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2005.
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Dopamine (DA) is a key striatal neuromodulator central to normal functioning of the basal ganglia. Identifying and characterizing circuits governing striatal DA transmission is necessary for understanding DA involvement in adaptive behaviour and pathology. Properties of evoked striatal DA release can be examined using fast-scan cyclic voltammetry at carbon fibre microelectrodes, a technique enabling live monitoring of transmitter release events with sub-millisecond resolution. Experimental work presented in this thesis employed this approach to study regulation of striatal DA by acetylcholine (ACh) in health and disease in acute brain slices. Synchronous activity in a small population of striatal cholinergic interneurons (ChIs) was previously shown to directly drive striatal DA release. Here using optogenetic approach I explore physiological relevance of ChI-evoked drive of striatal DA by examining whether corticostriatal and thalamostriatal afferents to ChIs can trigger ACh-evoked DA events. Following floxed vector injections in motor cortex or caudal intralaminar thalamus of CaMK2a-Cre mice I examine the properties of evoked DA upon light activation of channelrhodopsin-2-transduced inputs to striatal ChIs. These experiments revealed that both cortical and thalamic afferents are capable of driving ACh-evoked DA release, but operate using a different complement of post-synaptic ionotropic glutamate receptors and display distinct release recovery profiles. I further explore if rebound excitation in a population of striatal ChIs could drive DA events by examining whether ACh-evoked DA release follows optical inhibition of striatal ChIs selectively expressing hyperpolarizing halorhodopsin 3.0 or archaerhodopsin 3.0 in ChAT-Cre mice. This work showed that hyperpolarizing ion pumps were not successful in triggering ChI-evoked DA release. I also investigate whether cholinergic brainstem innervation of striatum could contribute to or drive ACh-evoked striatal DA events in ChAT-Cre rat, concurrently showing that ChI-evoked DA release is not a species artefact, and is present in mouse and rat alike. Current results also suggest that cholinergic brainstem afferents do not drive or contribute to striatal ACh-evoked DA events. Close interaction between DA and ACh systems further indicates that ACh could impact dopaminergic dysfunction. To explore this I examined the state of ACh transmission in a mouse model of Parkinson’s disease overexpressing human wild type alpha–synuclein protein. These animals present with impaired striatal DA release from young age, but DA deficits could be mediated by changes in ACh tone. Here I show that impaired striatal DA release is the results of primary DA axon dysfunction, although in ventral striatum DA release deficits could be partially compensated by increased ACh tone at nicotinic receptors. I further show that the functional state of muscarinic ACh receptors in not altered following decreased DA transmission, although the data from aged animals suggest that alpha–synuclein-dependent changes in vesicle handling could contribute to impaired DA releasability. Finally, I show that vesicle handling may indeed be altered in this mouse model as impaired DA release is evident with short stimulation protocols, while with prolonged depolarization of DA axon terminals alpha–synuclein-overexpressor mice are better able to sustain evoked DA release. Overall, the main body of work presented in this thesis examined the processes regulating striatal DA transmission via ACh system. In particular, I show that ChI-evoked drive of striatal DA release can be recruited physiologically and further establish that changes in ACh transmission are not the primary drivers of impaired DA releasability in a mouse model of Parkinson’s disease overexpressing human alpha–synuclein protein.

Parkinson’s disease (PD) is characterized by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Mechanisms regulating this neurodegeneration, however, are unclear. Evidence from PD pathology and models of PD, indicate mitochondrial disfunction triggers several death signalling pathways. Accordingly, in vivo and in vitro mitochondrial stress models of PD were employed to explore the role of two divergent molecular influences on dopaminergic neuronal survival. We examined neuroinflammatory and death signalling pathways arising from MPTP-induced mitochondrial stress. Interferon-gamma (IFN-ɣ) is a cytokine known to activate cellular components of inflammation, including microglia of the central nervous system (CNS). Results of a screen for cytokines in PD patient plasma revealed elevated levels of IFN-ɣ, suggesting a correlation between IFN-ɣ and PD associated DA cell death. In an MPTP mouse model of PD, germline deletion of IFN-ɣ improved survival of DA neurons and the nigrostriatal system, along with a reduction in microglia activation. Employing a survival co-culture system of neurons and microglia, it was found that neutralizing IFN-ɣ reduced DA cell loss induced by the mitochondrial complex I inhibitor, rotenone. DA cell death required localized microglia, activated through the IFN-ɣ-receptor (IFN-ɣ-R), with DA survival inversely proportional to IFN-ɣ expression, found to be up-regulated following rotenone. Investigation of the calpain-Cdk5-MEF2 signalling pathway in the MPTP model of DA cell death, motivated an examination of the nuclear orphan receptor, Nur77, following a review of potential MEF2 regulatory targets. MPTP induced a reduction in Nur77 mRNA from basal ii levels in SNc tissue, further regulated by ectopic Nur77 expression. These results strengthened our new model of MEF2 Nur77 regulation in DA neurons. In MPP+/MPTP DA survival experiments, loss in germline Nur77 expression presented an elevation in DA neuronal death both in vitro and in vivo, with a greater impairment in the nigrostriatal circuitry in comparison with normal expressing animals and cells. Dopaminergic supersensitivity related to Nur77 deficiency was attenuated with the ectopic expression of AV-Nur77 in vivo. These opposing mediators of survival yield new mechanisms by which DA neurons die, suggesting a mutitargeting approach to halt the progression of DA cell death.

Thesis (Ph.D.)--Boston University
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In Parkinson's disease (PD), dopaminergic (DA) neurons of the substantia nigra (SN) often degenerate years before those of the ventral tegmental area (VTA). The DA neurons in VTA and SN diverge both in their intrinsic biophysical features and in their embeddings within forebrain circuits. Improved PD therapies may depend on a deeper understanding of the implications of these intrinsic features and embeddings. This thesis surveyed intrinsic characteristics of the least and most vulnerable DA subpopulations. To evaluate how differences may effect neural excitability and signal integration, two computational models of the biophysical mechanisms and types of inputs that characterize these classes of DA neurons were developed, simulated, and compared. The two multi-compartment models were constructed using the NEURON simulator. An SN model was built by extending a prior model of DA neurons. The new model includes 20 ionic currents and receives synaptic input from simulated spike trains via glutamatergic and GABAergic receptors. Then a VTA model was built by modifying features of the SN model, in accord with published data. These mathematical models enabled simulations of current injection experiments and arbitrary synaptic input patterns. With realistic inputs, previously reported differences between VTA and SN, in firing rate and bursting, emerged in the simulations. Neurodegeneration research suggests that persistent elevated calcium levels can induce excitotoxic cell death. Beyond excessive calcium influx through NMDA-type glutamatergic receptors, a constellation of factors, including calcium-binding proteins and release of calcium from intracellular stores, may contribute to prolonged increases of calcium. To assess complex emergent interactions, the models included explicit representations of several such factors. The simulations revealed that the higher expression of calcium mechanisms in the SN model, together with higher autoinhibition, via somatodendritic DA release and type-two DA-receptor-induced activation of potassium channels, result in lower SN excitability, relative to the VTA. The SN model generates stronger pacemaking and a higher effective threshold for excitatory inputs to generate bursting. Such differences in electrophysiological properties covary with other factors, such as energy demands and mitochondria levels, and further extensions to these models will enable weighing their roles in the differential vulnerabilities of DA neurons.
2031-01-01

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.

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

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.

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) have a key role in regulation of voluntary movement control. Their death is a hallmark of Parkinson’s disease, characterised by inhibited motor control, including muscle rigidity and tremor. Excitatory input to SNc-DA neurons is primarily from the subthalamic nucleus, and in PD these afferents display a higher frequency firing, as well as increased burst firing, which could cause increased excitatory activity in SNc-DA neurons. NMDA receptors (NMDARs) bind the excitatory neurotransmitter glutamate, and are essential for learning and memory. In SNc-DA neurons, NMDARs have a putative triheteromeric subunit arrangement of GluN1 plus GluN2B and/or GluN2D. Wild type (WT) mice, and those lacking the gene for GluN2D (Grin2D-null), were used to explore its role in various aspects of DA neuronal function and dysfunction using patch-clamp electrophysiology, viability assaying, and immunofluorescence. Pharmacological intervention using subunit-specific inhibitors ifenprodil and DQP-1105 on elicited NMDAR-EPSCs suggested a developmental shift from primarily GluN2B to GluN2B/D. Activity dependent regulation was assessed by high frequency burst stimulation of glutamatergic afferents: in comparison to controls, significant downregulation of NMDARs was observed in SNc-DA neurons, though no differences were observed based on genotype. This regulatory function may be a neuroprotective or homeostatic response. Ambient extracellular glutamate elicits tonic NMDAR activity in SNc-DA neurons, which may be important for maintaining basal levels of excitability: the role of GluN2D was assessed by recording the deflection in baseline current caused by application of competitive NMDAR antagonist D-AP5. There was a significantly larger NMDAR-mediated current in WT vs Grin2D-null mice, indicating that GluN2D has a role in binding ambient glutamate. Dysfunction of glutamate uptake could be a secondary pathophysiological occurrence in the SNc, leading to increased ambient glutamate: the effect of this was explored by application of the competitive glutamate transporter blocker TBOA. Here, the NMDAR-mediated portion of this current was significantly higher in WT mice in comparison to Grin2D-null. Interestingly, dose-response data obtained from bath application of NMDA showed significantly larger currents in Grin2D-null animals vs WT, but only at the top of the response curve (~1-10 mM), which may indicate a capability for larger conductance in Grin2D-null animals at high NMDAR saturation due to replacement of GluN2D with GluN2B. GluN2D may therefore be neuroprotective, by attenuating peak current flow in response to very high agonist concentrations. Lastly, GluN2D has been found to decrease NMDAR open probability under hypoxic conditions, potentially conferring resistance to hypoxia / ischemia related excitotoxicity. Therefore, low (15% O2 / 80% N2 / 5% CO2) vs high (95% O2 / 5% CO2) oxygen conditions were used along with immunofluorescent propidium iodide cell death assaying and immunofluorescent labeling for DA neurons in order to compare levels of DA neuronal death in the SNc based on oxygen status and genotype. Whilst there was a significant submaximal effect based on O2 status, genotype did not confer a practical resistance under these conditions. In summary, NMDARs have diverse roles in SNc-DA neurons which may both serve to maintain normal function and protect the cell against potentially pathological conditions.

Les signaux neuromodulateurs induisent une adaptation des fonctions neuronales par le biais de mécanismes d’intégration dynamiques complexes. Parmi les voies de signalisation intracellulaires, celle de l’AMPc/PKA joue un rôle essentiel dans la réponse cellulaire à la dopamine. Pour analyser ces processus d’intégration, nous combinons l’imagerie de biosenseurs dans des préparations ​ex vivo de tranches de cerveau de souris avec de la modélisation de la signalisation intracellulaire dans les neurones D1 et D2 striataux. Dans une première partie de mon travail de thèse, nous analysons la dynamique de la signalisation striatale en réponse à des stimulations dopaminergiques transitoires telles celles associées aux récompenses. Nous montrons par imagerie que, contrairement à ce qui est communément admis, les récepteurs D​2 à la dopamine permettent la détection de dopamine phasique au niveau de l’AMPc. De plus, les simulations suggèrent que les neurones D2 pourraient détecter une diminution du niveau de dopamine tonique, indicateur d’une situation aversive chez l’animal. Ce travail a fait l’objet d’une publication (​Yapo et al., ​J. Physiol 2017​). Dans une deuxième partie, nous avons analysé l’effet dans le noyau de ces stimulations dopaminergiques rapides. En comparaison avec les neurones du cortex, nous montrons que les neurones du striatum disposent d’un mécanisme de contrôle en-avant (“​feed forward​”) qui renforce les réponses PKA nucléaires. Cette situation originale, à l’opposé des rétrocontrôles homéostatiques habituels en biologie, amène à une réponse du noyau tout ou rien, extrêmement sensible. Nous pensons que ce mécanisme est impliqué dans la détection des signaux dopaminergiques transitoires. Ce travail a été publié dans un article (​Yapo et al., ​J Cell Science​ 2018​). Enfin une troisième partie, sous forme de résultats préliminaires, consistait à analyser l’adaptation des neurones du striatum à la perte des afférences dopaminergiques, caractéristique de la maladie de Parkinson. Nous avons observé l’hypersensibilité à la dopamine affectant les neurones D1, largement décrite dans la littérature. De plus, nous montrons que les neurones du striatum présentent une activité phosphodiestérase accrue. Une meilleure compréhension de ces adaptations pathologiques pourrait mener à de nouvelles stratégies thérapeutiques
Neuromodulatory signals trigger adaptations in neuronal functions via complex integrative properties. Among the various existing intracellular signaling pathways, the cAMP/PKA cascade plays a critical role in the cellular response to dopamine. To analyze these integrative processes, we combine biosensor imaging in mouse brain slices with in silico modelisation of the intracellular signaling in D1 and D2 medium-sized spiny neurons. In a first part of my thesis work, we analyze the dynamics of cAMP/PKA signaling in striatal neurons stimulated by transient dopaminergic signals, such as those associated with reward. With imaging we show that the dopamine D​2 receptors can sense phasic dopamine signals at the level of cAMP, a thought that has been argued for long. Moreover ​in silico simulations suggest that D2 spiny neurons could sense the interruptions in tonic dopamine levels associated with aversion in the animal. This work was published in (​Yapo et al., ​J Physiol 2017​). In a second part, we analyzed the effect of such brief dopaminergic signals on the nuclear PKA-dependent signaling. In comparison to cortical neurons, we show that the striatal neurons display a positive feedforward mechanism which strengthens the nuclear responses. This peculiar situation, which contrasts with the usual homeostatic feedback mechanisms found in biology, leads to all-or-nothing and extremely sensitive responses. We believe that this mechanism allows for the detection of transient dopaminergic signals. This work was published in (​Yapo et al., ​J Cell Science​ 2018​). Lastly a third part, that will be introduced as preliminary data, consisted in analyzing the adaptations of the striatal neurons following a dopamine depletion, such as the one found in Parkinson’s disease. We observed in our mouse model an hypersensitivity of the D1 spiny neurons to dopamine, already described by other groups. Additionally we show that striatal neurons display an increased phosphodiesterase activity. A better understanding of these pathological adaptations could lead to the emergence of new therapeutic strategies

La maladie de Parkinson (MP) est caractérisée essentiellement par la perte progressive des neurones dopaminergiques (DA) de la substance noire parse compacte (SNpc) qui innervent le striatum et contrôlent les mouvements volontaires. Une des approches thérapeutiques de la MP est la transplantation ectopique de précurseurs DA fœtaux issus du mésencéphale ventral (MV) dans le striatum. Il est peu probable que la transplantation des neurones DA du MV humain devienne un traitement de routine de la MP en raison de problèmes d’approvisionnement et de standardisation de tissus pour la transplantation. L’avenir de ces greffes dépend donc de l’obtention de sources alternatives de tissus. L’objectif de ce projet est d’obtenir des neurones DA issus de cellules souches embryonnaires de souris et d’évaluer leur potentiel thérapeutique en les greffant dans le striatum ou la SN dans un modèle murin de la MP. Afin d’augmenter le nombre de neurones DA du type nigral, l’expression de LMX1A, un facteur de transcription jouant un rôle clé dans le développement embryonnaire des progéniteurs des neurones DA du MV, a été forcée. Nous avons montré que, in vitro, LMX1A induit une augmentation de précurseurs et de neurones DA du type nigral. Après transplantation dans la SN ou le striatum, les cellules survivent, expriment des marqueurs de neurones DA du type nigral et projettent vers le striatum. L’expression forcée de LMX1A semble augmenter, in vivo, la proportion de neurones DA matures responsables d’une réduction des déficits moteurs après transplantation dans le striatum
Parkinson’s disease (PD) is mainly characterised by the progressive loss of the dopaminergic (DA) neurons of the subtantia nigra pars compacta (SNpc) that are innervating the striatum and controlling voluntary movements. One of the therapeutical strategies of PD is the ectopic transplant of fetal DA precursors from the ventral mesencephalon (VM) into the striatum. It is unlikely that transplant of human DA neurons of the VM become a routine treatment for PD due to supply and tissues standardization problems for the transplant. The future of these transplants thus depends on the obtaining of alternative tissue sources. The aim of this project is to obtain DA neurons derived from mouse embryonic stem cells and to evaluate their therapeutical potential grafting them into the striatum or the SNpc in a mouse model of PD. In order to increase the number of DA neurons of the nigral subtype, the expression of LMX1A, a transcription factor playing a key role in the embryonic development of MV DA neuronal progenitors, was forced. We have shown that, in vitro, LMX1A induces an increase of nigral precursors and neurons. After transplantation into the SN or the striatum, the cells survive, express markers of DA neurons of the nigral subtype and project towards the striatum. The forced expression of LMX1A seems to increase, in vivo, the proportion of mature DA neurons responsible for reducing the motor deficits after transplantation into the striatum

Non-homogeneous degeneration within midbrain dopaminergic (DA) neurons is a histopathological hallmark of Parkinson’s disease (PD). Typically, DA neurons in the Substantia Nigra pars compacta (SNpc) are markedly more vulnerable than in the adjacent ventral tegmental area (VTA) (Brichta and Greengard 2014, Schapira and Jenner 2011). Numerous animal models, both toxin-based or transgenic, show non-uniform DA degeneration patterns, strongly suggesting that intrinsic cellular properties, rather than etiologic factors, underlie differential vulnerability between distinct subsets (Blesa and Przedborski 2014). For therapeutic prospects, understanding the molecular bases of this key pathogenic feature would dramatically improve our chances to develop neuroprotective, disease-modifying treatments. Comparative SNpc-VTA gene expression studies have revealed extensively overlapping signatures between the two DA populations (Grimm et al. 2004, Greene, Dingledine and Greenamyre 2005), suggesting that quantitative, rather than qualitative differences in the expression or function of a limited number of genes subtend selective vulnerability. Over the last decade, it has been suggested that intrinsic electrophysiological properties of specific DA subsets, such as the differential expression or function of selected ion channels, provide a physiological substrate for differential vulnerability (Liss et al. 2005, Guzman et al. 2009, Surmeier et al. 2012, Dragicevic, Schiemann and Liss 2015) In this regard, my colleagues previously demonstrated that MPP+, a neurotoxin able to cause selective nigrostriatal degeneration in animal rodents and primates, inhibits the Hyperpolarization-activated current (Ih) in SNpc DA neurons (Masi et al. 2013). The aim of this PhD project was to investigate the contribution of Ih loss of function (LOF) to the selective degeneration of SNpc DA neurons in PD. For first, we studied the impact of Ih inhibition at the cellular and molecular level, focusing on the electrical properties discriminating among differentially vulnerable subsets of midbrain DA neurons in TH-GFP mice. We showed that pharmacological suppression of Ih increases the amplitude and decay time of excitatory post-synaptic potentials (EPSPs), leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects was quantitatively more evident in SNpc DA neurons. Furthermore, we investigated the participation of VGCC-dependent calcium entry during evoked synaptic activity by combined electrophysiological and calcium fluorometry experiments in the SNpc and VTA DA neuron in wild-type rats. And, we showed that Ih block-induced synaptic potentiation leads to the amplification of somatic calcium responses (SCRs) in vitro. This effect was specific for the SNpc subfield and largely mediated by L-Type calcium channels, as indicated by sensitivity to the CaV 1 blocker isradipine. We showed that Ih is downregulated in presence of low intracellular ATP and that Ih suppression reduced the inhibitory effect of GABAergic transmission, suggesting the existence of a mechanistic link between disruption of mitochondrial homeostasis and abnormal synaptic excitability in SNpc DA neurons. Finally, we tested the effect of Ih suppression in vivo and found that intracerebral stereotaxic injection of the selective blockers ivabradine or ZD7288 causes a pattern of DA degeneration strikingly resembling that seen in MitoPark mice and MPP+-treated mice, two distinct PD models characterized by mitochondrial failure and SNpc-specific DAergic degeneration (Ekstrand et al. 2007, Blesa and Przedborski 2014) Overall, the present data support the hypothesis that Ih LOF may possibly be regarded as an acquired alteration, caused by disruption of mitochondrial metabolism, affecting specifically, or to a larger extent, DA neurons in the SNpc, where Ih is critical in the regulation of synaptic excitability. In vivo, Ih LOF may result from mitochondrial dysfunction, a key disease mechanism at the basis of extensively studied PD animal models, which is gaining increasing attention in the human pathology too. In this regard, there is increasing evidence linking mitochondrial damage to Ih function. Ih is suppressed by the mitochondrial toxin MPP+ in vitro (Masi et al. 2013) and lamotrigine (LTG), a commercial anticonvulsant agent reported to activate Ih (Poolos, Migliore and Johnston 2002, Friedman et al. 2014), is neuroprotective in MPTP-induced DA degeneration models (Archer and Fredriksson 2000, Lagrue et al. 2007). Furthermore, Ih current density is diminished in SNpc DA neurons of MitoPark mice at 6 weeks of age, well before the appearance of neurodegeneration (Good et al. 2011). This evidence supports the proposition that Ih LOF, which may result from mitochondrial failure during PD progression, leads to SNpc-specific DA degeneration through toxic calcium overload.Based on these premises, we tested the hypothesis that Ih LOF is a necessary pathogenic step in relevant PD animal models, and that reversion of this defect is neuroprotective. To test our hypothesis, we used The MitoPark mouse, a model based on a mitochondrial mutation expressed in DA neurons and featuring HCN LOF at an early disease stage (Ekstrand et al. 2007). This model shows late-onset, slow-progressing DA degeneration with differential vulnerability between SNc and VTA. furthermore, electrophysiological recordings in slices from mice at presymptomatic stage (6 weeks) have shown an aberrant activity pattern of SNc DA neurons, with a concomitant inactivation of the Ih (Branch et al. 2016) In this regard, we performed a pharmacological rescue of HCN channels using LTG in presymptomatic Mitopark mice (6 weeks). LTG, is generally considered as a voltage-gated sodium (Nav) channel blocker, as a anticonvulsivant that selectively reduced action potential firing from dendritic depolarization, while minimally affecting firing at the soma. Recent studies suggest that this regional and input-specific effect resulted from an increase in Ih, present predominantly in dendrites. These results demonstrate that neuronal excitability can be altered by drugs acting selectively on dendrites, and suggest an important role for Ih in controlling dendritic excitability (Poolos et al. 2002). LTG has already been used to functionally upregulate HCN function in DA neurons in vivo (Friedman et al. 2014). Based on the above hypothesis, we attempted a pharmacological rescue of Ih function by administering chronic LTG. LTG was administered daily starting from 6 weeks (presymptomatic stage), when Ih loss of function has been described, until 18 weeks (the beginning of the symptomatic stage). What we observed is a dramatic reduction of motor decay compared to MitoPark mice treated with vehicle. Pharmacological manipulation of Ih function in vivo suggests a possible link to DA vulnerability during disease progression. MitoPark mice has better face and construct validity compared with major toxin-induced models, the rat 6-OHDA model and the mouse MPTP-model (Terzioglu and Galter 2008). Indeed, MitoPark mice display a very slow progression of the symptoms, more similarly reflecting the disease progression in PD patients vs. the acute ablation of the DA system manifested in most lesion models. In addition, the slow progression of symptoms in MitoPark mice offers the opportunity to study the effects of chronic treatments for prolonged periods of time (i.e. several months) under conditions of a slow and gradual aggravation of symptoms in line with the situation in PD patients (Galter et al. 2010). To conclude, our research supports the hypothesis that Ih loss of function represents a bona fide pathogenic mechanism which, possibly in concert with SNpc-specific connectivity, may determine differential DAergic vulnerability during disease progression in relevant animal models and in human PD. Considering the total lack of neuroprotective medications in PD therapy and in consideration of the serious side effects associated with long-term levodopa therapy, the targets emerging from this research may be exploited to design protective therapies, in individuals with an early diagnosis of PD.

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.

In this study we aimed to investigate the effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone neurotoxins on dopaminergic (DAergic) neuronal survival using ventral mesencephalic (VM) organotypic cell culture derived from postnatal rat pups (P4-5) and immunocytochemistry for tyrosine hydroxylase (TH) as a marker of DAergic cells. In addition, we examined the neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) on TH-ir cells exposed to MPTP and rotenone as a possible treatment for PD. The TH-ir cells in co-cultures with striatum (ST) as a target grew better then when VM was cultured alone and that TH-ir cells in co-cultures could be maintained without using conditioned and trophic media. We treated 7 day and 14 day co-cultures at different times with varying MPTP and rotenone concentrations and found 14 day old cultures were more vulnerable than 7 day old co-cultures to the effects of either neurotoxin with TH-ir cell numbers significantly lower in 14 day cultures compared to 7 day cultures. Both neurotoxins induced a dose-dependent TH-ir cell reduction in the co-cultures. In addition we compared the toxicity of MPTP and its active metabolite 1-methyl-4- phenylpyridinium (MPP+) as the neurotoxic effects of MPTP on DAergic cells depends on its conversion to MPP+ by astrocytes. We found no significant difference in TH-ir cell reduction in co-cultures treated with MPTP and MPP+. Rotenone was more toxic than MPTP with less TH-ir cell survival in the weeks post treatment. GDNF exposure produced increased cell size and significant increases in TH-ir cell branching in cocultures in a dose-dependent manner. Post treatment of GDNF against MPTP and rotenone provided significant neuroprotection as TH-ir cell survival was at the lower neurotoxin doses and not at the higher doses.
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Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008

Dopamine (DA) is a key catecholamine neurotransmitter involved in motor control, cognition, and neuroendocrine regulation. Reduced DA transmission is associated with Parkinson’s disease, depression, and anhedonia. An overexpression of the dopamine transporter in mice (DAT-tg) results in a 40% reduction in extracellular DA, and can be classified as a genetic model of hypodopaminergia. Reserpine treatment depletes extracellular DA, and is a pharmacological model of hypodopaminergia. The aim of this study was to determine morphological and proteomic changes to medium spiny neurons (MSNs), which receive dopaminergic input, as a consequence of reduced DA transmission. To achieve this, MSNs were fluorescently labelled using a diolistics method and immunofluorescence. There were no observable changes to morphology or proteomic profile of MSNs in DAT-tg animals. Reserpine treatment resulted in reduced spine density in MSNs. DAT-tg animals may present a level of DA depletion that is below the threshold to induce morphological changes to MSNs.

Parkinson’s disease (PD) is a debilitating movement disorder. The cardinal symptoms of PD are bradykinesia, resting tremors and rigidity. PD is characterized by degeneration of dopaminergic neurons of A9 region, substantia nigra pars compacta (SNpc) and loss of dopaminergic terminals in striatum while the dopaminergic neurons of A10 region, ventral tegmental area (VTA) are relatively protected. Putative mechanisms, such as mitochondrial dysfunction, dysregulation of the ubiquitin proteasome system and increased oxidative stress have been hypothesized to mediate PD pathology. However, precise mechanisms that underlie selective vulnerability of SNpc dopaminergic neurons to degeneration are unknown. The aim of this thesis was to evaluate the pathological mechanisms that may contribute to degeneration of SNpc dopaminergic neurons in PD. Dopaminergic neurons of SNpc are pacemakers and constant calcium entry through L-type calcium channel, Cav1.3 has been reported in these neurons during pacemaking. In addition, these neurons have poor calcium buffering capacity. Together, this leads to dysregulation of calcium homeostasis in the SNpc dopaminergic neurons leading to increased oxidative stress. Gene expression of the full length channel and the variant was investigated in the mouse midbrain and further their presence was verified in mouse SNpc and VTA and also in SNpc and VTA in the MPTP mouse model of PD. Gene expression of Cav1.3 -42 and its variant was also studied in SNpc from autopsy tissue from PD patients and age matched controls. Having studied differential expression of the calcium channels, global changes in gene expression in SNpc from the MPTP mouse model of PD and PD autopsy tissues were next examined. This is the first report of transcriptome profile alterations from SNpc in mouse model and PD tissue performed using RNA-seq. Gene expression profiles were examined from SNpc 1 day post single exposure to MPTP, in which case there is no neuronal death and 14 days after daily MPTP treatment where SNpc has undergone ~50% cell death. Further, RNA- seq was performed to study gene expression alterations in SNpc from human PD patients and age- matched controls. The RNA-seq data was taken through extensive analyses; analysed for differential gene expression, gene-set enrichment analysis, pathway analysis and network analysis. Glutaredoxin 1 (Grx1) is a thiol disulfide oxidoreductase that catalyses the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. Down-regulation of Grx1 has been established to exacerbate neurodegeneration through impairment of cell survival signalling. Previous work from our laboratory has demonstrated that perturbation of protein thiol redox homeostasis through diamide injection into SNpc leads to development of PD pathology and motor deficits. It was therefore investigated if Grx1 down-regulation in vivo, leading to increased glutathionylation and protein thiol oxidation, could result in PD pathology. This work is thus the first study of RNA-seq based transcriptomic profile alterations in SNpc from human PD patients. This work also highlights several differences between mouse model and human PD tissue indicating that the underlying mechanisms of PD pathogenesis differ from mouse to humans in addition to developing a novel model for PD.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Indiana University-Purdue University Indianapolis (IUPUI)
Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue.