Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

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Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

Chemical-induced models have revealed the crucial role of oxidative stress and mito-chondrial dysfunction in the development of Parkinson’s Disease. In this project, firstly, we in-vestigated the mechanism of action of rotenone, a commercialized pesticide that was previously described to reproduce the bradykinetic dopaminergic neurodegeneration symptoms of Parkin-son’s Disease in zebrafish by inhibition of the mitochondrial complex I. We found out that rote-none caused change in the morphology of the zebrafish dopaminergic mitochondrial network. We also observed the altered expression of various genes involves in mitochondrial fusion and fission in response to rotenone exposure. Secondly, to develop the use of adult zebrafish as a toxin-based model for Parkinson’s Disease, we sought to minimize any off-target effects by exposure of rotenone specifically to the brain. We demonstrated that microinjection of rotenone into the forebrain ventricular zone of adult zebrafish decreases the number of dopaminergic neurons. However, behavioural tests suggested that did not translate into locomotor impairment in these fish. Taken together, these results gave us more information about the potential use of zebrafish to study the physiological mechanism leading to dopaminergic degeneration and allow for the development of therapeutic strategies for Parkinson’s Disease.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, featuring motor signs such as tremors, bradykinesia, and impaired gait that are often preceded by nonmotor symptoms such as anxiety/depression and olfactory dysfunction. Interestingly, significant olfactory loss was found to be manifested in the majority of PD patients and may precede motor symptoms by years, and thus can be used for the risk assessment of developing PD in asymptomatic individuals. The main pathological feature of PD is the progressive and irreversible loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain. Although the detailed etiology of PD remains unclear, most PD cases were found to be sporadic and can be associated with environmental factors. Only 5–10% of patients result from familial PD. With considerable effort in the past two decades, a number of genes associated with familial PD have been identified and interestingly, many of these genes are involved in regulating and maintaining mitochondrial function. The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we show novel information concerning the role of PARL in zebrafish by establishing a loss-of-function mutation in parla and parlb via CRISPR/Cas9- mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function, as well as loss of both parla and parlb function result in loss of DA neurons in the olfactory bulb and telencephalon of adult zebrafish brain. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants and in fish with loss of parla and parlb function. Furthermore, we showed that loss of parla and/or parlb function translates into altered locomotion parameters and that loss of parla but not parlb function results in impaired olfaction. Finally, increased susceptibility to neurotoxin exposure was identified in mutants with loss of both parla and parlb function but not with loss of parla or parlb function. These results suggest an evident role for parla in the development and/or maintenance of DA neuron function in zebrafish and confirm the existence of redundant and non-redundant functions for the two paralogs, parla and parlb.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20284号 / 医科博第75号 / 新制||医科||5(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 宮本 享, 教授 林 康紀, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The dopaminergic neurons form a brain-wide neuromodulatory system affecting reward, addiction, and motor behaviors. Alterations in dopamine signaling have been associated with many brain disorders, including schizophrenia and Parkinson’s disease. Over the last decade, it has been well-established that the dopaminergic system is capable of significant neurotransmitter phenotypic plasticity (NPP), which is defined as the activity-dependent loss or gain of a specific neurotransmitter usage by neurons. However, most of the related studies have focused on specific regions, providing important but limited insights into NPP. Attaining an unbiased mapping of brain-wide NPP and its relationship with behavioral changes has remained challenging. In this thesis, we first addressed the technical challenges by establishing a whole-brain phenotyping pipeline. Next, we utilized these tools to generate the first brain-wide map of NPP in response to chronic exposure to (R,S)-ketamine, revealing some of the underlying fundamental principles. Here, we describe a whole brain NPP mapping pipeline for studying dopaminergic phenotypic plasticity following chronic (R,S)-ketamine exposure. The pipeline includes 1) an optimized whole brain tissue clearing/immunostaining method for labeling the dopaminergic neurons by utilizing rate-limiting marker tyrosine hydroxylase (TH), 2) high-resolution whole-brain imaging with CLARITY optimized light-sheet microscopy (COLM) and light sheet theta microscopy (LSTM), and 3) a custom python-based data analysis pipeline for quantitative mapping of the brain-wide NPP. Our first key result is that chronic (R,S)-ketamine administration leads to divergent brain-wide changes in the dopaminergic system. Specifically, ten days (but not 1 or 5 days) of daily (R,S)-ketamine (100 mg/kg) administration resulted in a significant decrease in TH+ neurons in regions of the midbrain and a significant increase in areas of the hypothalamus. Second, chronic (R,S)-ketamine treatment also induced an increase in TH+ neuronal projections, including increases within associative cortical brain regions such as the prelimbic area, orbital area, and anterior cingulate area. Decreases in the density of TH+ neuronal projections were observed in the auditory (AUDd) and visual cortices (VISpl). Third, we performed mRNA expression mapping and before-after treatment comparison of the TH+ neuron population within the same animal to reveal that newly gained TH+ neurons are mainly recruited from the neuron pool that contained untranslated TH mRNA. Overall, this thesis provides a first brain-wide quantitative survey of the NPP caused by a specific drug (R,S)-ketamine, extending our fundamental understanding of the extent of adaptability of our brain in response to external stimuli.

Microglia-mediated neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Uncontrolled microglia activation produces major proinflammatory factors including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) that may cause dopaminergic neurodegeneration. Peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone has potent antiinflammatory property. We hypothesize pioglitazone protects dopaminergic neuron from lipopolysaccharide (LPS)-induced neurotoxicity by interacting with relevant signal pathways, inhibiting microglial activation and decreasing inflammatory mediators. First, the neuroprotection of pioglitazone was explored. Second, the signaling transductions such as jun N-terminal kinase (JNK) and the interference with these pathways by pioglitazone were investigated. Third, the effect of pioglitazone on these pathways-mediated PGE2 / nitric oxide (NO) generation was investigated. Finally, the effect of PPARγ antagonist on the inhibition of PGE2 / NO by pioglitazone was explored. The results show that LPS neurotoxicity is microglia-dependent, and pioglitazone protects neurons against LPS insult possibly by suppressing LPS-induced microglia activation and proliferation. Second, pioglitazone protects neurons from COX-2 / PGE2 mediated neuronal loss by interfering with the NF-κB and JNK, in PPARγ-independent mechanisms. Third, pioglitazone significantly inhibits LPS-induced iNOS / NO production, and inhibition of LPS-induced iNOS protects neuron. Fourth, inhibition p38 MAPK reduces LPS-induced NO generation but no effect is found upon JNK inhibition, and pioglitazone inhibits p38 MAPK phosphorylation induced by LPS. In addition, pioglitazone increases PPARγ phosphorylation, followed by the increased PI3K/Akt phosphorylation. Nevertheless, inhibition of PI3K increases LPS-induced p38 MAPK phosphorylation. Inhibition of PI3K eliminates the inhibitive effect of pioglitazone on the LPS-induced NO production, suggesting that the inhibitive effect of pioglitazone on the LPS-induced iNOS and NO might be PI3K-dependent.