Animals respond to changes in the environment and internal states to modify their behavior. The basal ganglia, including the striatum contribute to action selection by integrating sensory, motor and reward information. Therefore, dysregulation of striatal function is common in many neuropsychiatric disorders, including Parkinson’s disease, Huntington disease, schizophrenia, and addiction. Here, using fiber photometry, pharmacology, and behavioral approaches in transgenic mice, I explored the cellular and circuit mechanisms underlying key striatal functions. In Chapter 1, I begin by presenting the existing literature on the anatomy and physiology of the striatum. Next, I review the important functions of the striatum. Within this general review, I highlight the specific roles that striatal (DA) and acetylcholine (ACh) play in striatal circuitry and function. In Chapter 2, I demonstrate the naturally evoked ACh dip has a DA component and a non-DA component. Specifically, I show that DA via cholinergic DA D2 receptors (D2Rs) modulate the length of the ACh dip and rebound ACh levels following the dip. In addition, I show that DA coordinates the activity between DA and ACh during behavior. Finally, I present data that supports a role for ACh in motivated behavior. In Chapter 3, I show that cholinergic D2Rs are not necessary for reward learning but do facilitate reversal learning in a probabilistic choice task. In addition, I show that changes in DA and ACh levels contribute to reversal learning in a probabilistic choice task. Finally, in Chapter 4, I discuss the general conclusions and study implications, as well as future directions.

Indiana University-Purdue University Indianapolis (IUPUI) / In the age of big data, better use of large, real-world datasets is needed, especially ultra-large databases that leverage health information exchange (HIE) systems to gather data from multiple sources. Promising as this process is, there have been challenges analyzing big data in healthcare due to big data attributes, mainly regarding volume, variety, and velocity. Thus, these health data require not only computational approaches but also context-based controls.In this research, we systematically examined associations among select neural and dermal conditions in an ultra-large healthcare database derived from an HIE, in which computational approaches with epidemiological measures were used. After a systematic cleaning, a binary logistic model-based methodology was used to search for associations, controlling for race and gender. Age groups were chosen using an algorithm to find the highest incidence rates for each condition pair. A binomial test was conducted to check for significant temporal direction among conditions to infer cause and effect. Gene-disease association data were used to evaluate the association among the conditions and assess the shared genetic background. The results were adjusted for multiple testing, and network graphs of significant associations were created. Findings among methodologies were compared to each other and with prior studies in the literature. In the results, seemingly distant neural and dermal conditions had an extensive number of associations. Controlling for race and gender tightened these associations, especially for racial factors affecting dermal conditions, like melanoma, and gender differences on conditions like migraine. Temporal and gene associations helped explain some of the results, but not all. Network visualizations summarized results, highlighting central conditions and stronger associations. Healthcare data are confounded by many factors that hide associations of interest. Triangulating associations with separate analyses helped with the interpretation of results. There are still numerous confounders in these data that bias associations. Aside from what is known, our approach with limited variables may inform hypothesis generation. Using additional variables with controlled-computational methods that require minimal external input may provide results that can guide healthcare, health policy, and further research.

The advent of recent high throughput sequencing technologies resulted in an unexplored big data of genomics and transcriptomics that might help to answer various research questions in Parkinson’s disease(PD) progression. While the literature has revealed various predictive models that use longitudinal clinical data for disease progression, there is no predictive model based on RNA-Sequence data of PD patients. This study investigates how to predict the PD Progression for a patient’s next medical visit by capturing longitudinal temporal patterns in the RNA-Seq data. Data provided by Parkinson Progression Marker Initiative (PPMI) includes 423 PD patients with a variable number of visits for a period of 4 years. We propose a predictive model based on a Recurrent Neural Network (RNN) with dense connections. The results show that the proposed architecture is able to predict PD progression from high dimensional RNA-seq data with a Root Mean Square Error (RMSE) of 6.0 and rank-order correlation of (r=0.83, p<0.0001) between the predicted and actual disease status of PD. We show empirical evidence that the addition of dense connections and batch normalization into RNN layers boosts its training and generalization capability.

We also investigated whether BDE-47 exposure could worsen PD situation by applying transgenic Drosophila (fly) model in which human α-synuclein (α-syn) was overexpressed in wide-type fly to simulate PD. BDE-47 (0, 2, 10 and 50 µM) was fed to flies continuously for 30 days. Integrated LC-MS and GC-MS profiling indicated metabolic changes in tryptophan, phenylalanine, purine, and alanine, aspartate and glutamate pathways, similar to those from mouse experiment. After quantified metabolites of interest by LC-triple quadrupole MS, we confirmed the slowed-down formation of KYNA (kynurenic acid, a neuro-protector) and speeded-up formation of 3HKYN (3-hydroxykynurenine, a neurotoxin) in all BDE-47 exposed groups on the 20th exposure day. The levels of SAM/SAH (methylation biomarker) and GSH/GSSG (oxidative stress biomarker) were found to decrease on the 30th exposure day. Collectively, we propose that BDE-47 could induce imbalance of kynurenine metabolism, insufficient methylation and oxidative stress, which might contribute to the PD progression. To further explore the underlying mechanism of 6-OH-BDE-47 induced neurotoxicity, we conducted omics study of metabolic changes induced by 6-OH-BDE-47 on N2a cells. Cells were exposed to 6-OH-BDE-47 (0, 0.5 and 1 μM) for 24 hours. Considerable metabolic changes in pyrimidine and purine metabolism were observed in high exposure condition while oxidative stress was appeared under low exposure condition. Moreover, 6-OH-BDE-47 was found to affect the dopamine production. iTRAQ proteomics was carried out and pinpointed the dysregulation of ribosome, proteasome, RNA metabolism, aminoacyl-tRNA biosynthesis, vesicular trafficking, purine pathway, and mitochondria electron transport. Immunocytochemistry and Western blot analysis further confirmed that 6-OH-BDE-47 could inhibit autophagy flux, which might result in the aberrant protein aggregation, a pathological hallmark of PD. We further investigated whether 6-OH-BDE-47 exposure could directly induce PD pathology in Sprague Dawley rat. 6-OH-BDE-47 (0.1, 1 and 10 µg) was stereotaxically injected into the right VTA and SNc regions in the midbrain of rat where there are abundant dopaminergic neurons. The apomorphine-induced rotation test indicated significant deterioration in motor function in the group receiving injection of 10 µg. Striatal dopamine was found to decline in a dose-dependent manner. Notably, 6-OH-BDE-47 also promoted the formation of α-syn aggregate, an important pathological hallmark of PD. Proteomics study revealed that protein degradation processes were crucial rather than oxidative stress in 6-OH-BDE-47 induced neurotoxicity in vivo. Mechanistic study based on Western blot further confirmed that 6-OH-BDE-47 could inhibit ubiquitination and autophagy. Collectively, the rat experiment demonstrated that 6-OH-BDE-47 administration could induce motor defect by impairing dopaminergic system and promote α-syn aggregation by inhibiting ubiquitination and autophagy, suggesting that 6-OH-BDE-47 could be a novel risk factor of PD.;Polybrominated diphenyl ethers (PBDEs), as one typical persistent organic pollutants (POPs), are widely spread in the environment and pose potential adverse impacts on human health. As a predominant congener of PBDEs, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) has been reported to affect habituation capability, synaptic plasticity, and vesicular neurotransmitter release. As an important in vivo metabolite derived from BDE-47, 6-hydroxy-BDE-47 (6-OH-BDE-47) was also reported as a neurotoxin. However, the possible linkages between BDE-47/6-OH-BDE-47 exposure and typical neurodegenerative diseases such as Parkinson's disease (PD) are still unclear. Mass spectrometry (MS) based omics integrated with bioinformatics is emerging as a powerful tool to evaluate metabolic changes occurred after different exposures. Here we developed non-targeted metabolomics, lipidomics, and isobaric tag for relative and absolute quantitation (iTRAQ) proteomics methods based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to depict BDE-47/6-OH-BDE-47 induced metabolic changes and to explore the possible contribution of their exposure to PD pathology/pathogenesis. BDE-47 dissolved in corn oil (0, 1, 10 and 100 mg/kg bwt) was orally administered to adult male C57BL/6 mice for 30 consecutive days. Results of global metabolomics and lipidomics studies of PD-related brain regions based on LC-orbitrap MS revealed significant metabolite changes between the exposed and control groups in purine pathway, glutathione pathway, tryptophan pathway, phenylalanine pathway, alanine, aspartate and glutamate pathway, and lipid composition, mainly involved in oxidative stress and neurotransmitter production. By further quantifying metabolites involved in tryptophan and phenylalanine pathways in mice serum, colon and brain samples by using LC-triple quadrupole MS, dysregulation of PD linked neurotransmitters dopamine and serotonin were confirmed. iTRAQ proteomics study of the striatum, the part of the brain that is most intensively studied in PD pathogenesis, revealed that BDE-47 could induce neurotransmitter system disturbance, mitochondrial dysfunction, oxidative stress and abnormal phosphorylation. Oxygen consumption rate after BDE-47 treatment (0, 1 and 10 μM) in mouse neuroblastoma (N2a) cells was measured for the confirmation. BDE-47 was demonstrated to impair mitochondrial function.

Respiratory-swallow coordination (RSC) is critical for safe and efficient swallowing. In healthy adults, RSC is most frequently characterized by an exhale-swallow-exhale pattern initiated within the mid-lung volume range with a respiratory pause of approximately one second. This combination in RSC behaviors is thought to be most optimal for swallowing-related bolus clearance and airway protection. Deviations from these RSC behaviors are observed at disproportionately higher rates in people with Parkinson’s disease (PD) when compared to non-dysphagic, healthy adults. However, little is known about which variables influence RSC in PD, if the RSC behaviors that are most optimal for swallowing in healthy adults are also most optimal for swallowing in PD, and if respiratory-swallow training can be used to successfully rehabilitate suboptimal RSC, swallowing safety, and swallowing efficiency in PD. This dissertation document includes a series of four studies that address these important clinical research questions. Chapter 1 will begin by reviewing the current body of literature as it relates to dysphagia in PD, RSC in healthy adults and PD, respiratory-swallow training as a skill-based treatment for dysphagia rehabilitation, and motor learning considerations for respiratory-swallow skill training in PD. Chapter 2 will be used to examine the relationships among RSC with patient- and swallowing-specific factors in PD. Chapter 2 will also be used to assess the influence of RSC behaviors on measures of swallowing safety (penetration-aspiration) and swallowing efficiency (pharyngeal residue) in PD. Chapter 3 will then evaluate the effects of verbal cueing on RSC in PD as a method of determining if RSC is stimulable for rehabilitative change. Chapter 4 will explore the effects of respiratory-swallow training on swallowing safety and efficiency rehabilitation in a person with mid-stage PD and severe dysphagia within the context of a single-subject experimental design. Chapter 5 will then examine the effects of respiratory-swallow training on dysphagia and RSC rehabilitation within the context of a cohort study. Chapter 5 will also be used to compare the effects of constant versus variable practice in order to explore how principles of motor learning can be used to enhance respiratory-swallow training outcomes. This document will then conclude by synthesizing the results from Chapters 2-5 and by discussing directions for future research.

Parkinson’s Disease is a disorder of the midbrain dopaminergic system with characteristic neurodegenerative patterns, recognized for its motor symptoms. The neurodegeneration is most prevalent in the substantia nigra pars compacta, while dopaminergic neurons in neighboring structures are comparatively spared. There are many possible explanations for this disparity, including differences in tolerance to oxidative stress, and vulnerability to α-synuclein aggregates. The substantia nigra is part of the basal ganglia, a network of nuclei in the midbrain and base of the forebrain which are responsible for coordinating voluntary movement. Dopamine has an inhibitory effect in the basal ganglia. It dampens signals to remove noise, so the basal ganglia circuitry is not hyperactive. In the absence of dopamine, the flow of information through the basal ganglia is disrupted. This results in tremor, bradykinesia, and rigidity, known as the classic triad. No cure currently exists and therapies are unable to slow disease progression, so treatments are aimed at symptom management. Degenerative processes in Parkinson’s Disease occur rapidly, early in the disease progression, with about 60% neuronal death in the substantia nigra prior to diagnosis. There is a need for biomarkers or other signs which can be used to clinically to diagnose the disease at an earlier stage. In conclusion this paper provides suggestions for future lines of research.

Parkinson’s disease is an incurable neurodegenerative disease characterized by motor deficits, owing to dopaminergic denervation in the nigrostriatal pathway. The abnormal formation of hallmark Lewy bodies underlies the disease process. The pre-synaptic protein alpha- synuclein (⍺-syn) has prion-like properties arising from its propensity to propagate, seed misfolding, and self-aggregate. Pathogenesis is postulated to arise in olfactory and enteric regions, exploiting connected neuronal pathways to ultimately propagate to the substantia nigra pars compacta. There is little known about the earliest stages of ⍺-syn aggregation and its prion-like propagation mechanisms. Bimolecular fluorescence complementation of ⍺-syn aggregates has allowed us to directly visualize aggregation in transgenic mice and mice transduced with an adeno-associated virus vector. Although our transgenic mice expressed BiSyn in a mosaic fashion that limited utility, we were successful in transducing neurons in the mouse striatum. This work has validated the AAV2/9-CMV-BiSyn approach as groundwork for future systematic studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in the gene encoding for leucine rich repeats kinase 2 (LRRK2) are commonly found in Parkinson’s disease. Recently, we found that the disease-associated point mutations at residue R1441 in the G domain (ROC) of LRRK2 resulted in perturbation of its GTPase activity. In this study, we compare the biochemical and biophysical properties of the ROC domain of LRRK2 carrying the PD-associated mutations at residue R1441 with those of the wild-type. We found that the disease-associated mutations (R1441C/G/H) showed marked quaternary structure compared to wild-type, in that the latter existed in solution in both monomeric and dimeric conformations dynamically regulated by GDP/GTP binding state, while we detected only monomeric conformation for three disease-associated mutants. To understand the structural basis for this plasticity and the activity reduction in the mutants, we solved a 1.6 Å crystal structure of the wild type ROC that shows a stable dimeric conformation in which the switch motifs and inter-switch regions mediate extensive interactions at the dimer interface. Residue R1441, where PD-associated mutations occur, forms exquisite interactions at the interface, thus suggesting a critical role of this residue in maintaining a dynamic dimer-monomer interconversion and conformational flexibility of the switch motifs. Consistently, substituting R1441 for other arbitrary mutations (R1441K/S/T) lead to similar perturbation of GTPase activity and dimerization defects as observed in the disease-associated mutants. Locking the ROC domain in either dimeric or monomeric conformations by engineered disulfide bond alters the binding affinity to GTP (but not GDP) and significantly reduce GTPase activity, thus suggesting that the dynamic dimer-monomer interconversion and conformational plasticity are essential for ROC function as a molecular switch modulating the kinase activity of LRRK2.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12945号 / 論医博第2097号 / 新制||医||1010(附属図書館) / 32204 / (主査)教授 髙橋 良輔, 教授 富樫 かおり, 教授 髙橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

This dissertation is a collection of a total of seven publications that deal with eye movement disorders in patients with basal ganglia disorders. We obtained normative data for videooculography in healthy individuals. We have described the eye movement evolution during a human life such as the increase of latency, movements become hypometric and antisaccadic error rate increases. We have shown that sex and education do not affect the eye movements. Our study highlighted the asymmetry in the eye movement performance. As the first, we studied the vergence in patients with Parkinson's disease (PN) using videooculography (VOG). We devised and defined a paradigm for this examination and saw that in patients with PN there is a prolonged latency and hypometry of divergence. In patients with ephedrone induced parkinsonism (EP), we were the first who examined eye movements and found that it was possible to identify between this toxic Parkinson's syndrome and PN on the basis of a videooculography. In EP patients, we described velocity decsrease and hypometry in horizontal saccades, prolonged latency in horizontal saccades, and higher error rate in the antisacadic task. Behavioral disorder in REM sleep (RBD) as a prodromal stage of PN leads to impaired eye movement. In the evaluation with PN patients, we...