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.

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En la presente Tesis Doctoral se describen los cambios celulares que ocurren en la retina en la enfermedad de Parkinson y su posible uso como biomarcadores tempranos de la enfermedad. Los pacientes con enfermedad de Parkinson poseen acumulaciones de alfa sinucleína fosforilada en la retina similares a las que se encuentran en el cerebro de los mismos pacientes. De hecho, la cantidad de alfa-sinucleína fosforilada en la retina correlaciona con la cantidad de alfa-sinucleína fosforilada en el cerebro, con el estadio de progresión de la enfermedad y con la severidad de los síntomas motores. Además, en la retina de enfermos de párkinson se describe una degeneración de las células ganglionares melanopsínicas de la retina, lo que podría explicar las alteraciones en los ritmos circadianos y los desórdenes del sueño que aparecen en pacientes. Finalmente, también se muestra la degeneración de las células amacrinas dopaminérgicas, que se reducen en un 45%. Este fallo en el sistema dopaminérgico de la retina provoca alteraciones morfológicas en las células amacrinas AII, sus principales postsinápticas, y podría explicar algunas alteraciones visuales descritas en la enfermedad como la disminución de la sensibilidad al contraste o de la agudeza visual. En global, los resultados muestran que la retina reproduce los procesos degenerativos que ocurren en el cerebro en la enfermedad de Parkinson y, por tanto, que es un tejido idóneo para el estudio de la enfermedad. Además, el estudio de la retina aporta información sobre el estadio de la enfermedad y puede ser empleado como un biomarcador temprano que ayude al diagnóstico y seguimiento de la misma.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21015号 / 医博第4361号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 渡邉 大, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Combining the nematode Caenorhabditis elegans with novel microfluidic technology has produced a phenotypic movement assay that is at once rapid, sensitive, and low-cost. The method is based on the neurophysiologic phenomenon of worms exhibiting robust, continuous, directed locomotion in response to mild electric fields inside a microchannel. As we demonstrate with the studies reported herein, our microfluidic electrotaxis platform is a unique tool for studying the effects of environmental and genetic manipulations on C. elegans’ movement behaviour, which in turn indicates the state of the organism’s neuronal and muscular systems. In one initiative to develop an inexpensive biosensor, we use the setup to measure the response of worms to common environmental pollutants. Results indicate that worms’ electrotactic swimming behaviour is particularly susceptible to metal salts. A comparison with traditional assays measuring fecundity, growth, and lifespan reveals that electrotactic speed shows a comparable level of sensitivity as a toxicity endpoint. Another study demonstrates that worms expressing a mutant form of α-synuclein, a familial Parkinson’s disease-related protein, show deficits in electrotactic swimming speed that coincide with dopaminergic neuron damage. We further show that both the electrotaxis and neuronal phenotypes can be ameliorated by treatment with curcumin, a putative neuroprotective agent. We have also used the platform to investigate the effects of other environmental and genetic stresses on electrotactic behaviour. Our findings indicate that the response can withstand many different insults but is affected by stresses that induce the mitochondrial and ER unfolded protein responses, which themselves play roles in preserving electrotactic swimming behaviour alongside the heat shock response. These data expand our knowledge of how the motor output component of C. elegans’ electrotactic response is perturbed by environmental and genetic manipulations, and also support the utility of microfluidic electrotaxis as a functional output of nematode locomotory circuits in a multitude of contexts. / Thesis / Doctor of Science (PhD)

神経変性疾患で蓄積する異常タンパク質の1つであるα-synは、PD、DLBおよびMSAの脳内に主に蓄積する。DLBやMSAの患者脳から解析可能な量のα-syn凝集体の増幅に成功した。増幅前後の凝集体のプロテイナーゼKコアのMS分析結果から、増幅による変化はないもののマウスとヒトのα-syn凝集体で切断パターンが異なることがわかった。これらの結果から、この方法が神経変性疾患の異常タンパク質研究の発展に貢献できることを示唆した。 / Pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils. One of the pathological proteins, α-syn, accumulates in the brains of PD, DLB and MSA. We first performed amplification of α-syn aggregates. We successfully amplified enough α-syn aggregates derived from α-syncleinopathies. We found that the MS analysis results of proteinase K-resistant cores of the aggregates before and after the amplification differ between mouse and human α-syn aggregates. The results suggest that structural properties of amplified α-syn fibrils are preserved and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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