Parkinson’s disease is a movement disorder characterized by nigrostriatal dopamine pathway degeneration and neuronal α-synuclein accumulation. Pathogenesis is associated with mutations in α-synuclein and Gba1 encoding alleles. Animal models created to date do not recapitulate the spectrum of clinical disease features. This thesis characterizes the bi-genic Synergy mouse, hypothesized to demonstrate motor behavioural and histological abnormalities downstream of α-synuclein overexpression and mutated Gba1. Synergy and SNCA mice (overexpressed α-synuclein with wild-type Gba1) have early onset deficits in motor coordination, muscle strength and nest building. Both exhibit increased α-synuclein concentration in the brain and cerebellar inclusions positive for two markers of pathological α-synuclein processing. Overall mutant Gba1 expression within Synergy mice does not worsen the behaviour or the histopathological findings associated with overexpression of human α-synuclein in SNCA mice. Future studies will determine whether mutant Gba1 expression alters cognitive behaviour and/or lipid homeostasis in this new bi-genic model of Parkinson’s disease.

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Les protéines alpha-synucléine (α-syn) et leucine-rich repeat kinase 2 (LRRK2) sont deux protéines ayant un rôle majeur dans la physiopathologie de la maladie de Parkinson (MP) et interviennent aussi bien dans les formes dites sporadiques que dans les formes familiales. La mutation G2019S du gène codant pour LRRK2 est la mutation la plus fréquente. Cette mutation induit une augmentation de l’activité kinase de LRRK2 qui conduit à sa toxicité. Plusieurs hypothèses convergent vers l’idée que LRRK2 et l’α-syn interagiraient pour conduire à la dysfonction et/ou la mort des neurones dopaminergiques (DA) de la substance noire (SNc) dans la MP. Dans la première partie de cette étude, différentes formes sauvage (WT) ou mutée (G2019S) de LRRK2 ont été surexprimées spécifiquement dans les neurones de la SNc via l’utilisation de vecteurs lentiviraux (LV) et adéno-viraux associés (AAV). La question principale de cette étude était d’évaluer si l’expression spécifiquement neuronale de LRRK2 induisait la dégénérescence des neurones DA de la SNc. Nous avons généré des constructions comportant uniquement la partie C-terminale de LRRK2 (ΔLRRK2) en aval du domaine LRR. In vitro, le fragment ΔLRRK2G2019S présente une activité kinase supérieure au fragment ΔLRRK2WT avec une augmentation d’activité comparable à la forme entière de LRRK2. In vivo, six mois après l’injection (PI) de ΔLRRK2 WT ou G2019S dans la SNc, les mesures du nombre de neurones montrent que seul le fragment ΔLRRK2G2019S induit une mort neuronale significative (30%) comparé à la forme ΔLRRK2WT, uniquement lorsque l’expression est générée via des vecteurs AAV. Ces résultats suggèrent que l’expression purement neuronale d’un fragment contenant le domaine kinase de LRRK2 est suffisante pour induire une dégénérescence de la SN. Dans la seconde partie du projet, nous avons étudié l’hypothèse que ΔLRRK2G2019S via son activité kinase amplifiée, pourrait augmenter la toxicité le l’α-syn mutée A53T. Pour répondre à cette question, les vecteurs AAV codant pour ΔLRRK2 G2019S ou une forme inactive de la kinase (ΔLRRK2G2019S/D1994A), et celui codant pour l’α-syn A53T ont été co-injectés dans la SNc. Les analyses réalisées à 6 et 15 semaines PI montrent que ΔLRRK2G2019S augmente la mort neuronale induite par l’α-syn A53T d’une manière kinase dépendante. Tous ces résultats supportent l’hypothèse que l’existence d’une interaction fonctionnelle entre LRRK2 et l’α-syn pourrait jouer un rôle fondamental dans la physiopathologie de la MP offrant des possibilités de stratégie de neuroprotection ciblant l’interaction LRRK2/α-syn. / Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) proteins are likely to play crucial roles both in sporadic and familial forms of Parkinson’s disease (PD). The most prevalent mutation in LRRK2 is the G2019S substitution which induces neurotoxicity through a marked increase of its kinase activity. A possible interplay between LRRK2 and α-syn may be involved in the dysfunction and/or in the death of dopaminergic (DA) neurons in the substantia nigra (SNc) in PD. In the first part of the study, we evaluated whether the overexpression of LRRK2G2019S using lentiviral vectors (LVs) and adeno-associated virus (AAV2/9), which can overexpress transgenes selectively in neurons could trigger neurodegeneration in the SNc, in other words, whether cell-autonomous mechanisms are sufficient to trigger the degeneration of DA neurons. We generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2) containing the kinase domain. Results of assays performed in vitro indicated that ΔLRRK2 retains biochemical properties of full length LRRK2. Six months after the stereotaxic injection of LV-ΔLRRK2G2019S in the SNc, the number of DA neurons was unchanged, however, the infection of the SNc with AAV-ΔLRRK2G2019S but not with AAV-ΔLRRK2WT induced a significant ~30% loss of DA neurons. These results suggested that neuronal overexpression of the mutant kinase domain of LRRK2 was sufficient to trigger neurodegeneration in the SNc in the adult brain. In the second part of the study, we aimed at studying whether ΔLRRK2G2019S could increase the neurotoxicity of a mutant form of α-syn (A53T mutation) in vivo in DA neurons. We used a co-infection approach with AAV vectors encoding the α-synA53T, and ΔLRRK2 G2019S alone or with the D1994A mutation (ΔLRRK2G2019S/D1994A) that inactivates the kinase activity of LRRK2. AAVs were stereotaxically co-injected into the rat SNc and histological evaluation was performed at 6 and 15 weeks (early and late time points) post-infection. Results showed that ΔLRRK2G2019S increased the toxicity of α-synA53T in a kinase-dependent manner. Altogether, the present study supports the hypothesis that a functional interaction between LRRK2 and α-syn may play a key role in PD pathogenesis. The new “double hit” model we developed in rats may be of interest to test novel neuroprotective strategies targeting LRRK2/α-syn in vivo.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23031号 / 理博第4708号 / 新制||理||1675(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺嶋 正秀, 教授 林 重彦, 教授 渡邊 一也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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Background In the as of yet uncurable Parkinson´s disease aggregation of α-syn is an accelerator of pathogenesis. Oligomers of α-synuclein is considered to be neurotoxic hence blocking the endocytosis of aggregated α-syn is possibly a way of preventing pathogenesis. With a protein construct of the Receptor X (RX) previously shown to bind α-syn, it can be possible to bind soluble aggregated α-syn and decrease neuron endocytosis.  Aim The aim of this study was to express, purify and trimerize two different protein constructs of RX to study the binding to α-syn monomers & oligomers and if the proteins have higher affinity to α-syn oligomers. Methods In this study two RX constructs was produced with mammalian cell transfection and purified with Strep-Tactin affinity chromatography; D1, D123mut and D123 which affinity to α-syn monomers and oligomers were studied with ELISAs. Indirect ELISAs were optimized and conducted, a competitive ELISA with D123 was tested with poor reliability.  Results The results show that D1 could not be determined pure enough to examine its α-syn binding ability. D123mut was pure enough for ELISAs but did not show adequate binding to α-syn. D123 did show binding to α-syn in an indirect ELISA.  Conclusion The results were not as promising as expected and did not distinctly help strengthen the theory of a recombinant RX protein as a viable drug. Although there is potential, optimization of both protein constructs and methods used is essential for future studies of RX as a therapeutic protein.

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...

α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with l-serine, l-proline, l-aspartate, or l-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or l-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

In this work two overall subjects were addressed. 1. In recent years high pressure perturbance has become a tool to investigate the folding energy landscape, the volumetric properties and the conformational equilibria of proteins. Conformational states which are not populated at ambient conditions thus become accessible to spectroscopic characterization. In this work a high pressure application was combined with EPR spectroscopy to investigate three spin labeled proteins, BSA from Bos taurus, HisJ from Salmonella enterica serovar Typhimurium and α-synuclein from Homo sapiens. The goal of these studies was to comprehend the influence of pressure on the respective EPR spectra and to identify changes in conformational equilibria and volumetric properties of the investigated proteins. Studies on BSA revealed a negative activation volume for rotational diffusion of the spin labeled site. Moreover, a rotameric equilibrium was derived from the pressure-dependent side chain dynamics and a correlating negative partial molar volume was observed, indicating a shift of the rotameric equilibrium to lesser order. In this regard it was also shown that a chaotropic medium (guanidine hydrochloride) supports the pressure-dependent effect. Spin labeled sites in the substrate binding protein HisJ revealed to be highly influenceable by low pressures between ambient conditions and 200 bar. Pressurization induced oligomerization and precipitation of the protein. Substrate binding revealed differences in pressure-dependence with regard to a decreased precipitation effect but not in relation to oligomerization. The natively unfolded protein α-synuclein plays a key role in Parkinson´s disease and is known for forming β-sheet rich aggregates, so called amyloid fibrils. The experimental data of this work revealed that hydrostatic pressure can induce a non-amyloid aggregation of monomeric α-synuclein which produces an unspecific oligomer. Furthermore, it was shown that α-synuclein amyloid fibrils can be dissolved by hydrostatic pressure. From the pressure dependent conformational equilibrium between the monomer and the fibril form the change of the partial molar volume of the investigated site was determined. 2. The second subject of this work was focused on different import systems, ATP-binding cassette (ABC) transporters and Energy-Coupling-Factor (ECF) transporters, for amino acids, vitamins and metal ions in prokaryotes. Studies on one bacterial ABC and two ECF transporter systems from two different organisms, the histidine ABC-type transporter HisQMP2 from Salmonella enterica serovar Typhimurium, the biotin ECF-type importer BioMNY from Rhodobacter capsulatus and the cobalt-specific ECF-type transporter CbiMNQO from Rhodobacter capsulatus, were performed using DEER and cw EPR spectroscopy. The goal of the studies on HisQMP2 and BioMNY was to shed light on conformations and dynamics connected to their transporter function. Studies on CbiMNQO aimed at the detection of the substrate in the transporter´s substrate binding unit. For HisQMP2 transport cycle dependent conformational changes and interactions with the substrate binding protein HisJ were revealed. Three different distance values between sites H101R1 and H101’R1 in the transporter´s nucleotide binding domains were assigned to the apo-, the ATP-bound and the posthydrolysis state. It was shown that the closed conformation of the nucleotide binding domains is achieved only in the presence of the ligand-bound HisJ which indicates a transmembrane communication of the association of HisJ to the transporter. Furthermore, interspin distances were determined between sites D86R1-A96R1, C197R1-C104R1 and A118R1-G123R1 in the transmembrane domains HisQ and HisM revealing distinguishable conformational states which correlate to the different states of the nucleotide binding sites during the hydrolysis cycle. Measured interspin distances between HisJ and HisM in the HisQMP2 complex showed that interaction only occurred in the closed state of the HisP2 dimer, the nucleotide bound state. Two different, substrate-dependent interactions between site G24R1 in HisJ and site A96R1 in HisQMP2 were observed, revealing that the substrate-free and substrate-bound form of HisJ both associate with HisQMP2. Distance measurements between sites G24R1 and T151R1 in HisJ in the presence and absence of its substrate revealed interspin distance changes that correlate with the proteins open and closed conformation. Investigations on the ECF transporter BioMNY, reconstituted into nanodiscs, revealed a closure and reopening of the nucleotide binding domains between sites H87R1 and H87’R1 using DEER spectroscopy which delivered interspin distance values that correlate with the apo-, the ATP-bound and the posthydrolysis state of the transporter. Further experiments were aimed to shed light on the transporters substrate-translocation mechanism with regard to the so called toppling over mechanism. Unfortunately, the experiments of this work were not able to give a distinct answer with respect to the proposed model because of the transmembrane domains tendency to oligomerize when reconstituted into nanodiscs. In this work we showed that substrate uptake by the substrate binding unit CbiM of the cobalt-specific ECF transporter CbiMNQO depends on the presence of the small transmembrane protein CbiN. Measurements of spin labeled CbiMN in detergent showed oligomerization of CbiM.