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

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...

No description available.

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

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24086号 / 医博第4862号 / 新制||医||1059(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 井上 治久, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pesticides have been suggested to play a role in the development of many neurodegerative diseases including Parkinson's disease and Alzheimer's disease. Additionally, it has been suggested that exposure to pesticides and other environmental chemicals during the early stages of life could result in an increased vulnerability to such substances that could lead to neurotoxicity and degeneration late in life. We hypothesized that exposure to mixtures of certain pesticides could change neurotransmitter levels and cellular oxidative stress and that this would be greater in mice exposed early and later in life than mice exposed only as adults. We studied the effects of permethrin (PR) (a pyrethroid type I) and endosulfan (EN) (an organochlorine) on the levels of catecholamines, indolamines, acetylcholinesterase, lipid peroxidation and α-synuclein in the brain of mice. These pesticides have different structures but both are known to modify the kinetics of voltage-sensitive ion channels and calcium ion flux/homeostasis that could affect the release of several neurotransmitters. The study consisted of two experiments: In the first experiment, adult C57Bl/6 mice (7-9 months old) were injected, intraperitoneally, with the following treatments: EN 4.3, 2.15 mg/kg; PR 150, 15 mg/kg and their mixtures EN 4.3 + PR 150 and EN 2.15 + PR 15 mg/kg. Mice were sacrificed 24 hrs after the last injection. In the second experiment, doses consisted of EN 0.7, 1.4 mg/kg, PR 1.5, 15 mg/kg and their mixtures EN 0.7 + PR 1.5 mg/kg and EN 1.4 + PR 15 mg/kg were given to juvenile mice intraperitoneally daily during a period of two weeks from postnatal day 5 to 19. Mice were then, left undisturbed with their dams. Re-challenge was performed when mice were 7-9 months old and dosages of EN 4.3, 2.15 mg/kg, PR 150, 15 mg/kg and their mixtures, EN 4.3 + PR 150 and EN 2.15 + PR 15 mg/kg were given intraperitoneally every other day during a period of two weeks to match the treatments when pesticide exposure was only as adults. Mice were sacrificed 24 hrs after the last injection. The corpora striatum was extracted and analyzed by HPLC for catecholamines (dopamine, DOPAC, homovalinic acid and norepinephrine) and indolamines (serotonin and 5-HIAA). In general low doses of permethrin and endosulfan alone and in combination (EN 2.15 + PR 15 mg/kg) altered the levels of catecholamines and indolamines in both studies with adult mice and mice dosed as juveniles and re-challenged as adults. Catecholamine and indolamines levels were affected to a greater extent in the adult mice than in mice dosed as juveniles and re-challenged as adults, when compared to controls. Acetylcholinesterase was increased under both exposure situations but again adult mice seemed to be more affected than mice dosed as juveniles and re-challenged as adults. Because reactive oxygen species have been implicated in the development of Parkinson's disease, and are known to cause degradation of certain neurotransmitters, we monitored the levels of lipid peroxides in brain cortex as an indicator of free radical tissue damage. The peroxide levels were measured by thiobarbituric acid reactive products (TBARS). Increased levels of lipid peroxides were significant in the low dose treatment groups of the adult study. However, there seemed to be a pattern between the levels of dopamine and DOPAC in the striatum and the levels of peroxidation in cortex. The presence of dopamine metabolites appeared to be related to high levels of peroxidation within the basal ganglia and up-regulation of proteins such as α-synuclein. Western blots of α-synuclein in both experiments of the study showed intense double and triple bands that corresponded to aggregated α-synuclein. In general, when compared with controls, mice dosed as juveniles and re-challenged as adults did not alter the above parameters as much as mice dosed only as adults. Instead, the mice first dosed as juveniles seemed to develop an adaptation response to the later exposure of these pesticides. Taking all these results into account, early exposure and re-challenge with permethrin and endosulfan in this study appeared to induce a protective response against neurochemical changes in the brain of these mice. In addition, low doses of these pesticides and the low dose combination mixture seem to exert an effect on the parameters studied. Therefore, exposure to pesticides such as endosulfan and permethrin and their combinations could make a contribution towards the initiation or aggravation of biochemical neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. / Master of Science

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.