Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is an adult-onset, progressive and fatal neurodegenerative disease. Pathologically, it is characterized by a loss of motor neurons in the spinal cord, brain stem and motor cortex leading to progressive muscle weakness, atrophy, and death. ALS presents as both a sporadic (SALS) and familial (FALS) illness. Interestingly, over 100 mutations of the CuZn-Superoxide Dismutase (SOD1) gene have been reported to be dominantly inherited in ALS families. SOD1 is a 17KD protein that contains one copper and one zinc atom. The known function of this enzyme is to convert superoxide to oxygen and hydrogen peroxide. It was first thought that the toxicity of different SOD1 mutants linked to ALS resulted from decreased free-radical scavenging activity. However, studies show that mutant SOD1 enzymes cause motor neuron degeneration via a gain of harmful properties. The nature of the gain-of-toxic function in mutant SOD1 is not clear. Recent studies suggest that SOD1 itself is a target of oxidative stress. Human SOD1 has four cysteine residues, Cys6, Cys57, Cys111, and Cys146. An internal disulfide bond exists between Cys57 and Cys146. This disulfide bond is highly conserved in SOD1, making the protein considerably strong, while the remaining two cysteine residues are free and prone to post-translational modifications. Here we show that free cysteine residues in SOD1 are available to be modified by mal PEG (Maleimide polyethylene glycol) and AMS, and that this modification decreases with disease progression. Our data suggests that cysteine residues in SOD1 are post-translationally modified and may play a significant role in the development of the disease.

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is an adult-onset, progressive and fatal neurodegenerative disease. Pathologically, it is characterized by a loss of motor neurons in the spinal cord, brain stem and motor cortex leading to progressive muscle weakness, atrophy, and death. ALS presents as both a sporadic (SALS) and familial (FALS) illness. Interestingly, over 100 mutations of the CuZn-Superoxide Dismutase (SOD1) gene have been reported to be dominantly inherited in ALS families. SOD1 is a 17KD protein that contains one copper and one zinc atom. The known function of this enzyme is to convert superoxide to oxygen and hydrogen peroxide. It was first thought that the toxicity of different SOD1 mutants linked to ALS resulted from decreased free-radical scavenging activity. However, studies show that mutant SOD1 enzymes cause motor neuron degeneration via a gain of harmful properties. The nature of the gain-of-toxic function in mutant SOD1 is not clear. Recent studies suggest that SOD1 itself is a target of oxidative stress. Human SOD1 has four cysteine residues, Cys6, Cys57, Cys111, and Cys146. An internal disulfide bond exists between Cys57 and Cys146. This disulfide bond is highly conserved in SOD1, making the protein considerably strong, while the remaining two cysteine residues are free and prone to post-translational modifications. Here we show that free cysteine residues in SOD1 are available to be modified by mal PEG (Maleimide polyethylene glycol) and AMS, and that this modification decreases with disease progression. Our data suggests that cysteine residues in SOD1 are post-translationally modified and may play a significant role in the development of the disease.

Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons. Symptoms begin focally in one muscle and then spread contiguously, resulting in progressive paralysis and death from respiratory failure. Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause, however, mutations in SOD1 were the first identified and are found in 1-9% of patients. Misfolded SOD1 aggregates in the CNS are hallmarks of ALS associated with SOD1 mutations. However, accumulation of misfolded or aggregated SOD1 protein has also been reported in sporadic and familial ALS without SOD1 mutations, suggesting that wild-type SOD1 could play a role in ALS pathology in general. The aims of this thesis are: 1) To describe the resulting disease phenotype and specific characteristics of the SOD1 protein carrying the stable disease- associated mutation L117V. 2) To set up cell-based in vitro models to study the mechanisms of SOD1 misfolding and aggregation under physiologically relevant expression levels. 3) To compare SOD1 activity in patient-derived samples and screen for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. 1) We identified a novel L117V SOD1 mutant in two families of Syrian origin that co-segregated with the disease. This mutation was associated with slow disease progression, reduced penetrance and a uniform phenotype. The L117V mutant protein was indistinguishable from wild-type SOD1 in terms of stability, dismutation activity and misfolding in patient-derived cell lines. 2) We established patient-derived fibroblast and iPSC-MN lines expressing mutant SOD1 at physiological levels as in vitro models to study misfolding and aggregation of SOD1. We investigated the effects of several cellular pathway disturbances on SOD1 misfolding. Misfolded SOD1 was increased by inhibition of the ubiquitin-proteasome pathway in fibroblasts derived from both patients and controls. An age-related decline in proteasome activity could contribute to the late onset of ALS. Next, we studied the effects of low oxygen tension on misfolding and aggregation of SOD1 in patient-derived cells. Low O2 tensions were found to markedly increase C57-C146 disulphide reduction, misfolding and aggregation of SOD1. Importantly, the largest effects were detected in iPSC-MNs. This suggests that motor neurons are specifically vulnerable to misfolding and aggregation of SOD1 under low O2 tension. 3) We compared the enzymatic activity of SOD1 in blood samples from a large number of ALS patients and controls. We screened for potential underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. No aberrations in copy number, other large structural changes in introns and exons or intronic mutations in the 30-50 bp flanking the exons were found in the 142 outliers, with either very low or very high SOD1 dismutation activities. However, hemoglobinopathies, including thalassemias and iron deficiency anemia, were associated with high SOD1/mg Hb ratios. Erythrocytes from patients with destabilizing SOD1 mutations showed half the normal activity. There were no significant differences in SOD1 activity between control individuals and ALS patients without a coding SOD1 mutation, or carriers of TBK1 mutations or the hexanucleotide repeat expansion in C9ORF72. Our result suggests that SOD1 enzymatic activity is not associated with the disease in non-SOD1 mutation ALS.

Made available in DSpace on 2018-08-01T21:35:05Z (GMT). No. of bitstreams: 1 tese_9880_Dissertação_Schalana Oliveira dos Reis20160617-151046.pdf: 1408582 bytes, checksum: 11818c24ffd0ed418eb5cd23315502f6 (MD5) Previous issue date: 2016-02-26 / O câncer colorretal (CCR) é um dos tipos mais frequentes de câncer no mundo e uma das mais importantes causas de morte no Brasil. É o terceiro tipo de câncer mais comum em homens e o segundo em mulheres em todo o mundo. A superfície do intestino grosso é revestida por criptas de Lieberkuhn, células absortivas invaginadas. O CCR é iniciado pelo aparecimento de Focos de Criptas Aberrantes (FCA) e alterações morfológicas nas criptas de Lieberkuhn. Estudos demonstram que antioxidantes presentes em frutas e vegetais podem contribuir para a prevenção de algumas doenças, como o câncer. Esta ação de prevenção é considerada direta quando o antioxidante atua na eliminação de espécies reativas e indireta, quando induz o aumento da atividade ou expressão das enzimas antioxidantes endógenas, como a SOD1. O açaí Juçara vem se destacando por sua composição rica em compostos antioxidantes, muito importantes na neutralização das espécies reativas. Com intuito de avaliar a ingestão de Juçara no número de FCA e na expressão de SOD1 no CCR, foi realizado estudo com 16 amostras de intestino grosso de ratos submetidos à carcinogênese com a substância 1,2-dimetilhidrazina (DMH). Foram avaliadas a quantidade de FCA, a área e o escore de expressão da SOD1, em ratos suplementados e não suplementados com Juçara. A suplementação com suco de açaí Juçara promoveu menor número de focos de criptas aberrantes na mucosa colorretal de ratos induzidos à carcinogênese. A superóxido dismutase 1 foi expressa na mucosa colorretal de ratos induzidos ou não à carcinogênese. A suplementação com o suco do açaí Juçara resultou na maior expressão e percentual de ocupação da SOD1 em intestino grosso. Assim, o açaí Juçara pode ser um aliado na prevenção do câncer colorretal, visto a redução do número de lesões no epitélio do cólon e o possível auxílio no estímulo à produção de enzimas antioxidantes. Estes resultados abrem perspectivas para o uso do açaí Juçara na suplementação dietética, na elaboração de produtos e medicamentos que favoreçam a prevenção e tratamento do câncer colorretal.

Oxidative stress and inflammation are often linked to the prognosis of diseases caused by chronic alcohol consumption. Chronic alcohol consumption plays a key role in brain tissue damage, often leading to the development of cognitive disorders and loss of brain function. In addition to the direct effects of alcohol on brain function, consumption of alcohol can lead to psychosocial stressors such as legal, financial, and interpersonal problems. It has been found that mice that overexpress C1q/Tumor Necrosis Factor-related protein-3 (CTRP3) and exposed to ethanol daily do not die like the mice who did not overexpress CTRP3 and fed the same diet. Although the specific physiological functions regulated by the CTRP family are largely unknown, there is evidence showing that they have diverse biological effects on inflammation, metabolism, and survival signaling in several different types of tissue. Postmortem brain tissue samples were collected from mice that were exposed to ethanol with transgenic overexpression of CTRP3 and from wild type mice that were only exposed to ethanol. Interestingly, previous immunoblotting of the cerebellum and the hippocampus using collected tissue demonstrated that glia activation was present in the CTRP3 overexpressing mice but not in the wild-type ethanol fed mice. This finding suggests that glia cells are either dying in the ethanol fed wild type mice or that CTRP3 protects and prolongs activated glia cells. The current study will determine if markers of oxidative stress and cell viability are altered in the CTRP3 overexpressing mice when compared to wild-type mice at the molecular level. RNA isolation using the Directzol system and cDNA synthesis using punch dissected homogenate tissue collected from the hippocampus was used for this investigation. Gene expression of BDNF, SOD1 and PARP1 in mouse tissue was determined using quantitative PCR. Immunoblotting of a small number of hippocampal tissue using PARP1 was performed. The mice that were CTRP3 overexpressed and fed ethanol will likely exhibit altered gene expression of cytokines and increased oxidative stress gene expression in postmortem hippocampal brain tissue when compared to wild-type ethanol fed mice. The current studies could contribute to the body of knowledge for the development of novel therapies that may alleviate the neuro-inflammatory effects of alcohol use.

La sclérose latérale amyothrophique (SLA) est une maladie neurodégénérative charactérisée par la perte des neurones moteurs menant à la paralysie et à la mort. Environ 20% des cas familiaux de la SLA sont causés par des mutations de la superoxyde dismutase 1 (SOD1), conduisant vers un mauvais repliement de la protéine SOD1, ce qui a comme conséquence un gain de fonction toxique. Plusieurs anticorps spécifiques pour la forme mal repliée de la protéine ont été générés et utilisés comme agent thérapeutique dans des modèles précliniques. Comment le mauvais repliement de SOD1 provoque la perte sélective des neurones moteurs demeure non résolu. La morphologie, le bilan énergétique et le transport mitochondrial sont tous documentés dans les modèles de la SLA basés sur SOD1, la détérioration des mitochondries joue un rôle clé dans la dégénération des neurones moteurs. De plus, la protéine SOD1 mal repliée s’associe sélectivement sur la surface des mitochondries de la moelle épinière chez les modèles de rongeurs de la SLA. Notre hypothèse est que l’accumulation de la protéine SOD1 mal repliée sur les mitochondries pourrait nuire aux fonctions mitochondriales. À cette fin, nous avons développé un nouvel essai par cytométrie de flux afin d’isoler les mitochondries immunomarquées avec des anticorps spécifiques à la forme malrepliée de SOD1 tout en évaluant des aspects de la fonction mitochondriale. Cette méthode permettra de comparer les mitochondries portant la protéine SOD1 mal repliée à celles qui ne la portent pas. Nous avons utilisé un anticorps à conformation spécifique de SOD1, B8H10, pour démontrer que la protéine mal repliée SOD1 s’associe avec les mitochondries de la moelle épinière des rat SOD1G93A d’une manière dépendante du temps. Les mitochondries avec la protéine mal repliée SOD1 B8H10 associée à leur surface (B8H10+) ont un volume et une production excessive de superoxyde significativement plus grand, mais possèdent un potentiel transmembranaire comparable aux mitochondries B8H10-. En outre, la présence de la protéine mal repliée SOD1 reconnue par B8H10 coïncide avec des niveaux plus élevés de la forme pro-apoptotique de Bcl-2. L’immunofluorescence de sections de moelle épinière du niveau lombaire avec l’anticorps spécifique à la conformation B8H10 et AMF7-63, un autre anticorps conformationnel spécifique de SOD1, démontre des motifs de localisations distincts. B8H10 a été trouvé principalement dans les neurones moteurs et dans plusieurs points lacrymaux dans tout le neuropile. Inversement, AMF7-63 a marqué les neurones moteurs ainsi qu’un réseau fibrillaire distinctif concentré dans la corne antérieure. Au niveau subcellulaire, SOD1 possèdant la conformation reconnu par AMF7-63 est aussi localisée sur la surface des mitochondries de la moelle épinière d’une manière dépendante du temps. Les mitochondries AMF7-63+ ont une augmentation du volume comparé aux mitochondries B8H10+ et à la sous-population non marquée. Cependant, elles produisent une quantité similaire de superoxyde. Ensemble, ces données suggèrent qu’il y a plusieurs types de protéines SOD1 mal repliées qui convergent vers les mitochondries et causent des dommages. De plus, différentes conformations de SOD1 apportent une toxicité variable vers les mitochondries. Les protéines SOD1 mal repliées réagissant à B8H10 et AMF7-63 sont présentes en agrégats dans les fractions mitochondriales, nous ne pouvons donc pas prendre en compte leurs différents effets sur le volume mitochondrial. Les anticorps conformationnels sont des outils précieux pour identifier et caractériser le continuum du mauvais repliement de SOD1 en ce qui concerne les caractéristiques biochimiques et la toxicité. Les informations présentes dans cette thèse seront utilisées pour déterminer le potentiel thérapeutique de ces anticorps. / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by the loss of motor neurons resulting in paralysis and death. Approximately 20% of familial ALS cases are caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several antibodies have been generated that are specific for the misfolded form of the protein, and have been used as therapeutics in pre-clinical models. How misfolded SOD1 provokes a selective loss of motor neurons remains unresolved. Mitochondrial morphology, bioenergetics and transport are all documented is SOD1-mediated ALS models, thus mitochondrial impairment plays a key role in motor neuron degeneration. Moreover, misfolded SOD1 selectively associates with the surface of spinal cord mitochondria in ALS rodent models. We hypothesize that the accumulation of misfolded SOD1 on mitochondria could impair mitochondrial function. To this end, we developed a novel flow cytometric assay to immunolabel isolated mitochondria with misfolded SOD1 antibodies while also evaluating aspects of mitochondrial function. This method will allow for a comparison of mitochondria bearing misfolded SOD1 to those without. We utilized the B8H10 conformation specific SOD1 antibody to demonstrate that misfolded SOD1 associates with SOD1G93A rat spinal cord mitochondria in a in a time dependent manner. Mitochondria with B8H10-reactive misfolded SOD1 associated with their surface (B8H10+) have a significantly larger volume and produce excessive amounts of superoxide, but have a similar transmembrane potential compared to B8H10- mitochondria. In addition, the presence of B8H10-reactive misfolded SOD1 coincides with higher levels of the pro-apoptotic form of Bcl-2. Staining of lumbar spinal cord sections with both B8H10 and another conformation specific SOD1 antibody, AMF7-63, yielded distinct localization patterns. B8H10 was found predominantly in motor neurons and numerous puncta throughout the neuropil. Conversely, AMF7-63 marked motor neurons as well as a distinctive fibrillar network that was concentrated in the anterior horn. At the subcellular level, AMF7-63-reactive misfolded SOD1 also localized to the mitochondrial surface of spinal cord mitochondria in a time-dependent manner. AMF7-63+ mitochondria have an increased volume compared to B8H10+ mitochondria and the unlabelled subpopulation. However, they produce similar amounts of superoxide. Together, these data suggest that there are multiple species of misfolded SOD1 that converge at the mitochondria to cause damage. Moreover, different SOD1 conformations may ellicit varying toxicities towards mitochondria. Both B8H10 and AMF7-63-reactive misfolded SOD1 are present in aggregates in mitochondrial fractions and can therefore not account for any different effects produced in terms of mitochondrial volume. Conformational antibodies are invaluable tools to identify and characterize the continuum of misfolded SOD1 species with regards to biochemical characteristics and toxicity. The information presented in this thesis will be used in determining the future therapeutic potential of these antibodies.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13163号 / 論医博第2150号 / 新制||医||1029(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 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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