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.

Protein deposition in the form of amyloid fibrils is the hallmark of more than 40 human pathologies, including Alzheimer's disease (AD) and Parkinson's disease (PD). Misfolded protein oligomers formed as intermediates during the aggregation process have been strongly implicated in the onset and progression of these diseases. In this thesis, I describe our efforts to uncover molecular agents that can reduce the toxicity caused by protein aggregation via targeting the generation, the physiochemical properties or the membrane affinity of oligomeric species. We employed an integrative approach combining in vitro techniques, including chemical kinetics, atomic force microscopy, and biophysical measurements, and in vivo methods, including neuroblastoma cells and C. elegans models of AD and PD, to identify a range of small molecules and antibodies that can suppress the toxicity related to protein aggregation through a variety of mechanisms. In Chapter 3, we show that the deleterious effects of protein aggregation can be suppressed in AD and PD worms by interfering with the aggregation rates of the amyloid-β peptide (Aβ) and the α-synuclein protein (αS). In Chapter 4, we resolve the mechanism of action for a molecule that enhances the rate of Aβ42 aggregation in AD worms with the result that toxicity is reduced, and find that it potentiates the secondary nucleation microscopic step in vitro. In Chapter 5, we characterize molecules and antibodies that modify the physiochemical properties and self-association of oligomers comprised of several proteins into clusters with reduced diffusibility. In Chapter 6, we classify a family of molecules that protect the cell by displacing several types of oligomeric species from the membrane through a generic mechanism. These results demonstrate strategies by which one can target the aggregation process to alter its resulting toxicity, provide insight into modifying the properties of the most deleterious species associated with protein aggregation and suggest that the protection of the cell from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases is a promising strategy to combat protein misfolding diseases.

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

Chronic neurodegenerative diseases, such as Alzheimer’s disease, prion diseases and many others are unified by the aberrant folding of a host encoded protein to a disease-associated isoform and the predictable cell-to-cell spread of disease-associated misfolded proteins via a putative prion-like mechanism. Prion diseases, for example, are associated with the aberrant folding of host encoded prion protein (PrPC) to a disease-associated isoform, which acts as a seed for the further conversion of PrPC to misfolded protein species. The role of misfolded prion protein in neurodegeneration remains unclear. Accumulation and spread of misfolded prion protein is typically slow and progressive, correlating with neurodegeneration. A number of studies show that mice are susceptible to prion disease with characteristic hallmarks of prion pathology but in the presence of little detectable misfolded prion protein (e.g. the GSS/101LL model). In this thesis I test the hypothesis that detectable species of misfolded prion protein correlate with neurodegeneration and spreads in a predictable, progressive fashion from one anatomically distinct brain region to the next. Using the GSS/101LL model, misfolded prion protein was detected as mostly PK-sensitive isoforms (PrPsen). The progression and pathological presentation is comparable to other prion diseases with larger quantities of PK resistant prion isoforms. A highly sensitive in vitro assay system (the QuIC assay) was subsequently used to establish the extent that misfolded protein was present within the brain. Amyloidogenic prion seeds were found to be widespread throughout the brain from an early stage and spread rapidly throughout the brain. Absence of neurodegeneration in certain brain regions is not due to differing quantities of prion seeds between regions or time exposed to prion seeds, as unaffected regions are exposed to comparative quantities of prion seeds for the same time-period as regions of the brain which eventually succumb to neurodegeneration. These results indicate a clear dissociation between prion seeds and neurotoxicity. They highlight the need to understand regional host responses to prion seeds that may evoke neurodegeneration in some but resilience in others. To test this, transcriptomic analysis was carried out on brain samples from regions undergoing neurodegeneration and unaffected regions. A gene profile signature of hybrid pro-and anti-inflammatory response was observed in regions undergoing neurodegeneration. However, large cohorts of genes were down-regulated across all regions tested, including pro-inflammatory genes and a large proportion of genes involved within transcriptional and translational regulation and function. These results highlight the possible molecular pathways in response to the presence of misfolded protein. In summary, misfolded prion protein accumulates rapidly across the CNS but only specific brain regions undergo neurodegeneration. In the presence of the misfolded protein, the host elicits a robust molecular response. The additional activation of glial cells within regions undergoing neurodegeneration highlights their importance in disease. It is therefore proposed that misfolded prion protein, alone, is not sufficient to trigger neurodegeneration. This gives rise to a “multi-hit” hypothesis whereby two or more factors, for example the accumulation of misfolded protein and glial cell response, are required to trigger neurodegeneration.

No description available.

Amyotrophic lateral sclerosis, ALS, is a progressive fatal neurodegenerative disorder affecting motor neurones in motor cortex, brain stem and spinal cord. This inevitably leads to paralysis, respiratory failure and death. In about 5% of patients with ALS there is an association with mutations in gene for the abundant intracellular scavenging enzyme superoxide dismutase1, SOD1. The noxious property of SOD1 is proposed to be due to gain of function. In familial cases the inheritance is most commonly dominant. This study focus on two disparate SOD1 mutations occurring in Scandinavia. The recessive D90A mutation which has properties similar to that of the normal wild-type human SOD1. The dominantly inherited G127insTGGG mutation, G127X, causes a C-terminal truncation of the last 21 amino acids and is a highly unstable protein. Transgenic mice were created expressing D90A and G127X mutated human SOD1. Results from studies of tissue from the central nervous system of patients carrying either of these mutations were compared with similar tissue collected from transgenic mice generated with the same mutations. Tissue from the mice were also compared to central nervous tissue from several other transgenic mouse strains expressing human wild type SOD1 as well as other ALS associated human SOD1 mutations. The transgenic mice expressing D90A respectively G127X mutated human SOD1 develop motor neurone disease. Microscopic studies of central nervous tissues from G127X transgenic mice reveals inclusions of aggregated misfolded SOD1 in motor neurones and adjacent supporting cells. These inclusions are composed of detergent resistant aggregates and preceded by accumulations of minute quantities of detergent-soluble aggregates. The inclusions mimic those found in G127X patients. In D90A transgenic mice the progression, as in the humans, was slower and the mice, as the patients, showed bladder disturbance. In the D90A patients, the SOD1 inclusions mimic those found in sporadic ALS patients. Aggregation of SOD1 in central nervous tissue appears to be related to severity of disease. Degenerative features as vacuolization and gliosis precedes phenotypic alterations. Changes are seen not only in motor areas but also in higher centres of the telencephalon.

学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(総合学術) / 甲第21931号 / 総総博第6号 / 新制||総総||1(附属図書館) / 京都大学大学院総合生存学館総合生存学専攻 / (主査)教授 阪井 康能, 教授 山口 栄一, 教授 積山 薫 / 学位規則第4条第1項該当 / Doctor of Philosophy / Kyoto University / DGAM

No description available.

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.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the spinal cord, brain stem and motor cortex, leading to paralysis, respiratory failure and death. In about 5% of ALS cases, the disease is associated with mutations in the CuZn-superoxide dismutase (hSOD1) gene. As a rule, ALS caused by hSOD1 mutations is inherited dominantly and the mutant hSOD1s cause ALS by the gain of a noxious property. The present study focused on two hSOD1 mutations with widely differing characters. In Scandinavia, ALS caused by the D90A mutation is inherited in a recessive pattern. Elsewhere, families with dominant inheritance have been found. The properties of D90A mutant hSOD1 are very similar to those of the wild-type protein. The G127insTGGG (G127X) mutation causes a 21 amino acid C-terminal truncation which probably results in an unstable protein. The aim of this thesis was to generate transgenic mice expressing D90A and G127X mutant hSOD1s and to compare these mice with each other and with mice expressing other mutant hSOD1s, in search of a common noxious property. The findings were also compared with the results from studies of human CNS tissue. The cause of the different inheritance patterns associated with D90A mutant hSOD1 was investigated by analyzing erythrocytes from heterozygous individuals from dominant and recessive pedigrees. There was no evidence that a putative protective factor in recessive pedigrees acts by down-regulating the synthesis of D90A mutant hSOD1. In cerebrospinal fluid, there was no difference in hSOD1 content between homozygous D90A patients, ALS patients without hSOD1 mutations and controls. hSOD1 cleaved at the N-terminal end was found in both controls and D90A patients, but the proportion was significantly larger in the latter group. This indicates a difference in degradation routes between mutant and wild-type hSOD1. Both D90A and G127X transgenic mice develop an ALS-like phenotype. Similar to humans, the levels of D90A protein were high. The levels of G127X hSOD1 were very low in the tissues but enriched in the CNS. Similarly, in an ALS patient heterozygous for G127X hSOD1, the levels of the mutant protein were overall very low, but highest in affected CNS areas. Despite the very different levels of mutant hSOD1, both D90A and G127X transgenic mice developed similar levels of detergent-resistant aggregates in the spinal cord when terminally ill. Surprisingly, mice overexpressing wild-type hSOD1 also developed detergent-resistant aggregates, although less and later. Most of the hSOD1 in the CNS of transgenic mice was inactive due to deficient copper charging or because of reduced affinity for the metal. The stabilizing intrasubunit disulfide bond of hSOD1 was partially or completely absent in the different hSOD1s. Both these alterations could increase the propensity of mutant hSOD1s to misfold and form aggregates. The results presented here suggest that the motor neuron degeneration caused by mutant hSOD1s may be attributable to long-term exposure to misfolded, aggregation-prone, disulfide-reduced hSOD1s and that the capacity to degrade such hSOD1s is lower in susceptible CNS areas compared with other tissues. The data also suggest that wild-type hSOD1 has the potential to participate in the pathogenesis of sporadic ALS.