La formation d'agrégats protéiques insolubles et fibreux, appelés fibrilles amyloïdes, est impliquée dans une large variété de maladies humaines. Parmi elles, figurent entre autres, le diabète de type II, l'arthrite rhumatoïde et, notamment, les atteintes neurodégénératives débilitantes, telles que les maladies d'Alzheimer, de Parkinson ou encore de Huntington. Actuellement, il n'existe ni traitement, ni diagnostic précoce pour aucune de ces maladies.De nombreuses études ont montré que la capacité à former des fibrilles amyloïdes est une propriété inhérente à la chaîne polypeptidique. Ce constat a conduit au développement d'un certain nombre d'approches computationnelles permettant de prédire les propriétés amyloïdogéniques à partir de séquences d'amino-acides. Si ces méthodes s'avèrent très performantes vis à vis de courts peptides (~ 6 résidus), leur application à des séquences plus longues correspondant aux peptides et protéines en lien avec les maladies, engendre un nombre trop élevé de faux positifs. Le principal objectif de cette thèse consiste à développer une meilleure approche bioinformatique, capable de prédire les régions amyloïdogéniques à partir d'une séquence protéique. Récemment, l'utilisation de nouvelles techniques expérimentales a permis de mieux appréhender la structure des amyloïdes. Il est ainsi apparu que l'élément caractéristique de la majorité des fibrilles amyloïdes impliquées dans les maladies, était constitué d'une structure étagée (β-arcade), résultant de l'empilement de motifs « feuillet β – coude – feuillet b » appelés « β-arches ». Nous avons mis à profit cette particularité structurale pour créer une approche bioinformatique permettant de prédire les régions amyloïdogéniques d'une protéine à partir de l'information contenue dans sa séquence. Les résultats provenant de l'analyse des structures de type β-arcade, connues et modélisées, ont été compilés et traités à l'aide d'un algorithme écrit en langage Java, afin de créer le programme ArchCandy.L'application de ce programme à une sélection de séquences protéiques et peptidiques, connues pour leur lien avec les maladies, a permis de démontrer qu'il était en mesure de prédire correctement la majorité de ces séquences, de même que les séquences mutées impliquées dans les maladies familiales. Outre la prédiction de régions à haut potentiel amyloïde, ce programme suggère la conformation structurale adoptée par les fibrilles amyloïdes. Le séquençage de génomes entiers devenant toujours plus abordable, notre méthode offre une perspective de détermination individuelle des profils à risque, vis à vis de maladies neurodégénératives, liées à l'âge ou autres. Elle s'inscrit ainsi pleinement dans l'ère de la médecine personnalisée. / A broad range of human diseases are linked to the formation of insoluble, fibrous, protein aggregates called amyloid fibrils. They include, but are not limited to, type II diabetes, rheumatoid arthritis, and perhaps most importantly, debilitating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. There currently exists no cure, and no means of early diagnosis for any of these diseases. Numerous studies have shown that the ability to form amyloid fibrils is an inherent property of the polypeptide chain. This has lead to the development of a number of computational approaches to predict amyloidogenicity by amino acid sequences. Although these methods perform well against short peptides (about 6 residues), they generate an unsatisfactory high number of false positives when tested against longer sequences of the disease-related peptides and proteins. The main objective of this thesis was to develop an improved bioinformatics based approach to predict amyloidogenic regions from protein sequence.Recently new experimental techniques have shed light on the structure of amyloids showing that the core element of a majority of disease-related amyloid fibrils is a columnar structure (β—arcade) produced by stacking of β-strand-loop-β-strand motifs called “β-arches”. Using this structural insight, we have created a bioinformatics based approach to predict amyloidogenic regions from protein sequence information. Data from the analysis of the known and modeled β-arcade structures was incorporated into a rule based algorithm implemented in the Java programming language to create the ArchCandy program. Testing it against a set of protein and peptide sequences known to be related to diseases has shown that it correctly predicts most of these sequences and a number of mutated sequences related to the familial diseases. In addition to the prediction of regions with high amyloidogenic potential, a structural arrangement of the amyloid fibril is also suggested for each prediction. As whole genome sequencing becomes cheaper, our method provides opportunity to create individual risk profiles for the neurodegenerative, age-related and other diseases ushering in an era of personalized medicine.

Friedreich ataxia (FRDA) is a neurodegenerative disorder that is inherited in an autosomal recessive pattern. The most common FRDA mutation is hyperexpansion of a GAA triplet repeat sequence in the first intron of the affected gene, frataxin (FXN), resulting in decreased frataxin protein expression. The hyperexpanded GAA repeats can adopt unusual DNA structures and induce aberrant epigenetic changes leading to heterochromatin mediated gene silencing. Several epigenetic changes, including increased levels of DNA methylation, histone modifications, repressive chromatin formation and elevated levels of non-coding RNA have been reported in FRDA. It has been reported that a novel FXN antisense transcript (FAST-1), is present at higher levels in FRDA patient-derived fibroblasts and its overexpression is associated with the depletion of CTCF, a chromatin insulator protein, and heterochromatin formation involving the critical +1 nucleosome. Previously, characteristics of FAST-1 were investigated in our lab and a full-length FAST-1 transcript containing a poly (A) tail was identified. To investigate any possible effects of FAST-1 on FXN expression, I first overexpressed this FAST-1 transcript in three different non-FRDA cell lines and a consistent decrease of FXN expression was observed in each cell type compared to control cells. I also identified that FAST-1 copy number is positively correlated with increased FAST-1 expression, which in turn is negatively correlated with FXN expression in FAST-1 overexpressing cells. Additionally, we found that FAST-1 overexpression is associated with increased levels of DNA methylation at CpG sites U6 and U11 of the FXN upstream GAA repeat region, together with CTCF depletion and heterochromatin formation at the 5'UTR of the FXN gene. To further investigate the role of FAST-1 in FXN gene silencing, I used a small hairpin RNA (shRNA) strategy to knock down FAST-1 expression in FRDA fibroblast cells. I found that knocking down FAST-1 increases FXN expression, but not to the level of control cells. Lastly, I investigated the pattern of FAST-1 expression and histone modifications at the FXN transgene in our new FRDA mouse model, designated YG8LR. The YG8LR mice showed decreased levels of FXN expression and H3K9ac and increased levels of FAST-1 expression and H3K9me3. Our data suggest that since FAST-1 is associated with FXN gene silencing, inhibition of FAST-1 may be an approach for FRDA therapy.

The pathological hallmark of many age-related neurodegenerative diseases is the presence of proteinaceous inclusions in nerve cells and glial cells. Alpha-synuclein is the main component of the inclusions of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, as well as of rarer diseases, collectively called synucleinopathies. For a long time, it was widely believed that neurodegenerative diseases were cell-autonomous; however, a more recent hypothesis has suggested that some misfolded proteins resemble prions. Thus, aggregated alpha-synuclein shares features of PrPSc, the scrapie form of the prion protein. The aim of this thesis was to further characterize the prion-like properties of aggregated alpha-synuclein by studying the pathways of seeded aggregation, and to identify the species of alpha-synuclein responsible. I present evidence, using a HEK 293T cell model, that filamentous protein was the most seed-potent form of alpha-synuclein. Recombinant aggregated protein, aggregated alpha-synuclein from mice transgenic for A53T alpha-synuclein, as well as alpha-synuclein aggregates from Parkinson’s disease and multiple system atrophy brains, seeded aggregation. The mechanisms of alpha-synuclein internalization and intracellular trafficking, and how these processes affect seeded aggregation, are not fully understood. I showed that internalization of alpha-synuclein aggregates occurs through clathrin- and dynamin-independent, Cdc42-, actin- and PI3K-dependent endocytosis. Alpha-synuclein aggregates are trafficked to the endolysosomal pathway; a small fraction of lysosomes ruptures, which induces aggregation of expressed cytoplasmic alpha-synuclein, and disruption of autophagy, which in turn enhances seeded aggregation. These findings expand knowledge of the prion-like properties of assembled alpha-synuclein and identify novel mechanisms with therapeutic potential.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease of motor neurons characterized by muscle paralysis and death within 3-5 years of onset. However, due to unknown mechanisms, the extraocular muscles (EOMs) remain remarkably unaffected. The EOMs are highly specialized muscles that differ from other muscles in many respects, including innervation and satellite cells (SCs). Understanding whether these factors play a role in the relative sparing of EOMs in ALS could provide useful clues on how to slow down the progression of ALS in other muscles. The EOMs and limb muscles from terminal ALS patients and age-matched controls as well as the commonly used SOD1G93A ALS mouse model were studied with immunofluorescence. Antibodies against neurofilament and synaptophysin were used to identify nerves and neuromuscular junctions (NMJs); against Pax7, NCAM, MyoD, myogenin, Ki-67, dystrophin and laminin, to identify SCs and their progeny in EOMs and limb muscles. The proportion and fiber size of myofibers containing myosin heavy chain (MyHC) slow tonic and MyHC slow twitch were also determined in human EOMs. The abundance of SCs differed extensively along the length of control human EOMs, being twice as abundant in the anterior portion. Pax7-positive cells were also detected in non-traditional SC positions. EOMs from terminal ALS patients showed similar numbers of resting and activated SCs as the controls. In limb muscles of ALS patients, the number of resting and activated SCs ranged from low (similar to normal aged, sedentary individuals) to high numbers, especially in muscles with long duration of disease and varied between the upper and lower limbs. The EOMs maintained a high degree of innervation compared to hindlimb muscles of symptomatic SOD1G93A mice. MyHC slow tonic fibers were less abundant in ALS patients than in controls. The change seemed more pronounced in bulbar onset patients, and in this group of subjects only, there was a strong association between decline in MyHC slow tonic fibers and age of death. Notably, the decline in MyHC slow tonic fibers was unrelated to disease duration. Our data suggested that SCs play a minor role in the progression of ALS in general and in the sparing of the EOMs in particular. The generally preserved innervation in the EOMs of G93A mice may reflect distinct intrinsic properties relevant for sparing of the oculomotor system.  Even though the EOMs are relatively spared in ALS, MyHC slow tonic myofibers were selectively affected and this may reflect differences in innervation, as these fibers are multiply innervated. / Amyotrofisk lateralskleros (ALS) är en obotlig neurodegenerativ sjukdom som främst påverkar kroppens viljestyrda motoriska nervceller. ALS leder till förlamning, muskelförtvining och slutligen döden genom andningssvikt, vanligen inom tre till fem år efter sjukdomsdebuten. Av okända anledningar så bibehålls ögonmusklernas funktion mycket bättre vid ALS i jämförelse med andra muskler och är hos merparten av patienter i stort sett opåverkade. Ögonmusklerna är mycket specialiserade muskler som skiljer sig från andra muskler i kroppen på flera sätt, bland annat genom deras unika nervförsörjning och genom de satellitceller – muskelspecifika stamceller, som finns i dem. En ökad förståelse för hur dessa faktorer inverkar på ögonmusklernas motståndskraft vid ALS skulle kunna ge värdefulla ledtrådar till hur man skulle kunna sakta ned sjukdomens fortskridande i andra muskler vid ALS. Ögonmuskler och extremitetsmuskler från avlidna ALS-patienter och åldersmatchade friska kontroller, tillsammans med transgena möss med den sjukdomsalstrande mutationen SOD1G93A, studerades genom immunfluorescens och efterföljande mikroskopering. Antikroppar mot molekylerna Pax7, NCAM, MyoD, myogenin, Ki-67, laminin och dystrofin användes för att identifiera satellitceller och deras dotterceller i ögonmuskler och extremitetsmuskler. Antikroppar mot neurofilament och synaptofysin användes för att identifiera nerver och neuromuskulära synapser hos transgena SOD1-möss. Antikroppar mot toniska (tonic) och ryckande (twitch) muskelmyosinkedjor användes för att bestämma proportionen av och storleken på dessa typer av muskelfibrer i ögonmuskler från avlidna ALS-patienter och friska kontroller. Mängden satellitceller varierade mellan de främre och de mer bakre delarna i friska, humana ögonmuskler och var dubbelt så många i den främre delen av muskeln jämfört med den mellersta och bakre delen av muskeln. Celler som uttryckte satellitcellsmarkören Pax7 hittades även i icke-traditionella satellitcellspositioner i ögonmusklerna. Mängden satellitceller i ögonmusklerna från ALS-patienter var samma som hos friska kontroller. I extremitetsmusklerna hos ALS-patienter varierade mängden satellitceller mellan låga nivåer (liknande de hos friska åldrade, inaktiva individer) till höga nivåer, särskilt i muskler där sjukdomen fortskridit under lång tid. Dessutom varierade mängden satellitceller mellan övre och nedre extremiteter. Hos symptomatiska SOD1G93A-möss hade ögonmusklerna en mycket välbevarad innervation jämfört med bakbensmusklerna, där många neuromuskulära synapser saknade kontakt mellan nerven och motorändplattan. Proportionen muskelfibrer med toniska muskelmyosinkedjor var lägre hos ALS-patienter jämfört med friska kontroller. Denna minskning var tydligare hos patienter där sjukdomssymtomen hade debuterat i tugg- och ansiktsmuskulaturen – så kallad bulbär ALS. Dessutom fanns det i den här gruppen, men ingen annan studerad grupp, en stark korrelation mellan nedgången i toniska fibrer och patientens ålder. Värt att notera är att minskningen av toniska muskelfibrer saknade korrelation med hur länge patienten hade varit sjuk i ALS. Den generellt välbevarade innervationen i ögonmusklerna hos SOD1G93A-möss kan spegla distinkta inneboende egenskaper hos ögonmusklerna som är av vikt för bevarandet av ögonrörligheten vid ALS. Gällande satellitceller så antyder våra data att satellitceller och deras regenerativa kapacitet spelar en försumbar roll vid ALS i allmänhet och vid ögonmusklernas bevarande i synnerhet. Slutligen, även om ögonmuskler generellt är välbevarade vid ALS så är toniska muskelfibrer märkbart påverkade och detta kan spegla skillnader mellan olika nervcellsgruppers känslighet vid ALS.

Background: Children with chromosome 22q11.2 deletion syndrome (22q11.2DS) exhibit nonverbal learning disability that may manifest in part because of working memory (WM) deficits. 22q11.2DS is a complex developmental disorder with serious physical, learning, cognitive, and psychiatric symptoms including a risk of developing schizophrenia 30 times that of the general population. WM impairment likely contributes to and exacerbates learning difficulties, school problems, existing neuropsychological disorders such as attention deficit hyperactivity disorder (ADHD); and a poor WM may be a biological risk marker for future mental illness. WM impairment is established in this population, but less is known about its neurological origins. Frontoparietal cortical development and function are key to WM processing. In the neurotypical developing brain, studies indicate activation associated with WM shifts from parietal to frontal regions with age. However, in children with 22q11.2DS, activation is restricted to the frontal cortex, and volumes are reduced in parietal regions where abnormal tractography abides. The overarching aim of this study was to determine the neural origins of WM impairment in people with 22q11.2DS. Methods: We measured WM in children and adolescents with (n = 29) and without (n = 27) 22q11.2DS using the WISC-IV and a computer-based spatial working memory task (SWMT) task. Participants’ brains were scanned using high-resolution magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Focusing on brain morphometry and structural connectivity within frontoparietal networks, we investigated neural underpinnings of WM processing in 22 children with 22q11.2DS and 19 typically developing (TD) controls ages 7 to 16 (M = 12.13 ± 2.41). A connectome mapping network involved in WM processing was constructed by superimposing cortical segmentations on white-matter tractography. Results: Children with 22q11.2DS had impaired working memory performance. Individuals’ performance on our SWMT moderated the association between diagnosis and gray and white matter macro and microstructure. Children with 22q11.2DS with better working memory had larger lateral orbitofrontal volumes, greater axial diffusivity in the left superior frontal to superior parietal tract, and smaller volume in the right superior frontal to lateral orbitofrontal tract. Poorer performance in children with 22q11.2DS was associated with smaller right superior parietal and superior frontal cortical volumes. Conclusions: Children with 22q11.2DS performed worse on measures of working memory. Their performance was related to regional cortical volume differences and white matter microstructure abnormalities in the frontal and parietal lobes. These are brain regions consistently implicated in WM processing.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19004号 / 工博第4046号 / 新制||工||1623(附属図書館) / 31955 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 渡辺 宏, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

TAR DNA Binding Protein-43 (TDP-43) proteinopathy is a disease pathology that underlies a broad field of neurodegenerative disorders. Most prominently, TDP-43 aggregates are the hallmark of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). The implication of TDP-43 in ALS, in particular, has helped initiate a cascade of research to determine the properties of the previously obscure protein. From these studies, it is now known that TDP-43 is a DNA and RNA binding protein, important for the splicing and regulation of many transcripts. In the disease state, TDP-43 is modified in a way that fuels its accumulation into cytoplasmic aggregates called inclusions. This paper will delineate the current understanding of the mechanisms behind TDP-43 proteinopathy and the resultant clinical conditions. The body of evidence firmly supports a clinical spectrum of TDP-43 proteinopathy that ranges between pure motor neuron disease (MND) and pure frontotemporal dementia (FTD). It also appears that the root cause of neurodegeneration in these disorders comes about through a combination of a gain of toxic function and a loss of normal TDP-43. Continued research into the molecular processes leading to the capitulation of TDP-43 holds great promise for the development of new drug targets to help treat the spectrum of TDP-43 proteinopathy.

Riboflavin transport is mediated, in part, by riboflavin transporter proteins 2 and 3, encoded by SLC52A2 and SLC52A3, respectively. Biallelic mutations in SLC52A2 and SLC52A3 impair riboflavin transporter protein function and riboflavin transport, causing disruptions to mitochondrial metabolism which result in sensory and motor neurodegeneration and give rise to riboflavin transporter deficiency (RTD) in humans. RTD is a rare neurodegenerative disease characterised by respiratory compromise, muscle and limb weakness, and vision and hearing impairments. RTD patients are treated with high-dose riboflavin supplementation which is effective in over 70% of cases but can be ineffective due to rapid excretion of riboflavin when its plasma concentration exceeds 0.5 μM. To address the need for alternative or supplemental RTD treatment, this study generated morpholino-mediated knockdown and CRISPR/Cas9 models of RTD in zebrafish. An RTD-like phenotype is observed in these RTD models including hearing loss, decreased motor axon length, and impaired locomotor activity. The slc52a3 morphant phenotype was found to be specific via coinjection of slc52a3 morpholino/human SLC52A3 mRNA, which achieved effective rescue of the morphant phenotype, as well as slc52a3 morpholino/p53 morpholino coinjection, which maintains the slc52a3 morphant phenotype. In line with clinical findings, riboflavin supplementation resulted in some improvement of the morphant phenotype. Probenecid was selected as a candidate drug due to its inhibitory effect on OAT-3, which mediates riboflavin excretion. However, supplementing riboflavin treatment with probenecid provided no additional benefit to the slc52a3 knockdown model. Further development of CRISPR/Cas9-knockout lines of slc52a2 and slc52a3, as well continued therapeutic screening of riboflavin and probenecid and consideration of alternative therapeutics will provide more opportunities to uncover novel therapeutic strategies to improve RTD treatment.

No description available.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the world. The most accepted hypothesis for the cause of this disease is the amyloid cascade hypothesis, which postulates that the formation of extracellular neurotoxic amyloid-beta binds specific receptors on the surface of neuronal and glial cells to increase cell stress leading to cell death. Our laboratory previously showed that treatment of cultured human SHSY neuronal cells with amyloid beta increases the cellular levels of two key components (caspases-2 and -3) of the extrinsic apoptotic pathway, leading to cell death. The amyloid beta induced caspase elevation was blocked by simultaneously treating the cells with a short mimetic of human ependymin neurotrophic factor, hEPN-1, and the hEPN-1 treatment also blocked cell death. This thesis extends the AD investigation to show that treatment of SHSY cells with amyloid beta may also activate an intrinsic apoptotic mitochondrial stress pathway (assaying caspase-9 as a marker enzyme), and that hEPN-1 treatment significantly lowers this activation. In addition, our laboratory previously showed that treating SHSY cells with amyloid beta increases TUNEL staining, an assay for DNA fragmentation (a hallmark of end stage of apoptosis, and a different apoptotic marker than caspase activation). Treatment with hEPN-1 simultaneously with the amyloid beta, or 6 hrs post amyloid beta, significantly lowered the amyloid beta induced TUNEL signal. This thesis extended the earlier TUNEL experiments to show that hEPN-1 treatment can significantly lower the amyloid beta induced TUNEL staining even when added 18 hrs post amyloid beta. With respect to caspase-8, an initiator caspase in the extrinsic pathway, immunoblot assays of brain lysates from 8 month old transgenic AD mice showed that a 2 week oral delivery of hEPN-1 (conjugated to a carrier to deliver it across the blood brain barrier) significantly lowered caspase-8 levels. Finally, an assay of cellular inhibitors of apoptosis (cIAP) showed a significant increase in their cellular levels in SHSY cells, and in transgenic AD mice treated with hEPN-1, showing for the first time that hEPN-1 may aid cell survival by upregulating proteins known to directly bind specific caspases to block their activity leading to their degradation. The cIAP upregulation occurred in the presence or absence of amyloid beta, indicating that hEPN-1 likely does not block cell death by directly interfering with the interaction of amyloid beta with its cell surface receptors, but instead hEPN-1 may activate an independent cell survival signal transduction pathway in neuronal cells.