神経変性疾患で蓄積する異常タンパク質の1つであるα-synは、PD、DLBおよびMSAの脳内に主に蓄積する。DLBやMSAの患者脳から解析可能な量のα-syn凝集体の増幅に成功した。増幅前後の凝集体のプロテイナーゼKコアのMS分析結果から、増幅による変化はないもののマウスとヒトのα-syn凝集体で切断パターンが異なることがわかった。これらの結果から、この方法が神経変性疾患の異常タンパク質研究の発展に貢献できることを示唆した。 / Pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils. One of the pathological proteins, α-syn, accumulates in the brains of PD, DLB and MSA. We first performed amplification of α-syn aggregates. We successfully amplified enough α-syn aggregates derived from α-syncleinopathies. We found that the MS analysis results of proteinase K-resistant cores of the aggregates before and after the amplification differ between mouse and human α-syn aggregates. The results suggest that structural properties of amplified α-syn fibrils are preserved and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

No description available.

Prionen sind das infektiöse Agens transmissibler spongiformer Enzephalopathien von Tieren und Menschen. Prionen bestehen hauptsächlich aus einer abnormal gefalteten und aggregierten Isoform des zellulären Prionproteins (PrP). Die Replikation von Prionen findet mutmaßlich durch keiminduzierte Polymerisation des Prionproteins statt. Es existieren verschiedene Prionstämme, die unterschiedliche Eigenschaften aufweisen, aber vom selben zellulären Prionprotein abstammen können. Neben PrP scheinen Kofaktormoleküle an der Prionreplikation beteiligt zu sein. Weiterhin wird angenommen, dass Kofaktoren bei der Definition von Stammeigenschaften beteiligt sind, sowie ein Einfluss auf die Infektiosität von Prionen besteht. In dieser Arbeit wurden die Auswirkungen verschiedener Kofaktoren auf die Replikation von vier Hamster-adaptierten Prionstämmen in vitro mittels der Methode der „Protein Misfolding Cyclic Amplification“ (PMCA) untersucht. Es wurden stammabhängige Unterschiede bezüglich der Anforderungen an die Replikationsbedingungen in der PMCA, sowie Kofaktor-Selektivitäten festgestellt. Der Einfluss von Kofaktoren wurde durch den Vergleich ausgewählter biologischer, biochemischer und biophysikalischer Eigenschaften von in vitro erzeugten PMCA Produkten (PrPres) mit denen nativer Prionkeime untersucht. Es zeigte sich, dass Kofaktoren Stammeigenschaften, wie die biologische Keimaktivität in primären Gliazellkulturen und biochemische Eigenschaften, wie die Migration in SDS-Gelen, beeinflussen können. Um festzustellen, ob unterschiedliche Kofaktorbedingungen während der PMCA messbare Veränderungen der Proteinkonformation hervorrufen, wurde PMCA generiertes PrPres mittels FT-IR Spektroskopie in einer Pilotstudie charakterisiert. Erste Befunde zeigten spektrale Unterschiede zwischen den Proteinkeimen und deren PMCA Produkten bei allen Stämmen, unabhängig von den Kofaktorbedingungen. / Prions are the causative agent of transmissible spongiform encephalopathies in animals and humans such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). Prions are thought to be composed essentially of a misfolded and aberrantly aggregated isoform of the cellular prion protein (PrP) and to replicate by seeded PrP polymerization. Prions may exist in the form of distinct strains that differ in their phenotypic characteristics although they are derived from the same cellular prion protein. Cofactor molecules other than PrP may be involved in prion replication and may be a determinant of strain properties. Furthermore, cofactors may also be required for conveying infectivity. The present study examined the effects of different cofactor molecules on the replication efficacy of four hamster adapted prion agents using the method of serial protein misfolding cyclic amplification (PMCA) as in vitro assay for PrP misfolding and aggregation. The study revealed strain dependent differences of PMCA conditions and cofactors required for efficient in vitro replication. The impact of cofactors was assessed by comparative analyses of selected biological, biochemical and biophysical properties of PMCA products (PrPres) and native prion seeds. The biological seeding activity as monitored in a primary hamster glial cell assay, and biochemical properties such as electrophoretic migration in SDS-gels, were affected differently by different cofactors. In order to define the impact of putative cofactors on the molecular conversion of PrP in more detail, changes in the spatial structure associated with different cofactor molecule conditions during amplification of PrPres in PMCA was monitored by Fourier transform-infrared (FT-IR) spectroscopic analysis. Largely preliminary data revealed spectral differences between native prion seeds and progeny PMCA generated PrPres for all prion strains, but no variations due to different cofactor conditions.

Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.

The ability to accurately predict danger and implement appropriate protective responses is critical for survival. Environmental fluctuations can cause damage at the cellular level, leading to the misfolding and aggregation of proteins. Such damage is toxic to cells: in age-related neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s and Huntington’s Diseases, the accumulation of damaged proteins in the brain ultimately leads to neuronal cell death and disease onset. To date, there is still no cure to combat the progressive degeneration and cell death seen in the brains of patients. Cells within an animal possess defense programs to minimize protein damage. One such defense mechanism is the activation of a program called the Heat Shock Response, which increases production of protective proteins known as heat shock proteins (HSPs). These HSPs act as molecular chaperones to assist with the clearing out of damaged proteins. This program is implemented by a conserved transcription factor, Heat Shock Factor 1 (HSF-1). However, in brains of patients with degenerative diseases, this protective mechanism, for reasons yet unknown, is not constantly activated. My thesis has involved the discovery of innate mechanisms that exist in organisms to activate this cellular protective mechanism against protein misfolding. My research, using the model organism Caenorhabditis elegans, has shown that the protective heat shock response in the cells of the animal can be triggered through neurohormonal signaling. The neurohormonal signaling that I am studying is one that is highly conserved across all organisms from plants to insects to mammals – serotonergic signaling. The stimulation of serotonergic signaling appears sufficient to activate the Heat Shock Response, even in the absence of real damage. In fact, the neuronal release of serotonin facilitates a pre-emptive upregulation of protective genes in the animal, which we have observed to be able to reduce the accumulation of damaged proteins in a C. elegans model of Huntington’s Disease. Additionally, I have seen that anticipating danger can enhance the animal’s stress response in a serotonin-dependent manner, thus facilitating better survival against a subsequent insult that can cause protein damage. Together, these studies present the novel possibility of protection against neurodegenerative disease via modulation of neurotransmission and/or neurosecretion. They also allow for understanding how sensory inputs are coupled to gene expression under stressful conditions. I hope to understand the mechanism by which animals adapt to changes in their environment by coordinating their sensory input with changes in behavior and gene expression.

Small fluorescent probes are important tools when studying protein aggregates involved in different neurodegenerative diseases, such as Alzheimer’s disease. Luminescent conjugated oligothiophenes have been developed and shown to be excellent ligands when studying morphology among amyloids, due to their conjugated thiophene backbone that provides them with unique photophysical properties. This kind of probes are being developed successively to enhance the specificity of their biological targets. In this project, luminescent conjugated oligothiophenes functionalized with amino acids, so called proteophenes, have been synthesized to investigate their optical properties. Since amino acids are chiral molecules, the possibility of induced chirality to the thiophene backbone was examined, as well as the proteophenes ability to work as amyloidospecific ligands for the study of protein aggregates. The synthesis of four different proteophenes are presented in this report, along with analysis results of their photophysical properties.

Amyotrophic lateral sclerosis (ALS) is a disease in which the motor neurons die in a progressive manner, leading to paralysis and muscle wasting. ALS is always fatal, usually through respiratory failure when the disease reaches muscles needed for breathing. Most cases are sporadic, but approximately 5–10% are familial. The first gene to be linked to familial ALS encodes the antioxidant enzyme superoxide dismutase-1 (SOD1). Today, more than 160 different mutations in SOD1 have been found in ALS patients.  The mutant SOD1 proteins cause ALS by gain of a toxic property that should be common to all. Aggregates of SOD1 in motor neurons are hallmarks of ALS patients and transgenic models carrying mutant SOD1s, suggesting that misfolding, oligomerization, and aggregation of the protein may be involved in the pathogenesis. SOD1 is normally a very stable enzyme, but the structure has several components that make SOD1 sensitive to misfolding. The aim of the work in this thesis was to study misfolded SOD1 in vivo. Small amounts of soluble misfolded SOD1 were identified as a common denominator in transgenic ALS models expressing widely different forms of mutant SOD1, as well as wild-type SOD1. The highest levels of misfolded SOD1 were found in the vulnerable spinal cord. The amounts of misfolded SOD1 were similar in all the different models and showed a broad correlation with the lifespan of the different mouse strains. The misfolded SOD1 lacked the C57-C146 intrasubunit disulfide bond and the stabilizing zinc and copper ions, and was prinsipally monomeric. Forms with higher apparent molecular weights were also found, some of which might be oligomers. Misfolding-prone monomeric SOD1 appeared to be the principal source of misfolded SOD1 in the CNS. Misfolded SOD1 in the spinal cord was found to interact mainly with chaperones, with Hsc70 being the most important. Only a minor proportion of the Hsc70 was sequestered by SOD1, however, suggesting that chaperone depletion is not involved in ALS.  SOD1 is normally found in the cytoplasm but can be secreted. Extracellular mutant SOD1 has been found to be toxic to motor neurons and glial cells. Misfolded SOD1 in the extracellular space could be involved in the spread of the disease between different areas of the CNS and activate glial cells known to be important in ALS. The best way to study the interstitium of the CNS is through the cerebrospinal fluid (CSF), 30% of which is derived from the interstitial fluid. Antibodies specific for misfolded SOD1 were used to probe CSF from ALS patients and controls for misfolded SOD1. We did find misfolded SOD1 in CSF, but at very low levels, and there was no difference between ALS patients and controls. This argues against there being a direct toxic effect of extracellular SOD1 in ALS pathogenesis. In conclusion, soluble misfolded SOD1 is a common denominator for transgenic ALS model mice expressing widely different mutant SOD1 proteins. The misfolded SOD1 is mainly monomeric, but also bound to chaperones, and possibly exists in oligomeric forms also. Misfolded SOD1 in the interstitium might promote spread of aggregation and activate glial cells, but it is too scarce to directly cause cytotoxicity.

No description available.

La pathogénèse de la plupart des maladies neurodégénératives incluant les maladies transmissibles comme les encéphalopathies à prion, les maladies génétiques de type maladie de Huntington et les maladies sporadiques comme les maladies d’Alzheimer et de Parkinson est directement liée à la formation d’agrégats protéiques fibrillaires. Pendant de nombreuses années, le concept de dissémination et d’infectivité de ces agrégats a été réservé aux maladies à prion. Cependant, de récents résultats montrent que ces protéines amyloidiques extracellulaires (β-amyloïde) comme intracellulaires (α-synucléine, tau, huntingtin) sont capables de bouger (et possiblement de se répliquer) d’une zone à l’autre du cerveau à la façon des prions (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Récemment une nouveau lien a été établie entre prions et différentes protéinopathies à agrégats. Il a été suggéré que le prion cellulaire, PrPC, dont la forme pathologique (PrPSc) est responsable des maladies à prion, pourrait servir de médiateur dans la toxicité de la protéine β-amyloïde impliquée dans la maladie d’Alzheimer comme dans d’autres conformations-β, indépendamment de la propagation des prions infectieux (revue de Biasini et al., 2012). Malgré une intense recherche sur les maladies neurodégénératives à prion ou non, de nombreuses questions restent ouvertes à la fois au niveau du mécanisme de dissémination des agrégats protéiques que du mécanisme de toxicité. Dans la première partie de ma thèse, j’ai contribué à étudier le rôle de cellules dendritiques (DCs) dans la dissémination de l’infection à prion aux neurones. J’ai démontré que le transfert de PrPSc des cellules dendritiques infectées par un homogénat de cerveau infecté par du prion vers les neurones était dû à contact direct entre ces cellules et a pour résultat la transmission de l’infectivité aux neurones en co-culture. Ces résultats confirment le possible rôle des cellules dendritiques dans la propagation du prion de la périphérie vers le système nerveux central. J’ai aussi trouvé un potentiel mécanisme de transfert de PrPSc des cellules dendritiques aux neurones via des nanotubes (TNTs) et exclu l’implication de la sécrétion de PrPSc dans notre système. Dans la seconde partie de ma thèse, j’ai étudié les mécanismes de dissémination et de toxicité des agrégats protéiques huntingtin et le possible rôle de PrPC dans ces évènements. J’ai démontré que les agrégats Htt sont transférés entre les lignées de cellules neuronales et les neurones primaires et qu’un contact direct cellule à cellule est requis. De même, j’ai montré l’implication des TNTs dans ce transfert et l’agrégation des Htt sauvages endogènes dans les neurones primaires, probablement en suivant le transfert des agrégats Htt. La dernière partie de mes résultats montre que PrPC est impliqué dans la propagation de la toxicité induite par les Htt mutants dans des neurones primaires en culture. / The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathies, inherited disorders like Huntington’s disease, and sporadic diseases like Alzheimer’s and Parkinson’s diseases, appear to be directly linked to the formation of fibrillar protein aggregates. For many years, the concept of aggregate spreading and infectivity has been confined to prion diseases. However, recent evidence indicate that both extracellular (e.g. amyloid-β) and intracellular (α- synuclein, tau, huntingtin) amyloidogenic protein are able to move (and possibly replicate) within the brains of affected individuals, thereby contributing to the spread of pathology in a prion-like manner (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Recently another intriguing connection has been made between prions and other aggregation proteinopathies, as it was suggested that the cellular prion protein, PrPC, whose pathological counterpart is responsible for prion diseases, possibly mediates the toxicity of Aβ, the pathogenic protein in Alzheimer’s disease, and of other β- conformers independently of the propagation of infectious prions (reviewed in Biasini et al., 2012). However, despite the intense research, many questions in prion and non-prion neurodegenerative diseases are still open regarding both the mechanism of protein aggregate spreading and the mechanism of toxicity. In the first part of my thesis, I contributed to investigate the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. I demonstrated that the transfer of PrPSc from DCs (loaded with prion infected brain homogenate) to primary neurons was triggered by direct cell–cell contact and resulted in transmission of infectivity to the co-cultured neurons. These data confirm the possible role of DCs in prion spreading from the periphery to the nervous system. I also provided a plausible transfer mechanism of PrPSc through tunneling nanotubes (TNTs) shown to connect DCs to primary neurons and excluded the involvement of PrPSc secretion in our system. In the second part of my thesis, I investigated the mechanisms of the spreading and toxicity of Htt aggregates and the possible role of PrPC in these events. I demonstrated that Htt aggregates transfer between neuronal cells and primary neurons and that cell-cell contact is required. I also showed the involvement of TNTs in the transfer and reported the aggregation of endogenous wild-type Htt in primary neurons, possibly following the transfer of Htt aggregates. Finally, the last part of my results provides evidences that PrPC is involved in the spreading of the toxicity mediated by mutant Htt in primary neuronal cultures.

While great strides have been made in treating many classes of human disease, the late-onset neurodegenerative diseases continue to elude modern medicine. These diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), the transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS), involve accumulation of insoluble aggregates of one or more causative proteins, leading to progressive loss of central nervous system neurons, progressively worsening neurological symptoms, and eventual patient death. All of these diseases are currently incurable and fatal. In the case of ALS, progressive death of upper and lower motor neurons leads to full-body paralysis, respiratory difficulty, and patient death. Of the subset of ALS cases showing familial inheritance, approximately 20% are caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). These mutations do not have the common property of impairing SOD1's normal function as a free radical scavenger. Instead, they are thought to increase the protein's likelihood of misfolding and aggregating via a poorly-understood aggregation cascade. It is believed that species populated along the misfolding and aggregation pathway may prove to be good targets for therapies designed to block accumulation of downstream toxic species, or to prevent aberrant protein-protein interactions responsible for neurotoxicity. In this thesis, several new techniques are developed to enable detailed elucidation of the SOD1 unfolding and misfolding pathways. Time-resolved measurements collected during SOD1 unfolding or misfolding of release of bound Cu and Zn, of changes in intrinsic fluorescence, of exposure of hydrophobic surface area, and of alterations in the chemical environment of histidine residues, are presented. A new mathematical analysis technique named the Analytical Laplace Inversion Algorithm is developed for rapid extraction of mechanistic information from these time-resolved signals. These tools are applied to the construction of the most detailed models to date of the unfolding and misfolding mechanisms of WT and ALS-causing mutant SOD1. The models presented identify several well-populated unfolding and misfolding intermediates that could serve as good targets for therapies designed to address the fundamental molecular mechanisms underlying SOD1-associated ALS, and to treat what is currently a devastating and incurable disease.