京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19935号 / 工博第4218号 / 新制||工||1652(附属図書館) / 33021 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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タウオパチー変性神経細胞では、本来軸索に局在する微小管結合タンパク質タウが細胞体や樹状突起に蓄積するとともにチューブリン・微小管が減少する。この広範な神経細胞骨格系の変性は、タウの異常リン酸化に起因すると考えられてきた。しかし、タウの異常性獲得と微小管の消失との関係は未だ不明な状態にある。本研究では、初代培養神経細胞でαチューブリンの分子シャペロンであるTubulin-specific chaperon Eの発現抑制を行うことで、チューブリンの恒常性破綻を誘導するともに、それとタウの異常性獲得の関連性について検討した。 / Tauopathy is a type of neurodegenerative disorder including Alzheimer's disease defined by formation of tau filamentous inclusion in neurons. Tau is a microtubule associated protein localized in axon and assumed to promote microtubule stabilization in healthy neuron. In contrast, accumulation of hyperphosphorylated tau in somatodendrite and loss of microtubules (tubulin) are observed in tauopathy neuron. Although it is believed that abnormal phosphorylation of tau results in neurodegeneration, the relation with tau abnormalities and microtubule loss remains unclear. To investigate whether disruption of tubulin homeostasis induce tau abnormalities, we performed a miRNA-mediated knockdown of tubulin-specific chaperon E, an essential factor for the formation of alpha and beta tubulin heterodimeric complex, in mouse primary hippocampal neuron. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

微小管結合タンパク質の1つであるタウは、神経細胞の軸索に特異的に局在している。タウの軸索局在化分子機序を解明するために、外因性タウを神経細胞の発達初期に一時的に発現させ、軸索特異的に局在させる方法を構築した。この方法を用い、proline rich region 2 (PRR2)がタウの軸索局在化に重要であること、PRR2のリン酸化が軸索への移動に関与することを示唆した。またこの系の確立は局在や細胞内動態などの検討を行うことを可能にした。 / Microtubule-associated protein tau localizes specifically to neuronal axons. In order to elucidate the molecular mechanism of the axon localization of tau, we constructed an expression system for axon specific localization of exogenous tau in immature neurons in culture. Using this system, it suggested that the proline rich region 2 (PRR2) and phosphorylation of PRR2, which contains important phosphorylation sites, is critical for the localization. In the future, this experimental system will contribute greatly to the study of tau in normal and in the pathology of Alzheimer's disease. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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Celogentin C and theonellamide F are a class of natural products that have potential antimitotic behavior. They both contain interesting bicyclic structures with unusual linkages within a central moiety. Celogentin C's highly functionalized tryptophan moiety has two unusual linkages, a β-substituted Leu connection to the C6 of the indole structure that makes up the left-hand ring, and a τ-N connection of the imidazole to the C2 of the indole constructing the right-hand ring. This right-hand ring connection was solved via a novel oxidative coupling procedure developed in our group and the left-hand ring was initially constructed via a radical conjugated addition of an isopropyl group. Due to stereoselective concerns, our group explored hydrogen bond donors as potential catalyst candidates. Unfortunately, there were challenges in limiting the background reaction and obtaining reproducible results. We then designed an alternative route to solve this left-hand ring connection which would have utilized MacMillan asymmetric hydrogenation and α-chlorination procedures. Further work towards a second generation synthesis of the β-Leu-(C6)Trp connection was halted with the publication of two formal syntheses of celogentin C. Theonellamide F contains a τ-L-histidino-D-alanine (τ-HAL) bridging unit that separates the left-and right-hand rings. Previous efforts in the synthesis of this natural product were hindered due to an inefficient regioselective synthesis of τ-HAL. Our proposed synthesis of τ-HAL began with commercially available L- and D-Ser methyl esters which were then chemically transformed and coupled to one another to create a bis-amino subunit. Further preparations afforded us with an important cyclic intermediate which should readily lead to the first regioselective synthesis of a τ-HAL.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurodegenerative disease histopathologically characterized by the coexistence of amyloid plaques and neurofibrillary tangles, mainly consisting of amyloid β peptides hyperphosphorylated tau proteins, respectively. Multiple proteins and pathways are involved in the pathogenesis of AD, including Aβ precursor protein (APP), β-site APP-cleaving enzyme (BACE1), neprilysin, endothelin converting enzyme (ECE), repressor element-1 silencing transcription factor (REST), microtubule-associated protein tau, glycogen synthase kinase, and pro-inflammatory cytokines. However, how these proteins and pathways are dysregulated and converge in AD pathogenesis remains unclear. Genetic, epigenetic and environmental factors play important roles in disease progression. MicroRNAs (miRNAs), a group of small noncoding RNAs, are important epigenetic regulators that participate in AD development. We have identified three miRNAs capable of targeting several proteins in different AD-related pathways: miR-181-5p, miR-153-3p and miR-101-3p. We tested miR-181 activity with recombinant reporter gene- MME 3’-UTR constructs. All four miR-181-5p (miR-181a, miR-181b, miR-181c and miR-181d) sequences downregulated the reporter signal. Human differentiated neural cells were transfected with miR-181d-5p mimics. miR-181d-5p treatment significantly reduced MME mRNA levels, protein levels and enzyme activity. In addition, miR-181d-5p increased tau and phosphorylated tau levels proportionally. We further demonstrate that miR-153-3p reduced REST 3’-UTR activities, mRNA and protein levels in multiple human cell lines. Moreover, we show that miR-153-3p, by knocking down REST protein, induces apoptosis in HeLa cells but not differentiated neural cells. In addition, miR-153-3p regulates neuronal differentiation in neuronal stem cells, potentially via REST knockdown. We further found that miR-153 levels were correlated with a reduced likelihood of developing AD. Last, we demonstrated that miR-101-3p reduced ECE1 and GSK3β protein levels in multiple cell lines. miR-101-3p increased REST and pro-inflammatory cytokine secretion in microglia cells. In sum, we tested the hypothesis that miRNAs can serve as the master regulator of AD pathogenesis. / 2024-07-01

<p>Tauopathies are neurodegenerative diseases defined by the accumulation of pathological tau protein in neurons and glia. Alzheimer’s disease (AD), the most common tauopathy, is characterized by the presence of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein aggregates in neurons. Emerging evidence suggests that the NFT burden correlates with neuron death and cognitive decline, contributing to disease progression. Besides AD, a similar deposition of tau inclusions is found to be associated with neurodegeneration in the brains of patients with other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. These diseases display clinical, biochemical, and neuropathological heterogeneity. Little is known about how tau aggregation can lead to varied phenotypes in tauopathies, and there is no disease-modifying treatment. Thus, it is necessary to understand the role of diverse tau proteoforms in tauopathies for the development of new therapeutics to treat tauopathies, including AD.</p> <p>In the studies summarized in Chapter 2, we investigated how the molecular diversity of tau proteoforms could impact antibody-based assays of a phospho-tau variant serving as an AD biomarker. A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential AD biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in NFTs of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibody’s cognate antigen, such effects have not been extensively studied. Here, we developed a bio-layer interferometry (BLI)-based analytical assay to assess the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated.</p> <p>In the studies outlined in Chapter 3, we examined the relative abilities of different tau proteoforms to induce seeded tau aggregation and to themselves undergo seeded aggregation in cultured cells. Accumulating evidence suggests that tau aggregates, including NFTs, spread in a stereotypical pattern across neuroanatomically connected brain regions. This spreading phenomenon is thought to occur via a prion-like mechanism involving the release of tau aggregates from a diseased neuron into the extracellular space, aggregate uptake by neighboring healthy neurons, and the formation of new aggregates in the cytosol of the recipient cells via a seeding process. Although research over the past decade has revealed key molecular events involved in the cell-to-cell transmission of tau aggregates, the impact of the protein’s domain structure and phosphorylation profile on the efficiency of prion-like propagation remains poorly defined. Here, we compared three tau variants – K18, 0N4R, and 2N4R – in terms of their aggregation and seeding efficiencies in recombinant protein solutions and in cell culture models. Our results revealed that K18 had the highest fibrillization rate and yield among the three tau variants. Recombinant preformed fibrils (PFFs) derived from all three variants had similar seeding efficiencies. Additionally, we investigated the relationship between tau phosphorylation and aggregation. We found that hyperphosphorylated tau did not undergo self-assembly in the absence of heparin, whereas it formed fibrils at low yield in the presence of the cofactor. Moreover, hyperphosphorylated tau PFFs produced under these conditions induced seeded tau aggregation in cell culture.</p> <p>Taken together, these results point to critical roles of tau proteoforms as both AD biomarkers and drivers of disease progression. Our results indicate that the presence of different combinations of phosphorylated residues near a target phospho-tau antigen can affect the accuracy of antibody-based biomarker assays. In addition, the domain structure and phosphorylation profiles of tau proteoforms associated with AD and other tauopathies likely have a profound influence on the evolution of tau pathology in these disorders. Our findings highlight the importance of accounting for the molecular diversity of tau proteoforms in tauopathies and provide valuable insights into molecular determinants influencing tau aggregation and propagation in the brains of patients.</p>