Alzheimer’s disease (AD) is the leading cause of dementia, accounting for 50 to 80 percent of dementia cases, and the prevalence of the disease is projected to increase significantly with time. AD is characterized by severe cognitive decline with age, ultimately requiring continued caregiving and eventually death. The pathology of AD is characterized by the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein, neuron loss, and evidence of inflammation indicated by the presence of reactive microglia and astrocytes. Frontotemporal Lobe Dementia (FTLD) is a rare form of dementia that is related to AD, most notably in the pathology of hyperphosphorylated tau and macroscopic brain shrinkage. It has been defined as one of a host of tauopathies, and has a more rapid onset than AD. Symptoms that resemble personality changes, moreso than memory loss, are characteristic of these other tauopathies (FTLD is a representative of a whole class of neurological disorders). Like AD, there are no known treatments or cures for FTLD. AD and FTLD are two manifestations of a class of diseases known as tauopathies, due to the presence of toxic forms of tau. Tau is a protein normally found in neurons. It functions as a stabilizer for microtubules, and has a role in the trafficking of materials from the cell body to the presynaptic terminal. In AD and FTLD, tau can become hyperphosphorylated, which causes it to form twisted fibrils called NFTs. An emerging area of research is to identify antibodies that target tau as a way to clear tau pathology and hopefully reduce synaptic and neuron loss (Boutajangout et al., 2011b). While these diseases have no known cure or treatment at present, immunotherapy is emerging as a promising approach for treatment. The studies presented here investigated a variety of antibodies directed against tau, and incorporated different timeframes and administration routes to identify the best candidate for future clinical investigation of tau immunotherapy. The mouse model rTg4510, known for expressing cognitive-related tauopathy, was primarily used to evaluate tau antibody effectiveness prior to clinical consideration. Our investigations began by utilizing a more familiar mouse which was also reported to express tau pathology. Our studies first examined intracranial injection of a variety of antibodies using a mouse model previously reported to demonstrate tau pathology, to identify short-term clearance of tau pathology and NFTs. Next, we examined a more robust tau-producing mouse line, to further identify a most effective antibody, as well as to examine the time course of effect, after administration. A longer-term administration, and different route of administration was tested using mini-osmotic pump implantation into the mice, which provided for 28-day continuous infusion. This approach was followed with administration of antibodies, systemically. Behavioral analysis, in addition to pathological testing, was incorporated into the longer-term administration studies.

Tauopathies are a class of neurodegenerative diseases characterized by aggregation of hyperphosphorylated microtubule associated protein tau (phospho-tau), resulting in neuroinflammation and neurodegeneration. Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in these disorders, leading to increased levels of neurodegeneration. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however its role in central neuroinflammation is unclear. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules. Haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to characterize the role of TIA1 in neuroinflammation, and determine its relationship with neurodegeneration in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation (reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress), and neurodegeneration (gross hippocampal atrophy, neuronal loss, synapse loss, and phospho-tau load) in wildtype and P301S transgenic mice expressing TIA1+/+, TIA1+/-, and TIA1-/- in both early (5 month) and advanced (9 month) disease states through biochemical, ultrastructural, and histological analyses. Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress. TIA1 haploinsufficiency and knockout do not reduce neurodegeneration in advanced disease, and importantly, TIA1 knockout exacerbates neuron and synapse loss in hippocampal regions. With both increased levels of neuroinflammation and neurodegeneration, P301S animals with TIA1 knockout are distinct from age-matched P301S and wildtype mice. This study demonstrates that TIA1 plays an important role in the regulation of innate immune response in neurodegenerative disease, and its expression significantly impacts the progression of tauopathy.

Microglia are essential to the brain’s innate immune response and play a vital role in neuropathology related to tauopathies. Understanding how microglia change in response to differential expression of RNA binding protein T-cell intracellular antigen 1 (TIA1) will lend insight to microglial function in tauopathy. In preliminary studies our laboratory has shown that decreasing the expression of TIA1 has an inverse and dose dependent effect on activated microglial density in the dentate gyrus of the P301S mouse (PhD Candidate Chelsey LeBlang, 2018). Here, we utilized serial sectioning electron microscopy to define whether this relationship between TIA1 level and microglia remains consistent in the hilus and granule cell layer (GCL) of the dentate gyrus. Our analyses of microglial volume and microglial interactions within the neuropil have yielded four conclusions. First, the hilus, but not the GCL, exhibited a significant decrease in microglial volume per volume of tissue with the knock out and heterozygous expression of TIA1. Second, the number of appositions on microglia steadily increased on AT8+ and AT8- presynaptic and postsynaptic appositions in the hilus with decreasing TIA1 expression. Third, with the exception of one AT8- somatic apposition, the surface area of microglia apposing AT8+ somata is greater than any other structure and exhibits a dose dependent decrease with decreasing TIA1 expression. Fourth, in the GCL there is a larger fractional surface area of AT8- presynaptic and AT8+ postsynaptic structures when compared to their respective synaptic counterparts. Though not entirely consistent with previous data, this study has important implications for microglial function in tauopathy and related diseases.

Les processus de neuro-inflammation jouent un rôle majeur dans la maladie d'Alzheimer (MA). Des études génétiques récentes démontrent cette association entre neuro-inflammation et MA et impliquent notamment un gène, TREM2, qui code pour un récepteur exprimé à la surface de la microglie. La tauopathie est une lésion caractéristique de la MA. Elle se traduit par l’hyperphosphorylation et l’agrégation intraneuronale de la protéine Tau. Les travaux sur le rôle de TREM2 dans le développement de la pathologie Tau sont peu nombreux et donnent des résultats contradictoiresAinsi, l’objectif de ma thèse est d’étudier le rôle de la neuroinflammation et de TREM2 dans la progression de la tauopathie, dans deux modèles différents. Le premier est obtenu par injection stéréotaxique de vecteurs AAV dans la couche CA1 de l’hippocampe de souris déficientes ou non en TREM2. Ces vecteurs entrainent la surexpression de différentes formes de la protéine Tau humaine et permettent de récapituler les différents stades de la tauopathie.Nous avons en parallèle utilisé un modèle transgénique plus progressif de tauopathie, la souris THY-Tau22, afin d’étudier l’influence du stade de la pathologie dans l’effet provoqué par une déficience en TREM2 sur l’évolution de la pathologie. Notre étude a mis en évidence la toxicité des formes solubles de Tau dans le modèle AAV par rapport à ses formes agrégées. Le modèle transgénique THY-Tau22 nous a permis de mettre en évidence une augmentation des lésions tauopathiques dans les souris déficientes en TREM2 par rapport aux souris qui ne le sont pas, uniquement à un stade avancé. Cela suggère que, à l’instar des modèles amyloïdes, l’effet de la déficience en TREM2 sur le décours de la tauopathie est différent en fonction du stade considéré. / Neuroinflammation processes appear to play a major role in Alzheimer's disease (AD). Recent genetic studies support this correlation between neuroinflammation and AD and include a gene, TREM2, expressed on microglial surface. Tauopathy is a characteristic lesion of AD. It results in hyperphosphorylation and intraneuronal aggregation of Tau protein. In the literature, only few articles describe the role of TREM2 in the development of Tau pathology, and they report contradictory results. We therefore do not know for sure whether a deficiency in TREM2 has a deleterious effect or not on tauopathy. Thus, the goal of my thesis is to study the role of neuroinflammation and TREM2 in the progression of tauopathy, in two different models. The first is obtained by stereotaxic injection of AAV vectors into the CA1 layer of the hippocampus of TREM2-deficient or non-deficient mice. These vectors lead to the overexpression of different forms of the human tau protein, thus making it possible to recapitulate the different tauopathy stages.In parallel, we used a more progressive trangenic model of tauopathy, the THY-Tau22 mouse, to study the influence of TREM2 deficiency at different stage of the pathology. Our study demonstrated the toxicity of Tau soluble forms in the AAV model compared to its aggregated forms. The THY-Tau22 transgenic model allowed us to demonstrate an increase in tauopathic lesions in TREM2 deficient mice compared to wild type mice, at late stage only. This suggests that, similar to amyloid models, the effect of TREM2 deficiency on the course of tauopathy is influenced by the stage of the disease.

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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

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No description available.

La maladie d’Alzheimer (MA), la forme la plus répandue de démence, est caractérisée par une accumulation cérébrale de plaques amyloïdes formées de peptide beta-amyloïde, et d’enchevêtrements neurofibrillaires (NFT) de protéine tau anormalement phosphorylée (P-tau). Depuis plusieurs années, l’évidence d’une implication majeure d’altérations synaptiques dans la pathologie a émergée. De plus, il a été observé dans les cerveaux MA que les héparanes sulfates (HS), normalement extracellulaires, accumulent à l’intérieur des neurones, où ils co-localisent avec tau. Le laboratoire CRRET a mis en évidence que la 3-sulfotransferase 2 (HS3ST2), enzyme prédominante dans le cerveau où elle génère des HS 3-O-sulfatés (3S-HS) de rôle inconnu, est impliquée dans les mécanismes à l’origine de la tauopathie. Puisque la HS3ST2 et les 3S-HS n’ont jamais été caractérisés à la synapse où ils pourraient participer au développement de la tauopathie, les objectifs de ce travail sont : 1) déterminer si la HS3ST2 et les 3S-HS sont présents à la synapse et étudier des possibles rôles physiologiques ; 2) déterminer si les 3S-HS accumulent au niveau intracellulaire dans des cellules neuronales et/ou dans de synaptosomes issus d’un modèle murin de tauopathie ; et 3) examiner si les 3S-HS intracellulaires produits par la HS3ST2 sont impliqués dans le développement ou évolution de la tauopathie au niveau synaptique.Dans ce travail, nous avons montré la présence des 3S-HS et de la HS3ST2 à la synapse de cellules hippocampiques et accumulé des preuves de leur implication dans la stabilité et l’activité synaptique, toutes deux altérées par des peptides se liant aux 3S-HS ont pu bloquer cette activité. Nous avons implémenté et caractérisé le modèle murin de tauopathie rTg4510 et mise en place les cultures primaires de leur neurones hippocampiques. Dans ces cellules, nous avons montré l’accumulation intracellulaire des 3S-HS et une surexpression de la HS3ST2 corrélant avec l’accumulation de P-tau. La digestion enzymatique des HS dans les synaptosomes a résulté dans l’inhibition de la tauopathie.Ce travail révèle pour la première fois un rôle fondamental de la 3-O-sulfatation des chaines d’HS à la synapse, aussi bien dans des conditions physiologiques que pathologiques. Pour la première fois, l’enzyme HS3ST2 est décrite à la synapse. De plus, ce travail donne la preuve d’un lien fort entre l’expression d’HS3ST2, l’accumulation de 3S-HS et la tauopathie au niveau synaptique, ouvrant de nouvelles opportunités pour mieux comprendre la MA. / Alzheimer’s disease (AD), the main form of dementia in the world, is characterized by brain accumulation of amyloid plaques formed of amyloid beta, and neurofibrillary tangles (NFT) made of tau protein in an abnormally hyperphosphorylated form (P-tau). Strong evidences show that synaptic changes are central to the disease process. Moreover, previous observations in AD have shown that heparan sulfates (HS), typically present outside the cell , accumulate inside neurons of AD in where they interact with tau. Recently, the CRRET laboratory demonstrated that the neural 3-O-sulfotransferase 2 (HS3ST2), which generates 3-O-sulfated HS (3S-HS) of still unrevealed physiological roles, is involved in the mechanisms leading to tauopathy. Since it was unknown whether HS3ST2 and 3S-HS are expressed at the synapse and if there they participate to tauopathy development and/or evolution, the objectives of this work were: 1) to determine if HS3ST2 and 3S-HS are present at the synapse and to get insights on their physiological role; 2) to investigate whether 3S-HS accumulate intracellularly in hippocampal cells and/or in synaptosomes from a mice model of tauopathy; and 3) to investigate whether intracellular 3S-HS made by HS3ST2 are involved in tauopathy development and/or evolution at the synaptic level.We described here the presence of 3S-HS and HS3ST2 at the synapse and the role that may play 3S-HS in maintaining synaptic transmission and stability in primary cell culture from mice. These roles are the results of potential multiple implications of 3S-HS in various processes. Secondly, we implement and characterized the rTg4510 mice model of AD-related tauopathy and set primary cultures of hippocampal cells from these mice. In the tauopathic cells, we showed the intracellular accumulation of 3S-HS and HS3ST2 overexpression. Finally, we cleaned P-tau in synaptosomes from the rTg4510 mice aged of 2 months by digesting HS.The present work reveals, for the first time, the presence and a possible fundamental role of HS3ST2 and 3S-HS at the synapse. We give evidences of an interplay between 3S-HS, produced by HS3ST2, and tau and the synaptic level, leading to its abnormal phosphorylation. The results of these work open a new way to understand the phenomenon leading to synaptic impairment in AD patients and could reveal new targets to elaborate protection strategies against the AD pathological lesions.

Research into the molecular pathology of prevalent public health epidemics such as neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer’s Disease (AD), non-medical and illicit opioid use (OU), and Human Immunodeficiency Virus-1 (HIV-1) has been hindered by a lack of systems that allow for rapid and high-throughput modeling of the complex multifactorial conditions in a human context. In this thesis we have addressed this challenge using a multi-pronged approach that encompasses single cell RNA sequencing (scRNA-seq) of three-dimensional (3D) human induced pluripotent stem cell (hiPSC) assembloid culture models and patient derived peripheral blood mononuclear cell (PBMC) samples. We describe the development of an iPSC derived neuron-astrocyte assembloid model of tauopathies, including FTD and AD (AstTau), that rapidly recapitulates propagation of toxic human oligomeric tau (oTau) and cell type specific pathology including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. scRNA-seq identified vulnerable excitatory neuron specific inflammatory pathways and a heat shock response in astrocytes, recapitulating transcriptomic signatures of adult neurodegeneration and supporting a hypothesis of cell type specific neuroinflammation in tau pathogenesis. To more completely model AD, we incorporated amyloid precursor protein (APP) mutant iPSCs into the assembloid model. These iPSCs contained the familial AD APP V717I mutation or the isogenic CRISPR corrected control, and were used to derived neurons, astrocytes, and microglia. This advanced combinatorial system (AstAD and MAstAD) enabled selective microglial incorporation, APP mutation expression, and oTau seeding allowing us to identify discrete contributions to AD pathogenesis. Ast/MAstAD developed extracellular amyloid-β (Aβ) and microglial activation in addition to the pathology observed in AstTau. scRNA-seq identified divergent microglial activation in response to Aβ and oTau pathology, with APP V717I mutation and oTau seeding synergistically exacerbating AD phenotypes. These assembloid models enable study of the cellular and molecular inflammatory mechanisms in multifactorial neurodegenerative diseases. To better understand disease signatures at the crossroads of multifactorial OU, HIV-1, and antiretroviral (ART) viral suppression we also produced a scRNA-seq data set of more than 100,000 peripheral blood mononuclear cells (PBMCs) from 75 study participants. We identified chronic immune activation and T cell activation dysfunction driven by interferon transcriptomic signatures that were elevated in people with HIV (PWH) with opioid use as compared to PWH without OU. We also identified a functional natural killer cell subtype that was depleted with OU in PWH. Cessation of OU reduced these potentially deleterious inflammatory transcriptomic profiles, supporting the hypothesis that OU in PWH amplifies a state of chronic immune activation. Taken together, single cell transcriptomic resolution has enabled the identification of cell type specific disease signatures in complex pathophysiologies. These data demonstrate the dynamic range of inflammatory signaling across multifactorial disease states and emphasize the need for disease- and cell- type specific approaches to therapeutic development. / 2025-02-05T00:00:00Z