Abnormal aggregation of microtubule associated protein tau is the defining pathological hallmark of tauopathies, which include Alzheimer’s disease (AD) and related frontotemporal dementias (FTLD-tau). However, the cellular events precipitating tau pathogenesis in disease are unknown. Here, we demonstrate a novel mechanism regulating tau aggregation in tauopathies. We have previously shown that RNA binding proteins (RBPs) associated with stress granules (SGs) progressively accumulate with tau in multiple mouse models of tauopathy, as well as in human AD and FTLD-tau brain tissue. We now present a novel functional role for tau in regulating the biology of SGs in neurons. Tau facilitates the rapid formation of SGs in the soma and dendrites in response to exogenous stress, which functions to transiently reprogram protein synthesis to promote cell survival (also known as the ‘translational stress response’). However, the chronic interaction of tau with SG proteins in disease, such as with the SG nucleating protein T cell intracellular antigen 1 (TIA1), promotes tau misfolding and neurotoxicity, which can be modulated in primary neurons by pharmacological or genetic manipulations that increase (i.e. puromycin, TIA1 overexpression) or decrease (i.e. cycloheximide, TIA1 knockdown or knockout) SG formation, respectively. In order to test whether SGs also mediate the progression of tauopathy in vivo, we crossed PS19 transgenic (P301S) tau mice with Tia1-/- or C57BL/6J (background strain) mice. PS19 mice with heterozygous reduction in TIA1 (P301S TIA1+/-) developed less SGs compared to P301S TIA1+/+ mice, which was associated with marked neuronal protection, improved cognitive function, and prolonged lifespan. The behavioral neuroprotection in P301S TIA1+/- mice was associated with decreased accumulation of soluble tau oligomers, and occurred despite the increased presence of neurofibrillary tangles. Our findings suggest that TIA1 stabilizes tau in its oligomeric state, preventing its further assembly into insoluble fibrils, which are less toxic. More importantly, the studies described in this dissertation identify modulation of RBP aggregation in SGs as a promising therapeutic strategy for the treatment of AD and FTLD-tau.

BACKGROUND: Mild Traumatic Brain Injury (mTBI) is increasingly recognized as an adverse health consequence for athletes who participate in contact sports, such as football or boxing, as well as military personnel who are exposed to concussive blasts during training and combat operations. A consequence of this repetitive brain injury can be the development of a number of neurodegenerative diseases, particularly chronic traumatic encephalopathy (CTE), a disease involving the buildup of toxic phosphorylated tau (p-tau) in the pre-frontal cortical tissue. Additionally, it has been found that military personnel suffering repeated mTBI from primary blast concussions are prone to development of post traumatic stress disorder (PTSD), a disease that is becoming increasingly common among returning service members. Because mTBI is a common cause for both PTSD and CTE, it is possible for the two diseases to manifest comorbidly in an individual. Though much is known about PTSD psychologically and CTE neuropathologically, little is known about the overlapping effect of the two diseases together as well as PTSD neuropathologically. What is known, however, is that aquaporin-4; a channel involved in the movement of water through the blood brain barrier, is often affected by CTE and may play a role in PTSD as well. OBJECTIVE: The objective of this study was to primarily to analyze the disruption of aquaporin-4 around cerebral blood vessels due to chronic traumatic encephalopathy. A secondary objective of this project was to determine if any unique physiopathological biomarkers exist in PTSD and if the effects of CTE are exacerbated when present comorbidly with PTSD. METHODS: This study involved the analysis of multiple cohorts that had suffered from CTE, PTSD and CTE comorbidly, or neither disease as a control. In order to assess the primary objective, two cohorts, a CTE-only and a control, were analyzed to determine the effect of p-tau on aquaporin-4 directly around cerebral vessels in the pre-frontal cortex. The samples were cut from blocks and stained for the desired markers. Following staining, images were taken using a confocal microscope and the images were analyzed using Amaris and FIJI. For the secondary objective, samples were prepared in a similar way with three cohorts: CTE-only, CTE+PTSD comorbid, and a control. Images were obtained and processed in the same way. RESULTS: It was found that aquaporin-4 density is significantly reduced around both arterial and venous lesional vessels. Additionally, it was found that p-tau was more readily deposited in the depths of the sulci of the pre-frontal cortex due to the unique forces caused by repeated mTBI. However, PTSD was not found to significantly compound the disease when comorbidly present with CTE nor to have a unique biomarkers present. CONCLUSION: P-tau present in CTE causes a significant reduction in aquaporin-4 around cerebral vessels in the pre-frontal cortex, thereby potentially inhibiting the movement of fluids and clearance of metabolites into and out of the brain. Additionally, p-tau is more readily deposited in the depths of the sulci of the pre-frontal cortex. However, PTSD does not compound the CTE disease process when comorbidly present. / 2018-07-13T00:00:00Z

xix, 149 p. ; ill. (some col.) A print copy of this title is available through the UO Libraries. Search the library catalog for the location and call number. / We report on a study of the invariant mass spectrum of the hadronic system in à - [arrow right] à - à 0 à à à à à ½ à decays. This study was performed using data obtained with the BABAR detector operating at the PEP-II e + e - collider. We present fits to phenomenological models in which resonance parameters associated with the à (770), à '(1450) and à "(1700) mesons are determined. We also discuss the implications of our data with regard to estimates of the hadronic contribution to the muon anomalous magnetic moment. / Adviser: Eric Torrence

Emaranhados neurofibrilares intraneuronais, juntamente com as placas beta-amilóide e astrogliose são importantes marcadores neuropatológicos da doença de Alzheimer (DA). Apesar dos mecanismos envolvidos na DA do tipo esporádica ainda não estarem bem esclarecidos, a hiperfosforilação da proteína tau é sugerida como grande fator para o desenvolvimento dos emaranhados neurofibrilares, que podem gerar disfunção neuronal e morte. A toxina ácido ocadáico (AO) é considerada um efetivo inibidor das fosfatases 1 e 2A, as quais podem gerar a hiperfosforilação da tau. Dessa forma, este trabalho tem como objetivo, avaliar alterações neurogliais em hipocampo e líquido cerebroespinhal (LCE) de ratos expostos ao AO intracerebroventricular após 3 e 12 semanas da infusão. E ainda, verificar alterações neurogliais de fatias hipocampais expostas de forma aguda ao AO (in vitro). Como resultados encontramos no modelo in vivo, declínio cognitivo, hiperfosforilação da tau (Ser 396) e alteração astroglial hipocampal principalmente devido a redução da captação de glicose e aumento da expressão da GFAP e ainda, redução da S100B no LCE, a qual pode atuar na sinalização neurônio-astrócito, em condições fisiológicas ou patológicas, como na DA. Ao observar as alterações neurogliais 12 semanas após a infusão, verificamos que o modelo é parcialmente reversível, uma vez que a fosforilação da proteína tau não mostrou alteração, mas foi observado declínio cognitivo em um dos comportamentos realizados e hipometabolismo da glicose. E ainda, in vitro, o AO foi capaz de hiperfosforilar a proteína tau (Ser 396), mas não alterou parâmetros astrogliais. Portanto, o modelo animal se mostra adequado para avaliação de alterações neuroquímicas. Mas nossos resultados também apontam para uma reversibilidade parcial do modelo a longo prazo, indicando a necessidade de cautela na avaliação de estratégias terapêuticas com este modelo. E ainda, nossos dados reforçam a importância de investigar alterações do metabolismo cerebral da glicose em indivíduos com déficit cognitivo. / Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are important markers of Alzheimer’s disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. The okadaic acid (OKA) toxin is a protein phosphatase 1 e 2A inhibitor and can lead to tau protein hyperphosphorylation. We have investigated the effects of intracerebroventricular (ICV) OKA on neuroglial alterations 3 and 12 weeks after OKA infusion. We have also researched the effects on neuroglial parameters on hippocampal slices treated with OKA in vivo. Our results have shown cognitive impairment, hippocampal astrogliosis, based on GFAP increment, decreased glucose uptake and increase on tau phosphorylation (at Ser396) in hipocamppus and decrease in S100B protein on cerebrospinal fluid 3 weeks after ICV OKA-infusion. Moreover, 12 weeks after ICV OKA infusion we also observed a cognitive impairment and decreased on glucose uptake. In vitro, exposure of hippocampal slices to OKA altered tau phosphorylation at Ser396 without any associated change in astroglial function. In conclusion, the OKA-animal model proved to be a suitable model for neurochemical parameters assessment. Our results also indicate a partial reversibility of long-term animal model, suggesting that therapeutics strategies evaluations must be caution on this model; and reinforce how important is to investigate on brain glucose metabolism alterations on cognitive impairment.

Emaranhados neurofibrilares intraneuronais, juntamente com as placas beta-amilóide e astrogliose são importantes marcadores neuropatológicos da doença de Alzheimer (DA). Apesar dos mecanismos envolvidos na DA do tipo esporádica ainda não estarem bem esclarecidos, a hiperfosforilação da proteína tau é sugerida como grande fator para o desenvolvimento dos emaranhados neurofibrilares, que podem gerar disfunção neuronal e morte. A toxina ácido ocadáico (AO) é considerada um efetivo inibidor das fosfatases 1 e 2A, as quais podem gerar a hiperfosforilação da tau. Dessa forma, este trabalho tem como objetivo, avaliar alterações neurogliais em hipocampo e líquido cerebroespinhal (LCE) de ratos expostos ao AO intracerebroventricular após 3 e 12 semanas da infusão. E ainda, verificar alterações neurogliais de fatias hipocampais expostas de forma aguda ao AO (in vitro). Como resultados encontramos no modelo in vivo, declínio cognitivo, hiperfosforilação da tau (Ser 396) e alteração astroglial hipocampal principalmente devido a redução da captação de glicose e aumento da expressão da GFAP e ainda, redução da S100B no LCE, a qual pode atuar na sinalização neurônio-astrócito, em condições fisiológicas ou patológicas, como na DA. Ao observar as alterações neurogliais 12 semanas após a infusão, verificamos que o modelo é parcialmente reversível, uma vez que a fosforilação da proteína tau não mostrou alteração, mas foi observado declínio cognitivo em um dos comportamentos realizados e hipometabolismo da glicose. E ainda, in vitro, o AO foi capaz de hiperfosforilar a proteína tau (Ser 396), mas não alterou parâmetros astrogliais. Portanto, o modelo animal se mostra adequado para avaliação de alterações neuroquímicas. Mas nossos resultados também apontam para uma reversibilidade parcial do modelo a longo prazo, indicando a necessidade de cautela na avaliação de estratégias terapêuticas com este modelo. E ainda, nossos dados reforçam a importância de investigar alterações do metabolismo cerebral da glicose em indivíduos com déficit cognitivo. / Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are important markers of Alzheimer’s disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. The okadaic acid (OKA) toxin is a protein phosphatase 1 e 2A inhibitor and can lead to tau protein hyperphosphorylation. We have investigated the effects of intracerebroventricular (ICV) OKA on neuroglial alterations 3 and 12 weeks after OKA infusion. We have also researched the effects on neuroglial parameters on hippocampal slices treated with OKA in vivo. Our results have shown cognitive impairment, hippocampal astrogliosis, based on GFAP increment, decreased glucose uptake and increase on tau phosphorylation (at Ser396) in hipocamppus and decrease in S100B protein on cerebrospinal fluid 3 weeks after ICV OKA-infusion. Moreover, 12 weeks after ICV OKA infusion we also observed a cognitive impairment and decreased on glucose uptake. In vitro, exposure of hippocampal slices to OKA altered tau phosphorylation at Ser396 without any associated change in astroglial function. In conclusion, the OKA-animal model proved to be a suitable model for neurochemical parameters assessment. Our results also indicate a partial reversibility of long-term animal model, suggesting that therapeutics strategies evaluations must be caution on this model; and reinforce how important is to investigate on brain glucose metabolism alterations on cognitive impairment.

A l’heure actuelle, le processus neurodégénératif responsable de la MA n’est pas complètement élucidé et ses causes sont probablement multiples. Cependant, on sait qu’il met en jeu Tau, une protéine associée aux microtubules et qui permet leur stabilisation ; dans les cerveaux des patients Alzheimer, Tau est présente sous un état anormalement phosphorylée, agrégeant en filaments appariés en hélice à l’intérieur des neurones. Notre modèle d’étude in vitro consiste en une protéine Tau recombinante phosphorylée par la CDK2/CycA3 sur deux phosphoépitopes spécifiques à la MA et reconnus par les anticorps AT8 et AT180. Ce profil de phosphorylation a été caractérisé de façon qualitative et quantitative par spectroscopie RMN (Résonance Magnétique Nucléaire). Nous avons ensuite tenté de cartographier ces phosphoépitopes par deux techniques complémentaires originales : la RMN et le FRET (Fluorescence Resonance Energy Transfer). Nous avons également montré que l’(hyper)phosphorylation de Tau ne menait pas forcément à son détachement des microtubules, hypothèse largement répandue dans la littérature. Finalement, nous avons pu mettre en évidence une interdépendance entre les deux phosphoépitopes AT8 et AT180 lors de la déphosphorylation de notre échantillon de protéine Tau phosphorylée par PP2AT55, une phosphatase trimérique majeure du cerveau. / The neurodegenerative process responsible for Alzheimer’s disease is not yet elucidated and its causes are probably multiple. Nevertheless, we know that it brings into play Tau, a microtubule associated protein that allows their stabilization; in Alzheimer patients’ brain; Tau proteins are present under an abnormally hyperphosphorylated state which aggregate in paired helical filaments inside the neurons. Our in vitro study model is based on a recombinant Tau protein phosphorylated by CDK2/CycA3 on two Alzheimer’s disease specific phosphoepitopes recognized by AT8 and AT180 antibodies. This phosphorylation pattern was qualitatively and quantitatively characterized by NMR (Nuclear Magnetic Resonance) spectroscopy. We tried then to map these phosphoepitopes by two original and complementary methods NMR and FRET (Fluorescence Resonance Energy Transfer). We also showed that (hyper)phosphorylation of Tau doesn’t lead necessarily to its detachment from microtubules, a hypothesis widely spread in literature. Finally, we highlighted that during the dephosphorylation of our phospho-Tau sample by PP2AT55, a major trimeric phosphatase in brain, the dephosphorylation of AT8 phosphoepitope was under AT180 phosphorylation status control.

Depuis sa détection par Altman et Das en 1965, l’existence d’une neurogenèse adulte universelle dans le règne animal a été l’objet de controverses. Elle a été étudiée dans de nombreuses espèces animales, en particulier chez l’un des mammifères possédant un cerveau extrêmement bien développé :l’Homme. Un cerveau bien développé ne signifie cependant pas nécessairement qu’il y existe une neurogenèse accrue, au contraire. Dans notre travail, nous avons étudié la neurogenèse hippocampique adulte dans un modèle de rongeur :la souris. Un petit cerveau, sans circonvolutions, mais présentant une neurogenèse adulte conséquente. La neurogénese hippocampique adulte est un processus impliqué dans le fonctionnement de la mémoire, dont le dysfonctionnement est un symptôme de plusieurs maladies neurodégénératives dont la maladie d’Alzheimer.Le premier objectif de cette thèse est consacré à l’analyse comparative de la neurogenèse hippocampique adulte dans le cerveau de souris wild-type (WT) et de souris transgéniques modélisant une maladie neurodégénérative (Tg30) :une forme familale de démence frontotemporale (FTLD-17) faisant partie du groupe des Tauopathies, dont fait aussi partie la maladie d’Alzheimer. Cinq modèles murins ont été étudiés :des souris WT, Tg30, Tg30/tauKO, tauKO et htau/tauKO. Ces trois dernières lignées n’expriment pas de protéines tau murines, et les souris htau/tauKO expriment uniquement les 6 isoformes WT de protéines tau humaines. Dans ces différents modèles, nos analyses immunohistochimiques ont démontré la présence de nombreuses cellules souches et de précurseurs neuronaux dans la zone sous-granulaire du gyrus denté, l’une des zones neurogéniques du cerveau murin, également présente chez l’Homme. Par comparaison avec les souris WT, la neurogenèse est augmentée dans la zone sous granulaire du gyrus denté chez les souris Tg30/tauKO, alors qu’elle est diminuée chez les souris Tg30. Nous avons démontré que le volume et le nombre de neurones granulaires sont significativement plus importants dans l’hippocampe des souris htau/tauKO et tauKO, alors que le nombre de neurones immatures y est réduit. Afin d’étudier la neurogenèse chez l’Homme, nous avons réalisé des marquages immunohistochimiques sur des coupes d’hippocampes humains de sujets contrôles et atteints de maladie d’Alzheimer. Toutefois, les conditions de fixation, les délais post-mortem ainsi que la variabilité intra-individuelle ne nous ont pas permis de visualiser cette neurogenèse ni de pouvoir affirmer ou réfuter son existence chez l’Homme. Ces résultats soulignent le rôle protecteur de la réduction d’expression de tau dans les déficits de la neurogenèse hippocampique adulte dans ces modèles de tauopathies.Le second objectif de ce travail était d’approfondir la compréhension des mécanismes de propagation et de nucléation de la protéine tau pathologique. En effet, l’injection stéréotaxique de fractions enrichies en agrégats fibrillaires (PHF) extraits du cerveau de patients atteints de la maladie d’Alzheimer dans le gyrus denté de souris exprimant uniquement les six isoformes de la protéine tau humaine (htau/tauKO) induit la formation d’agrégats neuronaux de protéines tau et leur propagation intra- et interhémisphérique. Ces inclusions présentent de nombreuses caractéristiques similaires à celles des dégénérescences neurofibrillaires retrouvées lors de la maladie d’Alzheimer. Cependant, les inclusions formées ici correspondent vraisemblablement à des stades précoces d’agrégation, car ils ne sont pas mis en évidence par des marqueurs identifiant des modifications post-traductionnelles plus tardives des dégénérescences neurofibrillaires. Ce modèle htau/tauKO injecté avec des PHF représente un modèle plus proche de la physiopathologie humaine et qui permettra de mieux comprendre les mécanismes impliqués dans la formation et la propagation des agrégats de proteines tau dans plusieurs maladies neurodégéneratives. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Das Tau-Protein ist ein Mikrotubuli-assoziiertes Protein und kommt im zentralen Nervensystem (ZNS) und im peripheren Nervensystem (PNS) vor. Die Hyperphosphorylierung und Aggregation des Tau-Proteins ist eine der zentralen Pathologien bei der Entstehung der Alzheimerschen Erkrankung (AD). Die AD ist die häufigste Form der progressiven Demenz und eine der häufigsten neuro-degenerativen Erkrankungen. Gegenstand dieser Arbeit ist die Kartierung der nicht-neuronalen Gewebe, die Tau-Protein enthalten können. Mit diesem Ziel wurden in den unterschiedlichen Geweben mit den Methoden SandwichELISA, Immunhistochemie und RT-PCR auf die Tau-Menge quantifiziert, alternatives Spleißen untersucht und die Zelltyp-spezifische Expression detektier. So konnte eine Liste der Gewebe, die Tau-Protein enthalten erstellt werden. Eine immunhistochemische Markierung erfolgte jeweils in den drei Geweben mit dem höchsten Anteil an Tau-Protein. Hier ließ sich das Protein vorrangig in Nervenfasern darstellen. Durch RT-PCR wurde eine Isoformen-Analyse erstellt. Dabei wurde ein ähnliches Muster wie im ZNS festgestellt mit zusätzlicher Expression des „Big-Tau“ Exons 4a, sowie einer weiteren Spleiß-Isoform mit Inklusion des Exon 8, welches bisher nur im Rind und im Rhesus-Affen nachgewiesen war.

Alzheimer’s disease is characterized by the formation of two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Although there have been many studies to understand the role of microglia in Alzheimer’s disease, it is not yet known how microglia can promote disease progression while actively phagocytosing amyloid plaques or phosphorylated tau (p-tau). Through stereotaxic injection of adeno-associated virus expressing mutant P301L tau (AAV-P301L-tau) into the medial entorhinal cortex (MEC) of both wild-type (WT) and APPNL-G-F mice, we demonstrate how amyloid plaques exacerbate p-tau propagation to the granule cell layer (GCL) of the hippocampus. However, in mice receiving the colony-stimulating factor 1 receptor inhibitor (PLX5622), ~95% of microglia were depleted, which dramatically reduced p-tau propagation to the GCL. Although microglia depletion curtailed p-tau propagation, it also led to reduced plaque compaction and an increase in overall amyloid-beta (Aβ) plaque presence. Additionally, we found microglia depletion resulted in greater p-tau aggregation in dystrophic neurites surrounding amyloid plaques. We investigated neurodegenerative microglia (MGnD), which are activated in response to amyloid plaques, for their propensity to release extracellular vesicles in comparison to homeostatic microglia. We discovered that MGnD, identified by Clec7a or Mac2 staining, strongly express Tumor susceptibility gene 101 (Tsg101), which is an ESCRT-1 protein and a marker for extracellular vesicles (EVs). To further investigate EV release and MGnD, a novel lentivirus expressing fluorescent mEmerald conjugated to CD9 (mE-CD9) was constructed and injected into the MEC of both WT and APPNL-G-F mice which allowed for visualization of mE-CD9+ puncta around individual microglia. CD9 is a tetraspanin and also a marker for EVs. We observed that the number of mEmerald+ particles surrounding MGnD was three-fold higher compared to non-diseased, homeostatic microglia. Sequential injection of mE-CD9 and AAV-P301L-tau into the MEC revealed that microglia-derived EVs encapsulate pathologic p-tau, which is augmented by the MGnD phenotype. Taken together, these data provide strong evidence that MGnD exhibit increased secretion of tau-containing EVs, providing a possible mechanism for how amyloid deposition indirectly exacerbates tau propagation.

Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at ∼80 nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.