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.

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.

Submitted by Karin Goebel (karing@fiocruz.br) on 2014-11-26T11:59:33Z No. of bitstreams: 1 Dissertação_Sharon Martins_versão biblioteca.pdf: 5639087 bytes, checksum: d97ea2343abfd37c4cdfe4960a4df639 (MD5) / Approved for entry into archive by Karin Goebel (karing@fiocruz.br) on 2014-11-26T11:59:43Z (GMT) No. of bitstreams: 1 Dissertação_Sharon Martins_versão biblioteca.pdf: 5639087 bytes, checksum: d97ea2343abfd37c4cdfe4960a4df639 (MD5) / Made available in DSpace on 2014-11-26T11:59:43Z (GMT). No. of bitstreams: 1 Dissertação_Sharon Martins_versão biblioteca.pdf: 5639087 bytes, checksum: d97ea2343abfd37c4cdfe4960a4df639 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Trypanosoma cruzi, o agente etiológico da doença de Chagas, é um organismoamplamente estudado devido a sua importância médica e também por possuircaracterísticas peculiares que o tornam um bom modelo de estudo para questõesbiológicas básicas. A repressão de RNAs mensageiros em grânulos citoplasmáticoscompostos de complexos mRNA-proteína (mRNPs) é uma importante via de regulaçãopós transcricional em eucariotos e, recentemente, foi demonstrado que grânulos deRNA estão presentes em T. cruzi. Alguns ortólogos de proteínas humanas envolvidasem mecanismos de regulação foram encontrados nestas estruturas e caracterizados,mas a função e composição de grânulos de mRNA neste modelo experimentalpermanecem desconhecidas. Em humanos e outros eucariotos, condições de estressecomo calor, radiação UV, presença de agentes citotóxicos ou deficiência de glucosepodem induzir a formação de grânulos de estresse, um tipo de estrutura citoplasmáticaenvolvida em repressão, separação e armazenamento de mRNA durante condiçõesadversas. Foram encontradas, em banco de dados de T. cruzi, três sequências deproteínas que possuem certa similaridade estrutural com as proteínas humanas TIA1 eTIAR. Os genes correspondentes foram clonados utilizando-se a tecnologia Gateway®,para a obtenção de proteínas recombinantes. As proteínas purificadas foram utilizadaspara produzir anticorpos policlonais em camundongos, os quais puderam elucidar alocalização celular e padrões de expressão destas proteínas durante o ciclo de vida doparasita. A caracterização destas proteínas pode ajudar a elucidar melhor osmecanismos de regulação pós transcricional em T. cruzi. / Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an organism widely studied due to its medical importance and particular features that make it an alternative model for basic biological studies. Repression of messenger RNAs in cytoplasmic granules composed of mRNA-protein (mRNP) complexes is an important pathway of posttranscriptional regulation in eukaryotes, and recently was shown that mRNA granules are present in T. cruzi. A few orthologs of human proteins involved in mRNA metabolism were found and characterized on these structures, but the function and composition of mRNA granules in this organism remain unknown. In humans and other eukaryotes, stress conditions such as heat, UV radiation, presence of cytotoxic agents or glucose starvation can induce the formation of stress granules, cytoplasmic structures involved in mRNA repression, sorting and storage during adverse conditions. Three protein sequences that have a certain structural similarity with the human TIA1/TIAR proteins were found in the T. cruzi data bank. The genes encoding these three proteins were cloned using the Gateway® technology and the recombinant proteins were obtained. The purified proteins were used to produce polyclonal antibodies in mice and the cellular localization and expression patterns of these proteins during the parasite’s life cycle were performed. The characterization of these proteins can help to elucidate the mechanisms of posttranscriptional regulation in T. cruzi.

The eukaryotic stress response involves translational suppression of non-housekeeping proteins, and the sequestration of unnecessary mRNA transcripts into stress granules (SGs). This process is dependent on mRNA binding proteins (RBPs), such as T- cell intracellular antigen (TIA-1). RBPs interact with unnecessary mRNA transcripts through prion and poly-glutamine like domains, and their aggregation mirrors proteins linked to neurodegenerative diseases. Recent advances in molecular genetics emphasize the importance of SG biology in disease by associating multiple RBPs linked to SGs with neurodegenerative disease. The major difference between SG proteins and aggregation prone proteins in neurodegeneration is that aggregation of SGs is transient and rapidly reverses when the stress is removed. In contrast, aggregates associated with disease are stable and accumulate over time. This study identifies overabundant SGs as a novel pathology in Alzheimer's disease and related tauopathies. The data suggest that TIA-1 is intimately linked to tau pathogenesis, acting as a modifier of tau aggregation and associated toxicity. TIA-1 is present in a protein complex with tau protein including hyper-phosphorylated and misfolded tau. The expression of WT or P301L mutant tau increases the formation and size of TIA-1 positive SGs, and the localization and dynamics of these SGs are altered. Conversely, the expression of TIA-1 increases the formation and stabilization of phospho- and misfolded tau inclusions, as well as visible alterations in microtubule morphology, perhaps reflecting a loss of tau function. The data further show that co-expression of TIA-1 and tau leads to dendrite shortening, increases in caspase cleavage, and apoptosis in primary neurons, suggesting that an interaction between TIA-1 and tau results in neurotoxicity. This toxicity is SG-dependent and is rescued by microtubule stabilizing drugs. The results of this thesis research suggest that the aggregation of tau may proceed through the SG pathway, with SG formation accelerating the pathophysiology of tau aggregation. These studies propose that these tau aggregates serve as a nidus for further accelerated aggregation of SGs, leading to formation of long-lived pathological SG.