Absence of Tau triggers age-dependent sciatic nerve morphofunctional deficits and motor impairment

Dementia is the cardinal feature of Alzheimer's disease (AD), yet the clinical symptoms of this disorder also include a marked loss of motor function. Tau abnormal hyperphosphorylation and malfunction are well-established key events in AD neuropathology but the impact of the loss of normal Tau function in neuronal degeneration and subsequent behavioral deficits is still debated. While Tau reduction has been increasingly suggested as therapeutic strategy against neurodegeneration, particularly in AD, there is controversial evidence about whether loss of Tau progressively impacts on motor function arguing about damage of CNS motor components. Using a variety of motor-related tests, we herein provide evidence of an age-dependent motor impairment in Tau-/- animals that is accompanied by ultrastructural and functional impairments of the efferent fibers that convey motor-related information. Specifically, we show that the sciatic nerve of old (17-22-months) Tau-/- mice displays increased degenerating myelinated fibers and diminished conduction properties, as compared to age-matched wild-type (Tau+/+) littermates and younger (4-6months) Tau-/- and Tau+/+ mice. In addition, the sciatic nerves of Tau-/- mice exhibit a progressive hypomyelination (assessed by g-ratio) specifically affecting large-diameter, motor-related axons in old animals. These findings suggest that loss of Tau protein may progressively impact on peripheral motor system.The work was supported by grants 'PTDC/SAU-NMC/113934/2009,' 'PTDC/SAU-NSC/118194/2010,' 'SFRH/BPD/97281/2013,' PTDC/SAU-NSC/118194/2010,' 'SFRH/BPD/80118/2011,' 'SFRH/BD/89714/2012' funded by FCT-Portuguese Foundation for Science and Technology and project DoIT-Desenvolvimento e Operacionalizacao da Investigacao de Translacao (No do projeto 13853), funded by Fundo Europeu de Desenvolvimento Regional (FEDER) throughout the Programa Operacional Fatores de Competitividade (POFC). In addition, this work was also co-financed by European Union FP7 project SwitchBox (NS) and the Portuguese North Regional Operational Program (ON.2 - O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER).info:eu-repo/semantics/publishedVersio

Constitutive deficiency of the neurogenic hippocampal modulator AP2γ promotes anxiety-like behavior and cumulative memory deficits in mice from juvenile to adult periods

The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.H2020 -“la Caixa” Foundation(101003187

A influência da gliotransmissão sobre as funções cognitivas superiores

Tese de Doutoramento em Ciências da SaúdeIn recent years, the understanding of synaptic modulation by neuron-astrocyte interactions has evolved considerably, contributing to build up the concept of the “tripartite synapse”. This concept is based on the dynamic dialogue between astrocytes and neurons that complements and modulates the communication between pre- and post-synaptic structures. However, it remains elusive how this interaction between neurons and astrocytes translates into network computation of behavior. Astrocytes were described to release gliotransmitters (such as glutamate, GABA, ATP or D-Serine) by means of several mechanisms, being exocytosis the more extensively studied. This process is mediated by the vesicular machinery and SNARE complex formation between vesicles and the target membrane, ultimately resulting in the release of the vesicular content. Transmitter release is essential for astrocyte signaling and a disruption of this phenomenon is expected to impact on the function of neuronal networks, with consequences for the computation of higher brain functions. In this thesis, our main goal was to evaluate the influence of transmitter release by astrocytes on brain circuits responsible for cognitive functions, such as learning and memory. Throughout this work, we studied the transgenic dnSNARE mouse model that displays a conditional blockade of transmitter release by exocytosis, selectively in astrocytes. This was achieved by allowing the conditional expression of the dominant negative domain of vesicular SNARE protein synaptobrevin II (dnSNARE), which interferes with the SNARE complex formation, impairing vesicular release. Four weeks after the induction of transgene transcription, the levels of transgenic protein reached its maximum. The dnSNARE transgenes are expressed exclusively by astrocytes and display inter-subject variability. The quantification of dnSNARE transgene expression levels allowed to discriminate high “expressor” subjects to be analyzed throughout. Mice were first evaluated by performing in vivo electrophysiological recordings of local field potentials from neuronal populations of cognitive related brain regions: dorsal hippocampus and prefrontal cortex. This functional network evaluation was followed by a thorough assessment of cognitive ability of these mice, by performing a battery of behavioral tests. These tests addressed different cognitive tasks mainly dependent from the performance of the hippocampus-prefrontal network. This functional assessment was complemented by a morphological characterization of neurons and astrocytes to address structural correlates of network function. Our findings demonstrated a specific neural desynchronization in the theta rhythm between the dorsal hippocampus and prefrontal cortex in the dnSNARE mice, without any alteration of levels of neuronal activity. Moreover, the blockade of gliotransmitter release in astrocytes triggers a critical cognitive impairment in tasks classically attributed to neuronal circuits of the hippocampusprefrontal cortex network. More specifically, dnSNARE mice faced an increased difficulty when performing in reference memory tasks of Morris water maze (MWM) and hole-board test (HB), and revealed a clear deficit in tasks involving spatial recognition and long-term memory, such as the novel object recognition (NOR) and two-trial place recognition (2TPR) tests. Further analysis of electrophysiological recordings showed a direct correlation between the loss of theta coherence in dorsal hippocampus-prefrontal link and poor consolidation of reference memory. The structure evaluation of the dorsal hippocampus and prefrontal cortex revealed that the neuronal dendritic trees appear to be intact in dnSNARE mice. However, astrocytes undergo drastic process atrophy, specifically in GFAP+ cells that also express dnSNARE transgenes. Interestingly, the intraperitoneal supplementation with the NMDAR co-agonist D-serine — that is known to be released by exocytosis in astrocytes and to be significantly decreased in the brains of dnSNARE mice — completely restored theta synchronization and rescued the learning and memory deficits in transgenic mice. In conclusion, this PhD thesis provides the evidence of a mechanism by which astrocytic signaling is required for entrainment of distant cortico-limbic circuits, being mandatory for cognitive performance. Moreover, our findings suggest that D-serine may be the gliotransmitter maintaining the synchronization of the theta rhythm between these circuits required for learning and memory consolidation. Further studies should be performed to unravel the astrocytic contribution to different cognitive tasks, as well as the therapeutic potential of astrocyte signaling to the development of new approaches to treat disorders of the central nervous system, characterized by cognitive decline.Nos últimos anos, a compreensão da modulação sináptica através de interações neurónio-astrócito tem evoluído consideravelmente, contribuindo para o aparecimento do conceito da "sinapse tripartida". Este conceito é baseado no diálogo dinâmico entre astrócitos e neurónios que complementa e modula a comunicação entre as estruturas pré e pós-sinápticas. No entanto, continua por esclarecer de que forma essa interação entre neurónios e astrócitos se traduz na produção de comportamentos pelo cérebro. Os astrócitos libertam gliotransmissores (tais como glutamato, GABA, ATP ou D-serina) através de diversos mecanismos, sendo a exocitose o mais amplamente estudado. Este processo é mediado pela maquinaria vesicular e pela formação do complexo SNARE entre vesículas e a membrana alvo, resultando, em última instância, na libertação do conteúdo vesicular para o exterior. A libertação de transmissores é essencial para a sinalização dos astrócitos e seria de esperar que a interrupção desse fenómeno afectasse a função das redes neuronais, com consequências para a computação de funções cerebrais superiores. Assim, o principal objetivo deste trabalho foi avaliar a influência da libertação de transmissores pelos astrócitos nos circuitos cerebrais responsáveis pelas funções cognitivas, como a aprendizagem e o processamento de memória. Ao longo deste trabalho utilizámos o modelo de murganho transgénico dnSNARE que apresenta um bloqueio condicional da libertação de transmissores por exocitose, selectivamente em astrócitos. A ocorrência deste bloqueio é devida à expressão condicional do domínio negativo dominante da sinaptobrevina vesicular II (dnSNARE), que interfere com a formação do complexo SNARE e, consequentemente, com a libertação vesicular. O transgene atinge o seu pico de expressão quatro semanas após a indução da sua expressão. A mesma é exclusiva dos astrócitos e apresenta variabilidade entre indivíduos. A quantificação da expressão do transgene dnSNARE permitiu identificar os animais com níveis elevados de expressão transgénica, que foram utilizados ao longo do trabalho para comparação com os respectivos controlos. Os murganhos dnSNARE, e respectivos controlos, foram primeiramente avaliados através da realização de registos electrofisiológicos de potenciais de campo locais, in vivo, de populações neuronais de regiões do cérebro implicadas na função cognitiva: o hipocampo e o córtex préfrontal. Esta análise funcional da integridade da rede foi complementada com uma avaliação completa da capacidade cognitiva destes animais, procedendo-se para esse fim, à realização de uma bateria de testes comportamentais. Os testes utilizados abrangiam diferentes funções, principalmente dependentes do desempenho da rede hipocampo–cortex pré-frontal. Além desta caracterização funcional, procedeu-se ainda a uma caracterização morfológica de neurónios e astrócitos, com o intuito de avaliar possíveis correlatos estruturais das funções da rede. Os resultados principais desta tese demonstram uma desincronização neuronal específica no ritmo teta, entre o hipocampo dorsal e o córtex pré-frontal em murganhos dnSNARE, sem qualquer alteração dos níveis basais de atividade neuronal. Além disso, o bloqueio da libertação de gliotransmissores em astrócitos desencadeia um défice cognitivo severo em tarefas classicamente atribuídas aos circuitos neuronais do hipocampo dorsal e do córtex pré-frontal. Mais especificamente, os murganhos dnSNARE enfrentaram uma maior dificuldade na realização de tarefas de memória de referência no teste da pisicna de Morris (MWM) e no teste do Hole-Board (HB). Estes animais revelaram ainda um claro défice em tarefas que envolviam o reconhecimento espacial e memória de longo prazo nos testes de reconhecimento de novos objectos (NOR) e espaços (2TPR). Análises adicionais de registros electrofisiológicos mostraram a existência de uma correlação significativa entre a perda de coerência teta na ligação hipocampo dorsal-cortex préfrontal e a consolidação de memória de referência. A análise estrutural do hipocampo dorsal e do córtex pré-frontal revelou que a estrutura neuronal permance intacta nos murganhos dnSNARE. No entanto, os astrócitos GFAP-positivos que também expressam transgenes dnSNARE parecem sofrer uma drástica atrofia. Curiosamente, a suplementação intraperitoneal com D-serina – um co-agonista dos receptores NMDA, libertado por exocitose nos astrócitos e significativamente diminuído nos cérebros de murganhos dnSNARE – restaurou completamente a sincronização em teta e, recuperou os défices de aprendizagem e memória nos mesmos animais. Em conclusão, esta tese de doutoramento apresenta resultados relativos à evidência de um mecanismo pelo qual a sinalização astrocítica é necessária para o estabelecimento da comunicação entre circuitos cortico-límbicos distantes, revelando a sua importância para o desempenho cognitivo. Estes resultados sugerem que a D-serina pode ser o gliotransmissor responsável pela sincronização do ritmo teta entre os circuitos necessários para os processos de aprendizagem e consolidação de memória. Estudos adicionais deverão ser realizados para dissecar a contribuição astrocítica para outras tarefas cognitivas e, para avaliar o potencial terapêutico da sinalização de astrócitos para o desenvolvimento de novas abordagens, capazes de tratar distúrbios do sistema nervoso central caracterizados pelo declínio cognitivo.The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), Minho University. Financial support was provided by a PhD grant (SFRH/BD/89714/2012) from the FCT – Foundation for Science and Technology, by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145- FEDER-007038; and by the project NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Adenosine A2A receptor regulation of microglia morphological remodeling-gender bias in physiology and in a model of chronic anxiety

Developmental risk factors, such as the exposure to stress or high levels of glucocorticoids (GCs), may contribute to the pathogenesis of anxiety disorders. The immunomodulatory role of GCs and the immunological fingerprint found in animals prenatally exposed to GCs point towards an interplay between the immune and the nervous systems in the etiology of these disorders. Microglia are immune cells of the brain, responsive to GCs and morphologically altered in stress-related disorders. These cells are regulated by adenosine A2A receptors, which are also involved in the pathophysiology of anxiety. We now compare animal behavior and microglia morphology in males and females prenatally exposed to the GC dexamethasone. We report that prenatal exposure to dexamethasone is associated with a gender-specific remodeling of microglial cell processes in the prefrontal cortex: males show a hyper-ramification and increased length whereas females exhibit a decrease in the number and in the length of microglia processes. Microglial cells re-organization responded in a gender-specific manner to the chronic treatment with a selective adenosine A2A receptor antagonist, which was able to ameliorate microglial processes alterations and anxiety behavior in males, but not in females.This work was supported by PEst-C/SAU/UI3282/2011-2013, POCI-01-0145-FEDER-007440 and UID/NEU/04539/2013 (FCT, Portugal, and COMPETE-FEDER). Catarina A. Gomes acknowledges a fellowship from Fundacao para a Ciencia e a Tecnologia, Portugal (SFRH/BPD/63013/2009. [Gomes] Filipa I. Baptista acknowledges a fellowship from Fundacao para a Ciencia e a Tecnologia, Portugal (SFRH/BPD/86830/2012)