Pharmacological modulation of GABAA receptor signaling in the Ts65Dn mouse model of Down syndrome: an electrophysiological and behavioral investigation.

Objetivos: Este trabalho está dividido em dois capítulos. O objetivo do primeiro capítulo é avaliar os efeitos do diazepam, um modulador alostérico positivo de receptores GABAA (GABAAR) comumente utilizado na prática clínica, sobre a atividade elétrica cerebral, plasticidade sináptica e comportamento de camundongos Ts65Dn, o modelo experimental mais utilizado para o estudo da síndrome de Down (SD). O objetivo do segundo capítulo é fornecer uma perspectiva de como a avaliação da dinâmica das redes neurais em SD, em especial por meio do estudo do acoplamento fase-amplitude (PAC) de frequências cruzadas, pode contribuir para a identificação de biomarcadores para o diagnóstico e/ou prognóstico da doença de Alzheimer na população com SD. Métodos: A atividade elétrica cerebral de camundongos controle (N=6) e Ts65Dn (N=7) foi registrada por vídeo eletroencefalograma. Eletrodos foram implantados em ambos os hemisférios do córtex frontal, bem como profundamente na região CA1 do hipocampo esquerdo. A análise de densidade espectral de potência dos registros corticais e hipocampais foi realizada pelo método de Welch, enquanto o PAC entre as oscilações teta e gama do hipocampo foi estimado pelas ténicas envelope-to-signal correlation e mean vector length. Efeitos de dose-resposta do diazepam na atividade elétrica cerebral e no comportamento dos camundongos foram avaliados após a administração intraperitoneal (i.p.) única das doses 1, 3 e 10 mg/kg. A susceptibilidade a convulsões induzidas pela administração i.p. única de 2 ou 4 mg/kg picrotoxina, um antagonista não competitivo de GABAAR, também foi avaliada. A potenciação de longa duração (LTP) induzida por trens de estimulação na frequência teta em fatias hipocampais (região CA1) obtidas de camundongos controle (N=12) e Ts65Dn (N=12) foi avaliada por meio de registros extracelulares de potenciais pós-sinápticos excitatórios de campo nas seguintes condições: solução de fluido cérebro espinhal artificial (aCSF), aCSF contendo 200 μM GABA, aCSF contendo 1 μM diazepam e aCSF contendo 10 μM diazepam. Por fim, o comportamento do tipo ansioso em camundongos controle (N=32) e Ts65Dn (N=28) tratados com solução salina ou 1 mg/kg de diazepam foi avaliado por meio do teste do labirinto em cruz elevado. Resultados: Observou-se um aumento da potência das oscilações beta-2, bem como uma diminuição da potência das oscilações beta-1 e gama em camundongos controle tratados com diazepam. Tais efeitos, entretanto, não foram observados em camundongos Ts65Dn. Por outro lado, enquanto foi observada uma lentificação das oscilações delta em ambos os camundongos controle e Ts65Dn tratados com diazepam, uma lentificação das oscilações teta fora observada apenas em camundongos controle. Observou-se também efeitos genótipo-dependentes do diazepam quanto a dinâmica da modulação da diferença de fase entre o córtex e o hipocampo, uma vez que o tratamento promoveu um aumento na sincronização de fase entre as atividades oscilatórias dessas regiões cerebrais apenas em camundongos controle. A eficácia do diazepam em reduzir tanto a suscetibilidade quanto a gravidade de crises convulsivas induzidas por picrotoxina foi observada em ambos os camundongos controle e Ts65Dn. Efeitos genótipo-dependentes também foram observados sobre a magnitude da LTP, a qual fora reduzida pelos tratamentos com 200 μM GABA e 10 μM diazepam em fatias de camundongos controle quando comparadas às fatias de camundongos Ts65Dn. Enquanto o tratamento com o diazepam aumentou o número de entradas e o tempo de permanência nos braços abertos do labirinto em camundongos controle, tal tratamento não alterou as medidas espaço-temporais em camundongos Ts65Dn. Por outro lado, o tratamento com o diazepam alterou parâmetros etológicos em ambos os grupos, como o aumento no número de mergulhos de cabeça em espaços não protegidos do labirinto. Por fim, verificou-se a presença de PAC entre as oscilações teta e gama do hipocampo em camundongos controle e Ts65Dn. Entretanto, o padrão e a intensidade desse fenômeno foram diferentes entre os grupos, visto que uma maior intensidade de acoplamento fora observada em camundongos Ts65Dn. Conclusões: Este estudo revelou diferenças significativas nos efeitos do diazepam sobre as propriedades eletrofisiológicas cerebrais e fenótipos comportamentais de camundongos controle e Ts65Dn, as quais sugerem uma redução na sensibilidade aos efeitos do diazepam sobre a modulação da sinalização mediada por GABAAR no camundongo Ts65Dn. Adicionalmente, diferenças quanto aos índices de PAC sugerem um padrão alterado na dinâmica das redes neuronais no hipocampo de camundongos Ts65Dn. Tais parâmetros podem contribuir para a identificação de biomarcadores electroencefalográficos não-invasivos com potencial diagnóstico e/ou prognóstico da doença de Alzheimer (DA) na população com SD, cujo risco para a DA é conhecidamente aumentado.Objectives: This work is divided in two chapters. Chapter 1 consists of evaluating the effects of diazepam, a highly prescribed positive allosteric modulator of GABAA receptors (GABAAR), on brain activity, synaptic plasticity, and behavior of the wellstudied mouse model of Down syndrome (DS), the Ts65Dn mouse. Chapter 2 aims to provide a perspective of how the study of brain network dynamics in DS, especially by studying mechanisms of cross-frequency phase-amplitude coupling (PAC), may contribute to the discovery of non-invasive biomarkers with potential clinical application. Methods: Brain electrical activity was recorded by videoelectroencephalography (video-EEG), which was performed in control (N=6) and Ts65Dn (N=7) male mice. Electrodes were implanted bilaterally in the frontal cortex, as well as deeply into the left hippocampal CA1 area. Power spectral analysis of cortical and hippocampal recordings were carried out by Welch's method, while hippocampal theta-gamma PAC was estimated by envelope-to-signal correlation and mean vector length. Dose-response relationship of diazepam on brain activity and behavior was evaluated following a single intraperitoneal (i.p.) administration at 1-, 3- , and 10-mg/kg doses. The effects of a single i.p. injection of 2 and 4 mg/kg picrotoxin, a noncompetitive antagonist of GABAAR, on seizure susceptibility were also investigated. Theta-burst-induced long-term potentiation (LTP) in hippocampal CA1 region of control- (N=12) and Ts65Dn-derived (N=12) slices was evaluated by extracellular recordings of field excitatory postsynaptic potential under the following experimental conditions: artificial cerebral spinal fluid (aCSF), aCSF with 200 μM GABA, aCSF with 1 μM diazepam, and aCSF with 10 μM diazepam. Finally, anxietylike behaviors in saline- and diazepam-treated control (N=32) and Ts65Dn (N=28) male mice were evaluated by the elevated plus-maze test. Results: We found that diazepam neither increased beta-2 activity nor decreased the power of beta-1 and gamma oscillations in Ts65Dn mice compared to control mice. Although we observed slowing effects of diazepam on delta oscillations in both control and Ts65Dn mice, a similar effect on frequencies within the theta range was only found in controls. Our findings also revealed that diazepam modulated the dynamics of phase relationships between cortical and hippocampal areas in a genotype-dependent manner, with increased phase synchronization between oscillatory activities in control mice and opposite effects in Ts65Dn mice. In a separate set of experiments, it was found that diazepam was similarly effective in reducing susceptibility and severity to picrotoxininduced convulsive seizures in both control and Ts65Dn mice. Differences in diazepam effects were also observed in the magnitude of CA1 TBS-induced LTP, which was depressed by 200 μM GABA and 10 μM diazepam in control- but not in Ts65Dn-derived slices. Diazepam increased both time spent and number of entries into the open arms in control mice, while it had no major effects on spatiotemporal patterns of exploratory activity in Ts65Dn mice. In contrast, we observed that diazepam altered risk assessment behaviors such as head dipping in both groups. Finally, we found that hippocampal theta-gamma PAC was present in control and Ts65Dn mice, but the pattern and intensity of this phenomenon were different between groups, with Ts65Dn mice showing higher theta-gamma PAC than controls. Conclusions: This study unveiled significant genotype-dependent differences in the effects of diazepam on the electrophysiological and behavioral phenotypes of control and Ts65Dn mice, wherein Ts65Dn mice appear less sensitive to diazepammediated modulation of GABAAR signaling. In addition, altered dynamics in hippocampal networks of Ts65Dn mice as suggested by measures of PAC may contribute to the development of non-invasive EEG biomarkers for the diagnosis and prognosis of Alzheimer's disease in the DS population, which is at a higher risk for developing this neurodegenerative disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2016/17746-3Bolsa Estágio de Pesquisa no Exterior (BEPE) - 2018/10131-

Genetic Polymorphisms Involved in Folate Metabolism and Maternal Risk for Down Syndrome: A Meta-Analysis

Inconclusive results of the association between genetic polymorphisms involved in folate metabolism and maternal risk for Down syndrome (DS) have been reported. Therefore, this meta-analysis was conducted. We searched electronic databases through May, 2014, for eligible studies. Pooled odds ratios with 95% confidence intervals were used to assess the strength of the association, which was estimated by fixed or random effects models. Heterogeneity among studies was evaluated using Q-test and I2 statistic. Subgroup and sensitivity analyses were also conducted. Publication bias was estimated using Begg’s and Egger’s tests. A total of 17 case-controls studies were included. There was evidence for an association between the MTRR c.66A>G (rs1801394) polymorphism and maternal risk for DS. In the subgroup analysis, increased maternal risk for DS was found in Caucasians. Additionally, the polymorphic heterozygote MTHFD1 1958GA genotype was associated significantly with maternal risk for DS, when we limit the analysis by studies conformed to Hardy-Weinberg equilibrium. Finally, considering MTR c.2756A>G (rs1805087), TC2 c.776C>G (rs1801198), and CBS c.844ins68, no significant associations have been found, neither in the overall analyses nor in the stratified analyses by ethnicity. In conclusion, our meta-analysis suggested that the MTRR c.66A>G (rs1801394) polymorphism and MTHFD1 c.1958G>A (rs2236225) were associated with increased maternal risk for DS

Over‐expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice

International audienceDown syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins