Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons

Growing evidences have associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly. Nonetheless, signaling mechanisms that promote the disease outcome are far from being understood, affecting the development of suitable therapeutics. In this study, we applied shotgun mass spectrometry (MS)-based proteomics combined with cell biology approaches to characterize altered molecular pathways on human neuroprogenitor cells (NPC) and neurons derived from induced pluripotent stem cells infected by ZIKV-BR strain, obtained from the 2015 Brazilian outbreak. Furthermore, ZIKV-BR infected NPCs showed unique alteration of pathways involved in neurological diseases, cell death, survival and embryonic development compared to ZIKV-AF, showing a human adaptation of the Brazilian viral strain. Besides, infected neurons differentiated from NPC presented an impairment of neurogenesis and synaptogenesis processes. Taken together, these data explain that CNS developmental arrest observed in Congenital Zika Syndrome is beyond neuronal cell death

A phenotypic and pathogenic analysis of Zika virus infection in human neural cells in vitro

O Zika vírus (ZIKV) é um flavivírus transmitido pelo mosquito Aedes aegypti e que se espalhou rapidamente pelas Américas, causando uma epidemia no Brasil em 2015 . Um número crescente nos casos de infecções veio acompanhado de um aumento no número de fetos e bebês nascidos com microcefalia, levando a um chamado de emergência mundial de saúde. Históricamente, o ZIKV não havia causado infecções de destaque em humanos e a reermegência dessa ameça viral associada à defeitos do nascimento foi logo relacionada à evolução e consequente distinção entre os genótipos virais, o original Zika africano e seu descendente Zika asiático, que chegou ao Brasil. A hipótese da cepa brasileira do ZIKV ser a causadora de microcefalia e de outros defeitos do nascimento ganhou mais respaldo após a identificação do vírus em amostras de tecido cerebral e líquido amniótico de fetos. Posteriormente, a associação direta entre microcefalia e a síndrome congênita com o ZIKV foi confirmada por meio da aplicação de modelos biológicos experimentais que se revelaram susceptíveis à infecção viral, como células do sistema nervoso central em sistemas 2D e 3D in vitro e camundongos prenhês. Esse trabalho teve como objetivo investigar a infecção da cepa brasileira do ZIKV (ZIKVBR) em diferentes células humanas neurais in vitro diferenciadas a partir de células-tronco pluripotentes induzidas, além de criar uma plataforma para teste de fármacos in vitro contra o vírus. Nossos resultados comprovaram a susceptibilidade e permissividade celular à infecção do ZIKVBR em células neuronais e, em especial, progenitoras neurais, causando morte celular por apoptose. Além disto, quando células progenitoras neurais foram cultivadas em suspensão, formando neuroesferas, o ZIKVBR foi capaz de causar uma redução na população de células, gerando uma anormalidade morfológica semelhante à microcefalia. Além do mais, quando células progenitoras neurais infectadas com ZIKVBR foram diferenciadas em neurônios maduros, a análise da sinaptogênese revelou que esses neurônios apresentavam uma menor densidade de puncta sináptica, indicando um comprometimento no funcionamento das sinapses que pode estar contribuindo para os problemas associados com a síndrome congênita do ZIKV. Por fim, o tratamento dessas células com a droga Sofosbuvir, um inibidor de RNA polimerase dependente de RNA aprovado para uso clínico, foi capaz de resgatar NPCs e neurônios apoptóticos. Em suma, nossos dados indicam que o ZIKVBR infecta preferencialmente células progenitoras neurais, replicando-se eficientemente e causando morte por apoptose nessas células e neurônios maduros diferenciados de células progenitoras neurais infectadas apresentam uma menor desidade de puncta sináptica. Finalmente, a reutilização de compostos farmacêuticos já aprovados para uso clínico pode acelerar o tratamento para indivíduos infectados pelo ZIKV onde a prevenção já não é mais opção, como no caso de mulheres grávidas.Zika virus (ZIKV) is a mosquito-borne flavivirus transmitted by Aedes aegypti that has rapidly spread through the Americas, causing a widespread epidemic in Brazil in 2015. A increasing number of infection cases was followed by a rise in the number of fetuses and babies born with microcephaly, leading to a global health emergency call. Up to then, ZIKV had not caused meaningful infections in humans and the reemergency of this viral threat associated with birth defects was soon related to viral genotype mutations and its consequent distinction from the original african Zika strain to its descendent asian Zika strain, which reached Brazil. The hypotesis of the brazilian ZIKV strain being responsible for microcephaly and other birth defects gained support after the isolation and identification of the virus in samples of cerebral tissue and amniotic fluid of fetuses. Subsequently, the direct association between microcephaly and congenital syndrome with ZIKV was confirmed through the application of biologic experimental models which proved susceptible to viral infection, as for cells from the central nervous system cultured in 2D and 3D models as well as pregnant mice. The aim of this study was to investigate the brazilian ZIKV strain (ZIKVBR) infection in different human neural cells in vitro differentiated from induced pluripotent stem cells, as well as creating a platform for in vitro drug testing with antiviral capabilities. Our results showed cellular infection susceptibility and permissiveness to ZIKVBR in neurons and, specially, neural progenitor cells, displaying cell death by apoptosis. Futhermore, when neuronal progenitor cells cultured in suspension, forming neurospheres, were infected with ZIKVBR, it caused a reduction in cell population, displayed by evident morphological abnormalities resembling to microcephaly. Additionally, when neural progenitor cells infected with ZIKVBR were diferentiated further into mature neurons, synaptogenesis analysis revealed these neurons displayed fewer synaptic puncta density, indicating a compromise in synapse functioning that may be contributing to problems associated with ZIKV congenital syndrome. Moreover, cell treatment with Sofosbuvir, a RNA polymerase RNAdependent inhibitor approved for clinical use, was able to rescue apoptotic NPCs and neurons. In summary, our results reveal that ZIKVBR preferentially infects neural progenitor cells, efficiently replicating itself and causing death by apoptosis in these cells and mature neurons differentiated from infected neural progenitor cells display reduced synaptic puncta density. Lastly, the repurpose of FDA approved compounds may aid in accelerating treatment for infected individuals whose prevention is no longer an option, as it is for pregnant women