Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development. P rimary microcephaly is a severe brain malformation characterized by the reduction of the head circumference. Patients display a heterogeneous range of brain impairments that compromise motor, visual, hearing, and cognitive functions (1).Microcephaly is associated with decreased neuronal production as a consequence of proliferative defects and death of cortical progenitor cells (2). During pregnancy, the primary etiology of microcephaly varies from genetic mutations to external insults. The so-called TORCHS factors (toxoplasmosis, rubella, cytomegalovirus, herpes virus, and syphilis) are the main congenital infections that compromise brain development in utero (3).An increase in the rate of microcephaly in Brazil has been associated with the recent outbreak of Zika virus (ZIKV) (4, 5), a flavivirus that is transmitted by mosquitoes (6) and sexually (7-9). So far, ZIKV has been described in the placenta and amniotic fluid of microcephalic fetuses (10-13) and in the blood of microcephalic newborns (11, 14). ZIKV had also been detected within the brain of a microcephalic fetus (13, 14), and recently, direct evidence has emerged that ZIKV is able to infect and cause the death of neural stem cells (15).We used human induced pluripotent stem (iPS) cells cultured as neural stem cells (NSCs), neurospheres, and brain organoids to explore the consequences of ZIKV infection during neurogenesis and growth with three-dimensional culture models. Human iPS-derived NSCs were exposed to ZIKV [multiplicity of infection (MOI), 0.25 to 0.0025]. After 24 hours, ZIKV was detected in NSCs (Fig. 1, A to D); viral envelope protein was evident in 10.10% (MOI, 0.025) and 21.7% (MOI, 0.25) of cells exposed to ZIKV (Fig. 1E). Viral RNA was also detected in the supernatant of infected NSCs (MOI, 0.0025) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ( Fig. 1F), providing evidence of productive infection.To investigate the effects of ZIKV during neural differentiation, mock-and ZIKV-infected NSCs were cultured as neurospheres. After 3 days in vitro (DIV), mock-infected NSCs generated round neurospheres. However, ZIKVinfected NSCs generated neurospheres with morphological abnormalities and cell detachment ( Mock-infected neurospheres presented the expected ultrastructural morphology of the nucleus and mitochondria (Fig. 3A). Viral particles were present in ZIKV-infected neurospheres, similar to those observed in murine glial and neuronal cel...
The capacity of neurons to develop a long axon and multiple dendrites defines neuron connectivity in the CNS. The highly conserved microRNA-9 (miR-9) is expressed in both neuronal precursors and some post-mitotic neurons, and we detected miR-9 expression in the axons of primary cortical neurons. We found that miR-9 controlled axonal extension and branching by regulating the levels of Map1b, an important protein for microtubule stability. Following microfluidic separation of the axon and the soma, we found that miR-9 repressed Map1b translation and was a functional target for the BDNF-dependent control of axon extension and branching. We propose that miR-9 links regulatory signaling processes with dynamic translation mechanisms, controlling Map1b protein levels and axon development.
Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.
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