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...

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases.

Schizophrenia is a neurodevelopmental disease characterized by cerebral connectivity impairment and loss of gray matter. It was described in adult schizophrenia patients (SZP) that concentration of VEGFA, a master angiogenic factor, is decreased. Recent evidence suggests cerebral hypoperfusion related to a dysfunctional Blood Brain Barrier (BBB) in SZP. Since neurogenesis and blood-vessel formation occur in a coincident and coordinated fashion, a defect in neurovascular development could result in increased vascular permeability and, therefore, in poor functionality of the SZP’s neurons. Here, we characterized the conditioned media (CM) of human induced Pluripotent Stem Cells (hiPSC)-derived Neural Stem Cells of SZP (SZP NSC) versus healthy subjects (Ctrl NSC), and its impact on angiogenesis. Our results reveal that SZP NSC have an imbalance in the secretion and expression of several angiogenic factors, among them non-canonical neuro-angiogenic guidance factors. SZP NSC migrated less and their CM was less effective in inducing migration and angiogenesis both in vitro and in vivo. Since SZP originates during embryonic brain development, our findings suggest a defective crosstalk between NSC and endothelial cells (EC) during the formation of the neuro-angiogenic niche.

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca2+ effect in BAT mitochondria thermogenesis. We found that Ca2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 µM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN.Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca2+ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca2+ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.