The Brazilian Zika virus strain causes birth defects in experimental models.

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.

Modeling neuro-immune interactions during Zika virus infection.

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells.

BACKGROUND: Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta. METHODS: The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained. RESULTS: Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator. CONCLUSIONS: Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.

Genetic variations on SETD5 underlying autistic conditions.

The prevalence of autism spectrum disorders (ASD) and the number of identified ASD-related genes have increased in recent years. The SETD5 gene encodes a SET-containing-domain 5 protein, a likely reader enzyme. Genetic evidences suggest that SETD5 malfunction contributes to ASD phenotype, such as on intellectual disability (ID) and facial dysmorphism. In this review, we mapped the clinical phenotypes of individuals carrying mutations on the SETD5 gene that are associated with ASD and other chromatinopathies (mutation in epigenetic modifiers that leads to the development of neurodevelopmental disorders such as ASD). After a detailed systematic literature review and analysis of public disease-related databank, we found so far 42 individuals carrying mutations on the SETD5 gene, with 23.8% presenting autistic-like features. Furthermore, most of mutations occurred between positions 9,480,000-9,500,000 bp on chromosome 3 (3p25.3) at the SETD5 gene locus. In all males, mutations in SETD5 presented high penetrance, while in females the clinical phenotype seems more variable with two reported cases showing normal female carriers and not presenting ASD or any ID-like symptoms. At the molecular level, SETD5 interacts with proteins of PAF1C and N-CoR complexes, leading to a possible involvement with chromatin modification pathway, which plays important roles for brain development. Together, we propose that mutations on the SETD5 gene could lead to a new syndromic condition in males, which is linked to 3p25 syndrome, and can leads to ASD-related intellectual disability and facial dysmorphism. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 500-518, 2018.

Blocking Zika virus vertical transmission.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

Author Correction: Blocking Zika virus vertical transmission.

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Induced pluripotent stem cells for modeling neurological disorders.

Several diseases have been successfully modeled since the development of induced pluripotent stem cell (iPSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from iPSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific iPSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using iPSC modeling for Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, Phelan-McDermid, Rett syndrome as well as Nonsyndromic Autism.