Postnatal Zika virus infection leads to morphological and cellular alterations within the neurogenic niche.

The Zika virus received significant attention in 2016, following a declaration by the World Health Organization of an epidemic in the Americas, in which infections were associated with microcephaly. Indeed, prenatal Zika virus infection is detrimental to fetal neural stem cells and can cause premature cell loss and neurodevelopmental abnormalities in newborn infants, collectively described as congenital Zika syndrome. Contrastingly, much less is known about how neonatal infection affects the development of the newborn nervous system. Here, we investigated the development of the dentate gyrus of wild-type mice following intracranial injection of the virus at birth (postnatal day 0). Through this approach, we found that Zika virus infection affected the development of neurogenic regions within the dentate gyrus and caused reactive gliosis, cell death and a decrease in cell proliferation. Such infection also altered volumetric features of the postnatal dentate gyrus. Thus, we found that Zika virus exposure to newborn mice is detrimental to the subgranular zone of the dentate gyrus. These observations offer insight into the cellular mechanisms that underlie the neurological features of congenital Zika syndrome in children.

Amniotic fluid metabolomics identifies impairment of glycerophospholipid and amino acid metabolism during congenital Zika syndrome development.

PURPOSE: Our main goal is to identify the alterations in the amniotic fluid (AF) metabolome in Zika virus (ZIKV)-infected patients and their relation to congenital Zika syndrome (CZS) progression. EXPERIMENTAL DESIGN: We applied an untargeted metabolomics strategy to analyze seven AF of pregnant women: healthy women and ZIKV-infected women bearing non-microcephalic and microcephalic fetuses. RESULTS: Infected patients were characterized by glycerophospholipid metabolism impairment, which is accentuated in microcephalic phenotypes. Glycerophospholipid decreased concentration in AF can be a consequence of intracellular transport of lipids to the placental or fetal tissues under development. The increased intracellular concentration of lipids can lead to mitochondrial dysfunction and neurodegeneration caused by lipid droplet accumulation. Furthermore, the dysregulation of amino acid metabolism was a molecular fingerprint of microcephalic phenotypes, specifically serine, and proline metabolisms. Both amino acid deficiencies were related to neurodegenerative disorders, intrauterine growth retardation, and placental abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE: This study enhances our understanding of the development of CZS pathology and sheds light on dysregulated pathways that could be relevant for future studies.

Identification of an ex vivo inhibitor of the schizophrenia biomarker Ndel1 by high throughput screening.

Ndel1 oligopeptidase activity shows promise as a potential biomarker for diagnosing schizophrenia (SCZ) and monitoring early-stage pharmacotherapy. Ndel1 plays a pivotal role in critical aspects of brain development, such as neurite outgrowth, neuronal migration, and embryonic brain formation, making it particularly relevant to neurodevelopmental disorders like SCZ. Currently, the most specific inhibitor for Ndel1 is the polyclonal anti-Ndel1 antibody (NOAb), known for its high specificity and efficient anti-catalytic activity. NOAb has been vital in measuring Ndel1 activity in humans and animal models, enabling the prediction of pharmacological responses to antipsychotics in studies with patients and animals. To advance our understanding of in vivo Ndel1 function and develop drugs for mental disorders, identifying small chemical compounds capable of specifically inhibiting Ndel1 oligopeptidase is crucial, including within living cells. Due to challenges in obtaining Ndel1's three-dimensional structure and its promiscuous substrate recognition, we conducted a high-throughput screening (HTS) of 2,400 small molecules. Nine compounds with IC50-values ranging from 7 to 56 µM were identified as potent Ndel1 inhibitors. Notably, one compound showed similar efficacy to NOAb and inhibited Ndel1 within living cells, although its in vivo use may pose toxicity concerns. Despite this, all identified compounds hold promise as candidates for further refinement through rational drug design, aiming to enhance their inhibitory efficacy, specificity, stability, and biodistribution. Our ultimate goal is to develop druggable Ndel1 inhibitors that can improve the treatment and support the diagnosis of psychiatric disorders like SCZ.

Assessing the role of Ndel1 oligopeptidase activity in congenital Zika syndrome: Potential predictor of congenital syndrome endophenotype and treatment response.

Maternal infections are among the main risk factors for cognitive impairments in the offspring. Zika virus (ZIKV) can be transmitted vertically, causing a set of heterogeneous birth defects, such as microcephaly, ventriculomegaly and corpus callosum dysgenesis. Nuclear distribution element like-1 (Ndel1) oligopeptidase controls crucial aspects of cerebral cortex development underlying cortical malformations. Here, we examine Ndel1 activity in an animal model for ZIKV infection, which was associated with deregulated corticogenesis. We observed here a reduction in Ndel1 activity in the forebrain associated with the congenital syndrome induced by ZIKV isolates, in an in utero and postnatal injections of different inoculum doses in mice models. In addition, we observed a strong correlation between Ndel1 activity and brain size of animals infected by ZIKV, suggesting the potential of this measure as a biomarker for microcephaly. More importantly, the increase of interferon (IFN)-beta signaling, which was used to rescue the ZIKV infection outcomes, also recovered Ndel1 activity to levels similar to those of uninfected healthy control mice, but with no influence on Ndel1 activity in uninfected healthy control animals. Taken together, we demonstrate for the first time here an association of corticogenesis impairments determined by ZIKV infection and the modulation of Ndel1 activity. Although further studies are still necessary to clarify the possible role(s) of Ndel1 activity in the molecular mechanism(s) underlying the congenital syndrome induced by ZIKV, we suggest here the potential of monitoring the Ndel1 activity to predict this pathological condition at early stages of embryos or offspring development, during while the currently employed methods are unable to detect impaired corticogenesis leading to microcephaly. Ndel1 activity may also be possibly used to follow up the positive response to the treatment, such as that employing the IFN-beta that is able to rescue the ZIKV-induced brain injury.

Case report: Regression of Glioblastoma after flavivirus infection.

Glioblastoma is the most frequent and aggressive primary brain cancer. In preclinical studies, Zika virus, a flavivirus that triggers the death of glioblastoma stem-like cells. However, the flavivirus oncolytic activity has not been demonstrated in human patients. Here we report a glioblastoma patient who received the standard of care therapy, including surgical resection, radiotherapy and temozolomide. However, shortly after the tumor mass resection, the patient was clinically diagnosed with a typical arbovirus-like infection, during a Zika virus outbreak in Brazil. Following the infection resolution, the glioblastoma regressed, and no recurrence was observed. This clinical response continues 6 years after the glioblastoma initial diagnosis.

Centromere protein J is overexpressed in human glioblastoma and promotes cell proliferation and migration.

Glioblastoma is the most common and malignant type of primary brain tumor. Previous studies have shown that alterations in centrosome amplification and its components are frequently found in treatment-resistant tumors and may be associated with tumor progression. A centrosome protein essential for centrosome biogenesis is the centromere protein J (CENPJ), known to control the proliferation of neural progenitors and hepatocarcinoma cells, and also neuronal migration. However, it remains unknown the role of CENPJ in glioblastoma. Here we show that CENPJ is overexpressed in human glioblastoma cell lines in comparison to human astrocytes. Using bioinformatics analysis, we find that high Cenpj expression is associated with poor prognosis in glioma patients. Examining Cenpj loss of function in glioblastoma by siRNA transfection, we find impairments in cell proliferation and migration. Using a Cenpj mutant version with the deleted PN2-3 or TCP domain, we found that a conserved PN2-3 region is required for glioblastoma migration. Moreover, Cenpj downregulation modulates glioblastoma morphology resulting in microtubules stabilization and actin filaments depolymerization. Altogether, our findings indicate that CENPJ controls relevant aspects of glioblastoma progression and might be a target for therapeutic intervention and a biomarker for glioma malignancy.

Zika Virus Strains and Dengue Virus Induce Distinct Proteomic Changes in Neural Stem Cells and Neurospheres.

Brain abnormalities and congenital malformations have been linked to the circulating strain of Zika virus (ZIKV) in Brazil since 2016 during the microcephaly outbreak; however, the molecular mechanisms behind several of these alterations and differential viral molecular targets have not been fully elucidated. Here we explore the proteomic alterations induced by ZIKV by comparing the Brazilian (Br ZIKV) and the African (MR766) viral strains, in addition to comparing them to the molecular responses to the Dengue virus type 2 (DENV). Neural stem cells (NSCs) derived from induced pluripotent stem (iPSCs) were cultured both as monolayers and in suspension (resulting in neurospheres), which were then infected with ZIKV (Br ZIKV or ZIKV MR766) or DENV to assess alterations within neural cells. Large-scale proteomic analyses allowed the comparison not only between viral strains but also regarding the two- and three-dimensional cellular models of neural cells derived from iPSCs, and the effects on their interaction. Altered pathways and biological processes were observed related to cell death, cell cycle dysregulation, and neurogenesis. These results reinforce already published data and provide further information regarding the biological alterations induced by ZIKV and DENV in neural cells.

Neurodevelopment in Children Exposed to Zika in utero: Clinical and Molecular Aspects.

Five years after the identification of Zika virus as a human teratogen, we reviewed the early clinical manifestations, collectively called congenital Zika syndrome (CZS). Children with CZS have a very poor prognosis with extremely low performance in motor, cognitive, and language development domains, and practically all feature severe forms of cerebral palsy. However, these manifestations are the tip of the iceberg, with some children presenting milder forms of deficits. Additionally, neurodevelopment can be in the normal range in the majority of the non-microcephalic children born without brain or eye abnormalities. Vertical transmission and the resulting disruption in development of the brain are much less frequent when maternal infection occurs in the second half of the pregnancy. Experimental studies have alerted to the possibility of other behavioral outcomes both in prenatally infected children and in postnatal and adult infections. Cofactors play a vital role in the development of CZS and involve genetic, environmental, nutritional, and social determinants leading to the asymmetric distribution of cases. Some of these social variables also limit access to multidisciplinary professional treatment.

Microcephaly gene Cenpj regulates axonal growth in cortical neurons through microtubule destabilization.

Neocortex development comprises of a complex series of time- and space-specific processes to generate the typical interconnected six-layered architecture of adult mammals. Axon growth is required for the proper establishment of cortical circuits. Malformations in axonal growth and pathfinding might lead to severe neuropathologies, such as corpus callosum dysgenesis. Cenpj, a microcephaly gene, encodes a scaffold protein that regulates centrosome biogenesis and microtubule stabilization. During corticogenesis, Cenpj regulates progenitor division and neuronal migration. Since microtubule stabilization is crucial for axon extension, we investigated the role of Cenpj in axon growth during cortical development in a mouse model. Through loss- and gain-of-function assays ex vivo and in utero, we quantified callosal axonal length, branching, and growth cone size compared to controls. We observed that silencing Cenpj results in an increased axonal length. Ex vivo, we assessed the number of branches, the area of growth cones and the stability of microtubules. In silenced Cenpj axons, there were more branches, larger growth cone area, and more stable microtubules. Rescue experiments confirmed that neurons present axonal length comparable to controls. Here we propose that Cenpj regulates axon growth by destabilizing microtubules during cortical development. Finally, our findings suggest that Cenpj might be a novel target for axonal regeneration.

Proteomic profiles of Zika virus-infected placentas bearing fetuses with microcephaly.

PURPOSE: Zika virus (ZIKV) transmission to the fetus during pregnancy could enable a collection of severe fetal malformations like microcephaly (MC), termed Congenital Zika Syndrome (CZS). The mechanisms involved in ZIKV transplacental transmission are not fully understood. EXPERIMENTAL DESIGN: Here we aim to identify in placental tissues the deregulated proteins associated with ZIKV-induced MC using label-free proteomics. RESULTS: We found proteins associated with DNA damage and gene expression inhibition up-regulated in infected placentas with no MC fetuses (Z+) compared to the control group (Ctr). Actin filament organization and the immune response were also found deregulated in the Z+ group. In ZIKV-positive placentas bearing fetuses with MC (MC+) was detected an increase in T cell activation, indicating an elevated immune response. A comparison between MC+ and Z+ groups showed a higher abundance of proteins related to endocytosis and autophagy in MC+, suggesting a higher transcytosis of vesicles with ZIKV particles across the maternal-fetal interface. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that higher expression of integrins in MC+ might be associated with high internalization of the virus since these proteins are known as virus receptors. Similarly, an increased immune response in the placenta and higher infiltration of the virus to the fetus could contribute to the neurological malformation of the CZS.

Proteomics of ZIKV infected amniotic fluids of microcephalic fetuses reveals extracellular matrix and immune system dysregulation.

During pregnancy, the vertical transmission of the Zika virus (ZIKV) can cause some disorders in the fetus, called Congenital Zika Syndrome (CZS). Several efforts have been made to understand the molecular mechanism of the CZS. However, the study of CZS pathogenesis through infected human samples is scarce. Therefore, the main goal of this study is to identify and understand the biological processes affected by CZS development. We analyzed by a shotgun proteomic approach the amniotic fluid of pregnant women infected with Zika carrying microcephalic (MC+ ) or non-microcephalic (Z+ ) fetuses compared to Zika negative controls (CTR). Several groups of extracellular matrix (ECM) proteins were dysregulated in the Z+ and MC+ patients, triggering an opposite dysregulation. The down-regulation of the ECM proteins in the MC+ groups can be another factor that contributes to CZS. On the contrary, the Z+ group could be developing a neuroprotective response through ECM proteins up-regulation. The neutrophil degranulation process was disrupted in the Z+ and MC+ groups, where the MC+ groups showed a complex dysregulation. These results suggest that the microcephalic phenotypes are modulated by a down-regulation of the ECM and the impairment of the innate immune system processes.

The Dynamics of Axon Bifurcation Development in the Cerebral Cortex of Typical and Acallosal Mice.

The corpus callosum (CC) is a major interhemispheric commissure of placental mammals. Early steps of CC formation rely on guidance strategies, such as axonal branching and collateralization. Here we analyze the time-course dynamics of axonal bifurcation during typical cortical development or in a CC dysgenesis mouse model. We use Swiss mice as a typical CC mouse model and find that axonal bifurcation rates rise in the cerebral cortex from embryonic day (E)17 and are reduced by postnatal day (P)9. Since callosal neurons populate deep and superficial cortical layers, we compare the axon bifurcation ratio between those neurons by electroporating ex vivo brains at E13 and E15, using eGFP reporter to label the newborn neurons on organotypic slices. Our results suggest that deep layer neurons bifurcate 32% more than superficial ones. To investigate axonal bifurcation in CC dysgenesis, we use BALB/c mice as a spontaneous CC dysgenesis model. BALB/c mice present a typical layer distribution of SATB2 callosal cells, despite the occurrence of callosal anomalies. However, using anterograde DiI tracing, we find that BALB/c mice display increased rates of axonal bifurcations during early and late cortical development in the medial frontal cortex. Midline guidepost cells adjacent to the medial frontal cortex are significant reduced in the CC dysgenesis mouse model. Altogether these data suggest that callosal collateral axonal exuberance is maintained in the absence of midline guidepost signaling and might facilitate aberrant connections in the CC dysgenesis mouse model.

Myelination of Callosal Axons Is Hampered by Early and Late Forelimb Amputation in Rats.

Deafferentation is an important determinant of plastic changes in the CNS, which consists of a loss of inputs from the body periphery or from the CNS itself. Although cortical reorganization has been well documented, white matter plasticity was less explored. Our goal was to investigate microstructural interhemispheric connectivity changes in early and late amputated rats. For that purpose, we employed diffusion-weighted magnetic resonance imaging, as well as Western blotting, immunohistochemistry, and electron microscopy of sections of the white matter tracts to analyze the microstructural changes in the corticospinal tract and in the corpus callosum (CC) sector that contains somatosensory fibers integrating cortical areas representing the forelimbs and compare differences in rats undergoing forelimb amputation as neonates, with those amputated as adults. Results showed that early amputation induced decreased fractional anisotropy values and reduction of total myelin amount in the cerebral peduncle contralateral to the amputation. Both early and late forelimb amputations induced decreased myelination of callosal fibers. While early amputation affected myelination of thinner axons, late amputation disrupted axons of all calibers. Since the CC provides a modulation of inhibition and excitation between the hemispheres, we suggest that the demyelination observed among callosal fibers may misbalance this modulation.

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by Zika virus.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

Network of Interactions between ZIKA Virus Non-Structural Proteins and Human Host Proteins.

The Zika virus (ZIKV) is a mosquito-borne Flavivirus and can be transmitted through an infected mosquito bite or through human-to-human interaction by sexual activity, blood transfusion, breastfeeding, or perinatal exposure. After the 2015-2016 outbreak in Brazil, a strong link between ZIKV infection and microcephaly emerged. ZIKV specifically targets human neural progenitor cells, suggesting that proteins encoded by ZIKV bind and inactivate host cell proteins, leading to microcephaly. Here, we present a systematic annotation of interactions between human proteins and the seven non-structural ZIKV proteins corresponding to a Brazilian isolate. The interaction network was generated by combining tandem-affinity purification followed by mass spectrometry with yeast two-hybrid screens. We identified 150 human proteins, involved in distinct biological processes, as interactors to ZIKV non-structural proteins. Our interacting network is composed of proteins that have been previously associated with microcephaly in human genetic disorders and/or animal models. Further, we show that the protein inhibitor of activated STAT1 (PIAS1) interacts with NS5 and modulates its stability. This study builds on previously published interacting networks of ZIKV and genes related to autosomal recessive primary microcephaly to generate a catalog of human cellular targets of ZIKV proteins implicated in processes related to microcephaly in humans. Collectively, these data can be used as a resource for future characterization of ZIKV infection biology and help create a basis for the discovery of drugs that may disrupt the interaction and reduce the health damage to the fetus.

Dissecting the Toxic Effects of Zika Virus Proteins on Neural Progenitor Cells.

The mechanisms by which Zika virus (ZIKV) disrupts neurogenesis and causes microcephaly are poorly understood. In this issue of Neuron, Li et al. (2019) demonstrate that ZIKV protease NS2B-NS3 heterodimers cleave Septin-2 and lead to cytokinesis defects.

Loss of Cannabinoid CB1 Receptors Induces Cortical Migration Malformations and Increases Seizure Susceptibility.

Neuronal migration is a fundamental process of brain development, and its disruption underlies devastating neurodevelopmental disorders. The transcriptional programs governing this process are relatively well characterized. However, how environmental cues instruct neuronal migration remains poorly understood. Here, we demonstrate that the cannabinoid CB1 receptor is strictly required for appropriate pyramidal neuron migration in the developing cortex. Acute silencing of the CB1 receptor alters neuronal morphology and impairs radial migration. Consequently, CB1 siRNA-electroporated mice display cortical malformations mimicking subcortical band heterotopias and increased seizure susceptibility in adulthood. Importantly, rescuing the CB1 deficiency-induced radial migration arrest by knockdown of the GTPase protein RhoA restored the hyperexcitable neuronal network and seizure susceptibility. Our findings show that CB1 receptor/RhoA signaling regulates pyramidal neuron migration, and that deficient CB1 receptor signaling may contribute to cortical development malformations leading to refractory epilepsy independently of its canonical neuromodulatory role in the adult brain.

Zika virus disrupts molecular fingerprinting of human neurospheres.

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.