Patricia P. Garcez

Zika virus impairs growth in human neurospheres and brain organoids.

Zika virus tested in human brain organoids The pernicious and resilient Aedes mosquito is rapidly spreading Zika virus (ZIKV) through the Americas. ZIKV infection mostly causes mild disease, but in some patients, nervous system involvement is indicated. A particular worry is an observed correlation between infection of mothers in the first trimester of pregnancy and microcephaly in newborns. Garcez et al. tested the effects of ZIKV compared with dengue virus infection on human neural stem cells grown as organoids. ZIKV targeted the human brain cells, reduced their size and viability in vitro, and caused programmed cell death responses. Science, this issue p. 816 Zika virus infection in cell culture models damages human neural stem cells to limit growth and cause cell death. 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.

microRNA-9 regulates axon extension and branching by targeting Map1b in mouse cortical neurons

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.

Ephrin‐A5 acts as a repulsive cue for migrating cortical interneurons

Cortical interneurons are born in the germinative zones of the ganglionic eminences in the subpallium, and migrate tangentially in spatially and temporally well‐defined corridors into the neocortex. Because ephrin‐A5 is expressed in the ventricular zone (VZ) of the ganglionic eminences at these developmental stages, we examined the possible effects of this molecule on interneuron migration. Double‐immunocytochemistry of dissociated neurons from the medial ganglionic eminences (MGE) revealed that calbindin‐positive cells express the EphA4‐receptor. In situ, EphA4 is strongly expressed in the subventricular zone of the ganglionic eminences. Using different in vitro assays, we found that ephrin‐A5 acts as a repellent cue for MGE neurons. We then examined interneuron migration in slice overlay experiments, where MGE‐derived explants from enhanced green fluorescent protein‐expressing transgenic mice were homotopically grafted into host slices from wild‐type littermate embryos. In these in vitro preparations, interneurons recapitulated in vivo cell migration in several respects. However, interneurons in brain slices also migrated in the VZ of the ganglionic eminences, a region that is strictly avoided in vivo. In situ hybridizations revealed that ephrin‐A5 became downregulated in the VZ in vitro. When recombinant ephrin‐A5‐Fc was added to the slices, it preferentially bound to the VZ, and migrating MGE neurons avoided the VZ as in vivo. The restoration of the normal migration pathway in slices required ephrin‐A5 clustering and signalling of Src family kinases. Together, these experiments suggest that ephrin‐A5 acts as an inhibitory flank that contributes to define the pathway of migrating interneurons.

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

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.

The CB1 Cannabinoid Receptor Drives Corticospinal Motor Neuron Differentiation through the Ctip2/Satb2 Transcriptional Regulation Axis

The generation and specification of pyramidal neuron subpopulations during development relies on a complex network of transcription factors. The CB1 cannabinoid receptor is the major molecular target of endocannabinoids and marijuana active compounds. This receptor has been shown to influence neural progenitor proliferation and axonal growth, but its involvement in neuronal differentiation and the functional impact in the adulthood caused by altering its signaling during brain development are not known. Here we show that the CB1 receptor, by preventing Satb2 (special AT-rich binding protein 2)-mediated repression, increased Ctip2 (COUP-TF interacting protein 2) promoter activity, and Ctip2-positive neuron generation. Unbalanced neurogenic fate determination found in complete CB1−/− mice and in glutamatergic neuron-specific Nex–CB1−/− mice induced overt alterations in corticospinal motor neuron generation and subcerebral connectivity, thereby resulting in an impairment of skilled motor function in adult mice. Likewise, genetic deletion of CB1 receptors in Thy1–YFP–H mice elicited alterations in corticospinal tract development. Altogether, these data demonstrate that the CB1 receptor contributes to the generation of deep-layer cortical neurons by coupling endocannabinoid signals from the neurogenic niche to the intrinsic proneurogenic Ctip2/Satb2 axis, thus influencing appropriate subcerebral projection neuron specification and corticospinal motor function in the adulthood.

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.

Axons of callosal neurons bifurcate transiently at the white matter before consolidating an interhemispheric projection

The main alternative output routes of adult cortical axons are the internal capsule and the corpus callosum. How do callosal axons choose their trajectories? We hypothesized that bifurcation followed by elimination of one branch is a developmental strategy for accomplishing this aim. Using embryonic and postnatal mice, we labelled cortical projecting neurons and quantified their axonal bifurcations in correlation with the mediolateral position of their somata. Bifurcating axons were numerous in the younger brains but declined during further development. Most bifurcating axons pertained to neurons located in the dorsolateral cortex. Moreover, callosal neurons bifurcate more often than subcortically projecting cells. We then quantified bifurcations formed by dissociated green fluorescent cells plated onto cortical slices. Cells grown over dorsolateral cortex bifurcated more often than those grown over medial cortex, irrespective of their positional origin in the donor. Removal of intermediate targets from the slices prevented bifurcation. We concluded that transient bifurcation and elimination of the lateral branch is a strategy employed by developing callosal axons in search of their targets. As cell body position and intermediate targets determine axon behaviour, we suggest that bifurcations are regulated by cues expressed in the environment.

Chloroquine inhibits Zika Virus infection in different cellular models

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. In adults, cases of Guillain-Barré syndrome and meningoencephalitis associated with ZIKV infection have been reported, and no specific therapy is available so far. There is urgency for the discovery of antiviral agents capable of inhibiting viral replication and its deleterious effects. Chloroquine is widely administered 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, human brain microvascular endothelial, and neural stem cells. We demonstrated in vitro that chloroquine reduces the number of ZIKV-infected cells, virus production and cell death promoted by ZIKV infection without cytotoxic effects. Our results suggest that chloroquine is a promising candidate for ZIKV clinical trials, since it is already approved for clinical use and can be safely administered to pregnant woman.

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

Abstract 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.

Temporal and spatial regulation of interneuron distribution in the developing cerebral cortex—an in vitro study

GABAergic interneurons are local circuit cells that control the excitatory balance in most regions of the nervous system, particularly the cerebral cortex. Because they are integrated in every cortical module, we posed the question whether interneuronal precursors would display some topographic specificity between their origin at the ventral telencephalon and their cortical location after migration. If this was true, GABAergic cells would have to be provided with intrinsic features that would make them able to perform specific functional roles in each specific module. On the other hand, if no topography was found, one would conclude that inhibitory precursors would be functionally naive, being able to integrate anywhere in the cortex, with equal capacity of performing their functions. This issue was approached by use of organotypic cultures of wild mice embryonic slices, into which fragments of the ganglionic eminence taken from enhanced green fluorescent protein (eGFP) mice were implanted, observing the topographic location of both the implant and its destination. Despite the existence of different genetic domains in the ventricular zone of the medial ganglionic eminences (MGE), we found that cells originating in different regions spread in vitro all over the mediolateral axis of the developing cortical wall, independently of their sites of origin. Results favor the hypothesis that GABAergic precursors are functionally naive, integrating into modules irrespective of which cortical area they belong to.