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In recent years, we are living through different viral pandemics that result in neurological impairments. Given the human-specific nature of brain development, physiology, and pathology, it is imperative to use human models to investigate the neurological impact of viral infections, such as Zika virus and SARS-CoV-2. Brain organoids are powerful in vitro platforms for the analysis of the effects of viral infections on brain development and function, with prospective application to new emerging viral threats. Using brain organoids, it was possible to show that Zika virus infects neural stem cells, disrupting the cell cycle and neurogenesis, leading to microcephaly, a severe reduction of the brain. On the other hand, while it is still under investigation how SARS-CoV-2 might enter and alter the brain, organoid studies are helping to characterize its neurotropism and potential mechanisms of neurovirulence. Here, we describe a method for the infection of human brain organoid cultures with Zika and Sars-CoV-2 viruses that can be used to study neurodevelopmental phenotypes, alteration in neuronal functionality, host–pathogen interactions, as well as for drug testing.

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

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Viral infection in early pregnancy is a major cause of microcephaly. However, how distinct viruses impair human brain development remains poorly understood. Here we use human brain organoids to study the mechanisms underlying microcephaly caused by Zika virus (ZIKV) and herpes simplex virus (HSV-1). We find that both viruses efficiently replicate in brain organoids and attenuate their growth by causing cell death. However, transcriptional profiling reveals that ZIKV and HSV-1 elicit distinct cellular responses and that HSV-1 uniquely impairs neuroepithelial identity. Furthermore, we demonstrate that, although both viruses fail to potently induce the type I interferon system, the organoid defects caused by their infection can be rescued by distinct type I interferons. These phenotypes are not seen in 2D cultures, highlighting the superiority of brain organoids in modeling viral infections. These results uncover virus-specific mechanisms and complex cellular immune defenses associated with virus-induced microcephaly.

Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling.