Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome.

Zika virus (ZIKV) infection was first associated with Central Nervous System (CNS) infections in Brazil in 2015, correlated with an increased number of newborns with microcephaly, which ended up characterizing the Congenital Zika Syndrome (CZS). Here, we investigated the impact of ZIKV infection on the functionality of iPSC-derived astrocytes. Besides, we extrapolated our findings to a Brazilian cohort of 136 CZS children and validated our results using a mouse model. Interestingly, ZIKV infection in neuroprogenitor cells compromises cell migration and causes apoptosis but does not interfere in astrocyte generation. Moreover, infected astrocytes lost their ability to uptake glutamate while expressing more glutamate transporters and secreted higher levels of IL-6. Besides, infected astrocytes secreted factors that impaired neuronal synaptogenesis. Since these biological endophenotypes were already related to Autism Spectrum Disorder (ASD), we extrapolated these results to a cohort of children, now 6-7 years old, and found seven children with ASD diagnosis (5.14 %). Additionally, mice infected by ZIKV revealed autistic-like behaviors, with a significant increase of IL-6 mRNA levels in the brain. Considering these evidence, we inferred that ZIKV infection during pregnancy might lead to synaptogenesis impairment and neuroinflammation, which could increase the risk for ASD.

An update on preclinical pregnancy models of Zika virus infection for drug and vaccine discovery.

INTRODUCTION: Congenital Zika syndrome is caused by Zika virus (ZIKV) infection during pregnancy and can culminate in structural and neurological defects in the fetus, including a spectrum of symptoms such as brain calcifications, hydrocephalus, holoprosencephaly, lissencephaly, ventriculomegaly, and microcephaly. Using animal models to study ZIKV infection during pregnancy represents a critical tool for understanding ZIKV pathophysiology, drug testing, vaccine development, and prevention of vertical transmission. AREAS COVERED: In this review, the authors cover state-of-the-art preclinical pregnancy models of ZIKV infection for drug discovery and vaccine development to prevent vertical transmission. EXPERT OPINION: The discovery of drugs against ZIKV infection represents an urgent necessity, and until now, no effective drug that can prevent the effects of vertical transmission has been tested in humans. Even after six years of the ZIKV outbreak in Brazil, no drugs or vaccines have been approved for use in humans. In part, this failure could be related to the lack of translatability from available preclinical models to humans.

Morphological and biochemical repercussions of Toxoplasma gondii infection in a 3D human brain neurospheres model.

BACKGROUND: Toxoplasmosis is caused by the parasite Toxoplasma gondii that can infect the central nervous system (CNS), promoting neuroinflammation, neuronal loss, neurotransmitter imbalance and behavioral alterations. T. gondii infection is also related to neuropsychiatric disorders such as schizophrenia. The pathogenicity and inflammatory response in rodents are different to the case of humans, compromising the correlation between the behavioral alterations and physiological modifications observed in the disease. In the present work we used BrainSpheres, a 3D CNS model derived from human pluripotent stem cells (iPSC), to investigate the morphological and biochemical repercussions of T. gondii infection in human neural cells. METHODS: We evaluated T. gondii ME49 strain proliferation and cyst formation in both 2D cultured human neural cells and BrainSpheres. Aspects of cell morphology, ultrastructure, viability, gene expression of neural phenotype markers, as well as secretion of inflammatory mediators were evaluated for 2 and 4 weeks post infection in BrainSpheres. RESULTS: T. gondii can infect BrainSpheres, proliferating and inducing cysts formation, neural cell death, alteration in neural gene expression and triggering the release of several inflammatory mediators. CONCLUSIONS: BrainSpheres reproduce many aspects of T. gondii infection in human CNS, constituting a useful model to study the neurotoxicity and neuroinflammation mediated by the parasite. In addition, these data could be important for future studies aiming at better understanding possible correlations between psychiatric disorders and human CNS infection with T. gondii.

Mesenchymal stem cells in dogs with demyelinating leukoencephalitis as an experimental model of multiple sclerosis.

Researchers have used dogs with neurological sequelae caused by distemper as an experimental model for multiple sclerosis, owing to the similarities of the neuropathological changes between distemper virus-induced demyelinating leukoencephalitis and multiple sclerosis in humans. However, little is known about the role of mesenchymal stem cells in treating such clinical conditions. Therefore, we investigated the use of mesenchymal stem cells in four dogs with neurological lesions caused by the distemper virus. During the first year after cellular therapy, the animals did not demonstrate significant changes in their locomotive abilities. However, the intense (Grade V) myoclonus in three animals was reduced to a moderate (Grade IV) level. At one year after the mesenchymal stem cell infusions, three animals regained functional ambulation (Grade I), and all four dogs started to move independently (Grades I and II). In two animals, the myoclonic severity had become mild (Grade III). It was concluded that the use of mesenchymal stem cells could improve the quality of life of dogs with neurological sequelae caused by canine distemper, thus presenting hope for similar positive results in human patients with multiple sclerosis.

The use of iPSC technology for modeling Autism Spectrum Disorders.

Autism Spectrum Disorders (ASDs) are a group of neurodevelopmental disorders that influence social skills, involving communication, interaction, and behavior, usually with repetitive and restrictive manners. Due to the variety of genes involved in ASDs and several possible environmental factors influence, there is still no answer to what really causes syndromic and non-syndromic types of ASDs, usually affecting each individual in a unique way. However, we know that the mechanism underlying ASDs involves brain functioning. The human brain is a complex structure composed of close to 100 billion cells, which is a big challenge to study counting just with post mortem tissue investigation or genetic approaches. Therefore, human induced pluripotent stem cells (iPSC) technology has been used as a tool to produce viable cells for understanding a working brain. Taking advantage of patient-derived stem cells, researchers are now able to generate neurons, glial cells and brain organoids in vitro to model ASDs. In this review we report data from different studies showing how iPSCs have been a critical tool to study the different phenotypes of ASDs.

Developing animal models of Zika virus infection for novel drug discovery.

INTRODUCTION: Just before the Brazilian outbreak, Zika virus was related to a mild infection, causing fever and skin rash. Congenital Zika Syndrome was first described in Brazil, causing microcephaly and malformations in newborns. Three years after the outbreak, the mechanisms of Zika pathogenesis are still not completely elucidated. Moreover, as of today, there is still no approved vaccine that can be administered to the susceptible population. Considering the unmet clinical need, animal models represent an unprecedented opportunity to study Zika pathophysiology and test drugs for the treatment and prevention of vertical transmission. Areas covered: The authors explore the current knowledge about Zika through animal models and advancements in drug discovery by highlighting drugs with the greatest potential to treat ZIKV infection and block vertical transmission. Expert opinion: Some drugs used to treat other infections have been repurposed to treat Zika infection, reducing the cost and time for clinical application. One promising example is Sofosbuvir, which protected mice models against Zika pathogenesis by preventing vertical transmission. Importantly, there is a lack on exploration on the long-term effects of Zika Congenital Syndrome, as well as the possible ways to treat its sequelae.

Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons.

Growing evidences have associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly. Nonetheless, signaling mechanisms that promote the disease outcome are far from being understood, affecting the development of suitable therapeutics. In this study, we applied shotgun mass spectrometry (MS)-based proteomics combined with cell biology approaches to characterize altered molecular pathways on human neuroprogenitor cells (NPC) and neurons derived from induced pluripotent stem cells infected by ZIKV-BR strain, obtained from the 2015 Brazilian outbreak. Furthermore, ZIKV-BR infected NPCs showed unique alteration of pathways involved in neurological diseases, cell death, survival and embryonic development compared to ZIKV-AF, showing a human adaptation of the Brazilian viral strain. Besides, infected neurons differentiated from NPC presented an impairment of neurogenesis and synaptogenesis processes. Taken together, these data explain that CNS developmental arrest observed in Congenital Zika Syndrome is beyond neuronal cell death.

Autism spectrum disorders and disease modeling using stem cells.

Autism spectrum disorders (ASD) represent a variety of disorders characterized as complex lifelong neurodevelopment disabilities, which may affect the ability of communication and socialization, including typical comportments like repetitive and stereotyped behavior. Other comorbidities are usually present, such as echolalia, hypotonia, intellectual disability and difficulties in processing figured speech. Furthermore, some ASD individuals may present certain abilities, such as eidetic memory, outstanding musical or painting talents and special mathematical skills, among others. Considering the variability of the clinical symptoms, one autistic individual can be severely affected in communication while others can speak perfectly, sometimes having a vocabulary above average in early childhood. The same variability can be seen in other clinical symptoms, thus the "spectrum" can vary from severe to mild. Induced pluripotent stem cell technology has been used to model several neurological diseases, including syndromic and non-syndromic autism. We discuss how modeling the central nervous system cells in a dish may help to reach a better understanding of ASD pathology and variability, as well as personalize their treatment.

Modeling the Interplay Between Neurons and Astrocytes in Autism Using Human Induced Pluripotent Stem Cells.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unclear etiology and imprecise genetic causes. The main goal of this work was to investigate neuronal connectivity and the interplay between neurons and astrocytes from individuals with nonsyndromic ASD using induced pluripotent stem cells. METHODS: Induced pluripotent stem cells were derived from a clinically well-characterized cohort of three individuals with nonsyndromic ASD sharing common behaviors and three control subjects, two clones each. We generated mixed neural cultures analyzing synaptogenesis and neuronal activity using a multielectrode array platform. Furthermore, using an enriched astrocyte population, we investigated their role in neuronal maintenance. RESULTS: ASD-derived neurons had a significant decrease in synaptic gene expression and protein levels, glutamate neurotransmitter release, and, consequently, reduced spontaneous firing rate. Based on co-culture experiments, we observed that ASD-derived astrocytes interfered with proper neuronal development. In contrast, control-derived astrocytes rescued the morphological neuronal phenotype and synaptogenesis defects from ASD neuronal co-cultures. Furthermore, after identifying interleukin-6 secretion from astrocytes in individuals with ASD as a possible culprit for neural defects, we were able to increase synaptogenesis by blocking interleukin-6 levels. CONCLUSIONS: Our findings reveal the contribution of astrocytes to neuronal phenotype and confirm previous studies linking interleukin-6 and autism, suggesting potential novel therapeutic pathways for a subtype of individuals with ASD. This is the first report demonstrating that glial dysfunctions could contribute to nonsyndromic autism pathophysiology using induced pluripotent stem cells modeling disease technology.

Zika infection and the development of neurological defects.

Starting with the outbreak in Brazil, Zika virus (ZIKV) infection has been correlated with severe syndromes such as congenital Zika syndrome and Guillain-Barré syndrome. Here, we review the status of Zika virus pathogenesis in the central nervous system (CNS). One of the main concerns about ZIKV exposure during pregnancy is abnormal brain development, which results in microcephaly in newborns. Recent advances in in vitro research show that ZIKV can infect and obliterate cells from the CNS, such as progenitors, neurons, and glial cells. Neural progenitor cells seem to be the main target of the virus, with infection leading to less cell migration, neurogenesis impairment, cell death and, consequently, microcephaly in newborns. The downsizing of the brain can be directly associated with defective development of the cortical layer. In addition, in vivo investigations in mice reveal that ZIKV can cross the placenta and migrate to fetuses, but with a significant neurotropism, which results in brain damage for the pups. Another finding shows that hydrocephaly is an additional consequence of ZIKV infection, being detected during embryonic and fetal development in mouse, as well as after birth in humans. In spite of the advances in ZIKV research in the last year, the mechanisms underlying ZIKV infection in the CNS require further investigation particularly as there are currently no treatments or vaccines against ZIKV infection.

The impact of Zika virus in the brain.

The recent outbreak of ZIKV in Brazil called the attention of the world because the effects of viral infection in the brain under development in fetuses. Consequences of vertical infection comprise brain malformation, especially microcephaly, eye and musculoskeletal abnormalities, among others. In adults, outcomes of infection include meningoencephalitis and Guillain-Barré Syndrome. Recent data specific suggest that neural progenitor cells are the main targets of ZIKV infection, causing massive cellular death and impairment in the neurogenesis process. Here we review the fetal and adult brain damage after ZIKV exposure, exploring models to study the mechanisms underlying the pathways related to microcephaly and cell death.

Macroscopic and microscopic analysis of 2 embryos and 1 foetus derived from a sheep (Ovis aries) without breed / Análise macroscópica e microscópica de 2 embriões e 1 feto derivados de ovelha (Ovis aries) sem raça

The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.(AU)

O interesse em Embriologia, a ciência do desenvolvimento de um zigoto em um feto completamente desenvolvido, tem aumentado consideravelmente nos últimos anos devido a uma série de estudos envolvendo células-tronco pluripotentes embrionárias e induzidas. Além disso, o desenvolvimento de técnicas como a clonagem tem ajudado a compreender os eventos críticos que ocorrem durante o desenvolvimento embrionário. Neste estudo, descrevemos a morfologia de dois embriões de ovinos e um feto utilizando técnicas macroscópicas e microscópicas. Obtivemos ovelhas sem raça definida com 24, 32 e 50 dias de gestação (estimado pelo método de Crown-Rump, CR). Os conceptos foram mensurados, pesados e caracterizados a olho nu. Macroscopicamente, observamos o desenvolvimento dos embriões E1 (24 dias), apresentando globo ocular sem pigmentação de retina e broto do membro torácico e pélvico. Já o E2 (32 dias), apresentava globo ocular com pigmentação na retina e os membros torácicos e pélvicos mais desenvolvidos. O F1 apresentou olhos cobertos com uma membrana e membros torácicos e pélvicos mais desenvolvidos. Enquanto isso, microscopicamente observamos no E1 somitos, ventrículo, átrio e cavidade oral ainda em desenvolvimento. Porém, no F1 já era possível observar ossificação da coluna espinhal, coração com estruturas mais complexas, como ventrículo, átrio, septo interventricular e saco pericárdio. Além disso, na cavidade oral observamos a formação da língua. Este trabalho fornece informações precisas e detalhadas sobre as características morfológicas dos principais órgãos dos sistemas (nervoso, circulatório, respiratório, digestivo e urinário) em cada fase embrionária e fetal analisadas.(AU)

Mice embryology: a microscopic overview.

In this work, we studied the embryology of mice of 12, 14, and 18 days of gestation by gross observation, light microscopy, and scanning electron microscopy. Grossly, the embryos of 12 days were observed in C-shaped region of the brain, eye pigmentation of the retina, first, second, and third pharyngeal arches gill pit nasal region on the fourth ventricle brain, cervical curvature, heart, liver, limb bud thoracic, spinal cord, tail, umbilical cord, and place of the mesonephric ridge. Microscopically, the liver, cardiovascular system and spinal cord were observed. In the embryo of 14 days, we observed structures that make up the liver and heart. At 18 days of gestation fetuses, it was noted the presence of eyes, mouth, and nose in the cephalic region, chest and pelvic region with the presence of well-developed limbs, umbilical cord, and placenta. Scanning electron microscopy in 18 days of gestation fetuses evidenced head, eyes closed eyelids, nose, vibrissae, forelimb, heart, lung, kidney, liver, small bowel, diaphragm, and part of the spine. The results obtained in this work describe the internal and external morphology of mice, provided by an integration of techniques and review of the morphological knowledge of the embryonic development of this species, as this animal is of great importance to scientific studies.