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.

Assessment of vulnerability dimensions considering Family History and environmental interplay in Autism Spectrum Disorder.

BACKGROUND: Despite previous studies have recently shown Autism Spectrum Disorders (ASD) as having a strong genetics background, over a minimum environmental background, no study up to date has investigated the interplay between genetics and environment. METHODS: We have collected data regarding Family History (FH) and Environmental Factors (EF) from 2,141 individuals with ASD and their caretakers throughout Brazil, based on an online questionnaire. Most of the ASD individuals were males (81%) and the average age was 02 years minimum for males and females, and the maximum age was 41 years for males and 54 for females. People from all states in Brazil have answered the questionnaire. Genetic inheritance was obtained based on the declared FH of Psychiatric and Neurological diagnosis. As for EF, exposure to risk factors during pregnancy was considered, like infections, diabetes, drugs/chemicals exposure, socioeconomic, and psychological factors. Respondents were invited to answer the questionnaire in lectures given throughout Brazil, and by the social networks of the NGO "The Tooth Fairy Project". A Multiple Correspondence Analysis (MCA) was conducted to search vulnerability dimensions, and a Cluster Analysis was conducted to classify and identify the subgroups. RESULTS: Regarding EF, social and psychological exposures contributed to the first two dimensions. Concerning FH, the first dimension represented psychiatric FH, while the second represented neurological FH. When analyzed together, EF and FH contributed to two new dimensions: 1. psychiatric FH, and 2. a psychosocial component. Using Cluster Analysis, it was not possible to isolate subgroups by genetic vulnerability or environmental exposure. Instead, a gradient of psychiatric FH with similar contributions of EF was observed. CONCLUSION: In this study, it was not possible to isolate groups of patients that correspond to only one component, but rather a continuum with different compositions of genetic and environmental interplay.

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.

Drug repositioning for psychiatric and neurological disorders through a network medicine approach.

Psychiatric and neurological disorders (PNDs) affect millions worldwide and only a few drugs achieve complete therapeutic success in the treatment of these disorders. Due to the high cost of developing novel drugs, drug repositioning represents a promising alternative method of treatment. In this manuscript, we used a network medicine approach to investigate the molecular characteristics of PNDs and identify novel drug candidates for repositioning. Using IBM Watson for Drug Discovery, a powerful machine learning text-mining application, we built knowledge networks containing connections between PNDs and genes or drugs mentioned in the scientific literature published in the past 50 years. This approach revealed several drugs that target key PND-related genes, which have never been used to treat these disorders to date. We validate our framework by detecting drugs that have been undergoing clinical trial for treating some of the PNDs, but have no published results in their support. Our data provides comprehensive insights into the molecular pathology of PNDs and offers promising drug repositioning candidates for follow-up trials.

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.

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.

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.

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.

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.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury.

PURPOSE:: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. METHODS:: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. RESULTS:: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. CONCLUSION:: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury

Abstract Purpose: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. Methods: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. Results: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. Conclusion: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

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)

Aquapuncture Using Stem Cell Therapy to Treat Mdx Mice.

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to absence or decrease of dystrophin protein generating progressive muscle degeneration. Cell therapy using mesenchymal stem cell (MSC) has been described as a treatment to DMD. In this work, MSC derived from deciduous teeth, called stem cells from human exfoliated deciduous teeth (SHED), were injected in acupoint as an alternative therapy to minimize muscle degeneration in twenty-two mdx mice. The treatment occurred three times with intervals of 21 days, and animals were analyzed four times: seven days prior treatment (T-7); 10 days after first treatment (T10); 10 days after second treatment (T31); and 10 days after third treatment (T52). Animals were evaluated by wire test for estimate strength and blood was collected to perform a creatinine phosphokinase analysis. After euthanasia, cranial tibial muscles were collected and submitted to histological and immunohistochemistry analyses. Treated groups presented improvement of strength and reduced creatinine phosphokinase levels. Also, a slight dystrophin increase was observed in tibial cranial muscle when aquapuncture was associated SHED. All therapies have minimized muscle degeneration, but the association of aquapuncture with SHED appears to have better effect, reducing muscle damage, suggesting a therapeutic value.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results / Sucesso no transplante de células tronco mesenquimais em ovinos com osteonecrose induzida da cabeça do fêmur: resultados preliminares

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

OBJETIVO: Verificar os efeitos das células-tronco mesenquimais da medula óssea de ovinos e da polpa dentária imatura humana em ovinos com osteonecrose induzida, da cabeça do fêmur. MÉTODOS: Oito ovelhas foram distribuídas em três grupos experimentais. O primeiro grupo foi composto por quatro animais com osteonecrose da cabeça do fêmur induzida por etanol através da descompressão central, que não receberam nenhum tratamento. O segundo e o terceiro grupo, cada um composto por dois animais, receberam transplante heterólogo de células tronco mesenquimais de ovinos e polpa dentária imatura humana seis semanas após a indução da osteonecrose da cabeça do fêmur, respectivamente. Em ambos os grupos experimentais as células foram transplantadas sem o uso de drogas imunossupressoras. RESULTADOS: Os achados demonstram que as células-tronco mesenquimais injetadas na cabeça do fêmur se encontravam viáveis após o transplante no novo sítio e proliferaram em pouco tempo. Os dados histológicos sugerem que a regeneração óssea nos animais transplantados com polpa dentária imatura humana foi mais rápida do que nos animais submetidos somente a descompressão central. CONCLUSÃO: O transplante de células tronco mesenquimais na osteonecrose da cabeça do fêmur induzida em ovinos através da técnica de descompressão central é um procedimento seguro, e aparentemente favorece a regeneração óssea de tecidos lesados.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results.

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.