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.

The Brazilian Zika virus strain causes birth defects in experimental models.

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.

microRNAs Control Antiviral Immune Response, Cell Death and Chemotaxis Pathways in Human Neuronal Precursor Cells (NPCs) during Zika Virus Infection.

Viral infections have always been a serious burden to public health, increasing morbidity and mortality rates worldwide. Zika virus (ZIKV) is a flavivirus transmitted by the Aedes aegypti vector and the causative agent of severe fetal neuropathogenesis and microcephaly. The virus crosses the placenta and reaches the fetal brain, mainly causing the death of neuronal precursor cells (NPCs), glial inflammation, and subsequent tissue damage. Genetic differences, mainly related to the antiviral immune response and cell death pathways greatly influence the susceptibility to infection. These components are modulated by many factors, including microRNAs (miRNAs). MiRNAs are small noncoding RNAs that regulate post-transcriptionally the overall gene expression, including genes for the neurodevelopment and the formation of neural circuits. In this context, we investigated the pathways and target genes of miRNAs modulated in NPCs infected with ZIKV. We observed downregulation of miR-302b, miR-302c and miR-194, whereas miR-30c was upregulated in ZIKV infected human NPCs in vitro. The analysis of a public dataset of ZIKV-infected human NPCs evidenced 262 upregulated and 3 downregulated genes, of which 142 were the target of the aforementioned miRNAs. Further, we confirmed a correlation between miRNA and target genes affecting pathways related to antiviral immune response, cell death and immune cells chemotaxis, all of which could contribute to the establishment of microcephaly and brain lesions. Here, we suggest that miRNAs target gene expression in infected NPCs, directly contributing to the pathogenesis of fetal microcephaly.

Zika Virus Infection Associated with Autism Spectrum Disorder: A Case Report.

INTRODUCTION: The aim of this case was to investigate the association of the Zika virus infection in utero with the autism spectrum disorder (ASD) as clinical outcome that presented no congenital anomalies. METHODS: ASD was diagnosed in the second year of life by different child neurologists and confirmed by DSM-5 and ASQ. After that, an extensive clinical, epidemiological, and genetic evaluations were performed, with main known ASD causes ruled out. RESULTS: An extensive laboratorial search was done, with normal findings. SNP array identified no pathogenic variants. Normal neuroimaging and EEG findings were also obtained. ZIKV (Zika virus) IgG was positive, while IgM was negative. Other congenital infections were negative. The exome sequencing did not reveal any pathogenic variant in genes related to ASD. CONCLUSION: Accordingly, this report firstly associates ZIKV exposure to ASD.

Modeling neuro-immune interactions during Zika virus infection.

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

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.

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.

Altered proliferation and networks in neural cells derived from idiopathic autistic individuals.

Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells, neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a ß-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1), a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

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.

NS1 codon usage adaptation to humans in pandemic Zika virus.

BACKGROUND: Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES: To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS: The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS: Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

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.

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells.

BACKGROUND: Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta. METHODS: The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained. RESULTS: Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator. CONCLUSIONS: Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

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.

Differentiation of peripheral sensory neurons from iPSCs derived from stem cells from human exfoliated deciduous teeth (SHED).

Peripheral nervous system (PNS) sensory alterations are present in several pathologies and syndromes. The use of induced pluripotent stem cell (iPSC) technology is an important strategy to produce sensory neurons in patients who are accomplished in terms of sensory symptoms. The iPSC technology relies on manipulating signaling pathways to resemble what occurs in vivo, and the iPSCs are known to carry a transcriptional memory after reprogramming, which can affect the produced cell. To this date, protocols described for sensory neuron production start using iPSCs derived from skin fibroblasts, which have the same ontogenetic origin as the central nervous system (CNS). Since it is already known that the cells somehow resemble their origin even after cell reprogramming, PNS cells should be produced from cells derived from the neural crest. This work aimed to establish a protocol to differentiate sensory neurons derived from stem cells from human exfoliated deciduous teeth (SHED) with the same embryonic origin as the PNS. SHED-derived iPSCs were produced and submitted to peripheral sensory neuron (PSN) differentiation. Our protocol used the dual-SMAD inhibition method, followed by neuronal differentiation, using artificial neurotrophic factors and molecules produced by human keratinocytes. We successfully established the first protocol for differentiating neural crest and PNS cells from SHED-derived iPSCs, enabling future studies of PNS pathologies.

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.

Modeling Inflammation on Neurodevelopmental Disorders Using Pluripotent Stem Cells.

Neurodevelopmental disorders (ND) are characterized by an impairment of the nervous system during its development, with a wide variety of phenotypes based on genetic or environmental cues. There are currently several disorders grouped under ND including intellectual disabilities (ID), attention-deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD). Although NDs can have multiple culprits with varied diagnostics, several NDs present an inflammatory component. Taking advantage of induced pluripotent stem cells (iPSC), several disorders were modeled in a dish complementing in vivo data from rodent models or clinical data. Monogenic syndromes displaying ND are more feasible to be modeled using iPSCs also due to the ability to recruit patients and clinical data available. Some of these genetic disorders are Fragile X Syndrome (FXS), Rett Syndrome (RTT), and Down Syndrome (DS). Environmental NDs can be caused by maternal immune activation (MIA), such as the infection with Zika virus during pregnancy known to cause neural damage to the fetus. Our goal in this chapter is to review the advances of using stem cell research in NDs, focusing on the role of neuroinflammation on ASD and environmental NDs studies.

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.

Can Paraplegia by Disruption of the Spinal Cord Tissue Be Reversed? The Signs of a New Perspective.

Central nervous system (CNS) trauma is often related to tissue loss, leading to partial or complete disruption of spinal cord function due to neuronal death. Although generally irreversible, traditional therapeutic efforts, such as physical therapy exercises, are generally recommended, but with a poor or reduced improvement of the microenvironment, which in turn stimulates neuroplasticity and neuroregeneration. Mesenchymal stem cells (MSCs) have paracrine, immunomodulatory, and anti-inflammatory effects. Here we use stem cells to see if they can promote not only physical but also the functional regeneration of neuronal tissue in dogs with CNS traumas. Two dogs, one with chronic spinal cord injury and one with subacute spinal cord injury, underwent infusion of autologous MSCs in association with physiotherapy. The two treatments in combination were able to partially or completely recover the dog's walking movement again. The treatment of MSCs in association with physical therapy improved the microenvironment, which could be evidence of a paradigm shift that the CNS is not capable of functional regeneration after aggressive traumas. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1812-1820, 2020. © 2019 American Association for Anatomy.

Antidepressant Paroxetine Exerts Developmental Neurotoxicity in an iPSC-Derived 3D Human Brain Model.

Selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat depression during pregnancy. Various concerns have been raised about the possible effects of these drugs on fetal development. Current developmental neurotoxicity (DNT) testing conducted in rodents is expensive, time-consuming, and does not necessarily represent human pathophysiology. A human, in vitro testing battery to cover key events of brain development, could potentially overcome these challenges. In this study, we assess the DNT of paroxetine-a widely used SSRI which has shown contradictory evidence regarding effects on human brain development using a versatile, organotypic human induced pluripotent stem cell (iPSC)-derived brain model (BrainSpheres). At therapeutic blood concentrations, which lie between 20 and 60 ng/ml, Paroxetine led to an 80% decrease in the expression of synaptic markers, a 60% decrease in neurite outgrowth and a 40-75% decrease in the overall oligodendrocyte cell population, compared to controls. These results were consistently shown in two different iPSC lines and indicate that relevant therapeutic concentrations of Paroxetine induce brain cell development abnormalities which could lead to adverse effects.