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Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines, however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus–host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

Williams syndrome (WS) is a neurodevelopmental disorder involving hemideletion of as many as 26-28 genes, resulting in a constellation of unique physical, cognitive and behavior phenotypes. The haploinsufficiency effect of each gene has been studied and correlated with phenotype(s) using several models including WS subjects, animal models, and peripheral cell lines. However, links for most of the genes to WS phenotypes remains unclear. Among those genes, general transcription factor 2I (GTF2I) is of particular interest as its haploinsufficiency is possibly associated with hypersociability in WS. Here, we describe studies of atypical WS cases as well as mouse models focusing on GTF2I that support a role for this protein in the neurocognitive and behavioral profiles of WS individuals. We also review collective studies on diverse molecular functions of GTF2I that may provide mechanistic explanation for phenotypes recently reported in our relevant cellular model, namely WS induced pluripotent stem cell (iPSC)-derived neurons. Finally, in light of the progress in gene-manipulating approaches, we suggest their uses in revealing the neural functions of GTF2I in the context of WS.

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host's first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution.

Monge's disease, also known as chronic mountain sickness (CMS), is a disease that potentially threatens more than 140 million highlanders during extended time living at a high altitude (over 2500m). The prevalence of CMS in Andeans is about 15-20%, suggesting that the majority of highlanders (non-CMS) are rather healthy at the high altitude; however, CMS subjects experience severe hypoxemia, erythrocytosis and many neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS, except for phlebotomy. In the current study, we reprogrammed fibroblast cells from both CMS and non-CMS subjects’ skin biopsies into the induced pluripotent stem cells (iPSCs), then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties, but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide, for the first time, evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects, hoping that such studies can pave the way to a better understanding of the neuropathology in CMS.

Anorexia nervosa (AN) is a complex and multifactorial disorder occurring predominantly in women. Despite having the highest mortality among psychiatric conditions, it still lacks robust and effective treatment. Disorders such as AN are most likely syndromes with multiple genetic contributions, however, genome-wide studies have been underpowered to reveal associations with this uncommon illness. Here, we generated induced pluripotent stem cells (iPSCs) from adolescent females with AN and unaffected controls. These iPSCs were differentiated into neural cultures and subjected to extensive transcriptome analysis. Within a small cohort of patients who presented for treatment, we identified a novel gene that appears to contribute to AN pathophysiology, TACR1 (tachykinin 1 receptor). The participation of tachykinins in a variety of biological processes and their interactions with other neurotransmitters suggest novel mechanisms for how a disrupted tachykinin system might contribute to AN symptoms. Although TACR1 has been associated with psychiatric conditions, especially anxiety disorders, we believe this report is its first association with AN. Moreover, our human iPSC approach is a proof-of-concept that AN can be modeled in vitro with a full human genetic complement, and represents a new tool for understanding the elusive molecular and cellular mechanisms underlying the disease.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1, 2, 3, 4, 5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

The cellular and molecular mechanisms of neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The unavailability of live patient neurons for research, however, has represented a major obstacle in the elucidation of the disease etiologies. Recently, the development of induced pluripotent stem cell (iPSC) technology allows for the generation of human neurons from somatic cells of patients. We review ongoing studies using iPSCs as an approach to model neurodevelopmental disorders, the promise and caveats of this technique and its potential for drug screening. The reproducible findings of relevant phenotypes in Rett syndrome iPSC-derived neurons suggest that iPSC technology offers a novel and unique opportunity for the understanding of and the development of therapeutics for other autism spectrum disorders.

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin−1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

Background Formalin fixation (FF) is the standard and most common method for preserving postmortem brain tissue. FF stabilizes cellular morphology and tissue architecture, and can be used to study the distinct morphologic and genetic signatures of different cell types. Although the procedure involved in FF degrades messenger RNA over time, an alternative approach is to use small RNAs (sRNAs) for genetic analysis associated with cell morphology. Although genetic analysis is carried out on fresh or frozen tissue, there is limited availability or impossibility on targeting specific cell populations, respectively. New method The goal of this study is to detect miRNA and other classes of sRNA stored in formalin or in paraffin embedded for over decades. Two brain samples, one formed by a mixed population of cortical and subcortical cells, and one formed by pyramidal shaped cells collected by laser-capture microdissection, were subjected to sRNA sequencing. Results Performing bioinformatics analysis over the sequenced sRNA from brain tissue, we detected several classes of sRNA, such as miRNAs that play key roles in brain neurodevelopmental and maintenance pathways, and hsa-mir-155 expression in neurons. Comparison with existing method: Our method is the first to combine the approaches for: laser-capture of pyramidal neurons from long-term formalin-fixed brain; extract sRNA from laser-captured pyramidal neurons; apply a suite of bioinformatics tools to detect miRNA and other classes of sRNAs on sequenced samples having high levels of RNA degradation. Conclusion This is the first study to show that sRNA can be rescued from laser-captured FF pyramidal neurons.