ABSTRACT
Elevated interleukin (IL-)6 levels during prenatal development have been linked to increased risk for neurodevelopmental disorders (NDD) in the offspring, but the mechanism remains unclear. Human-induced pluripotent stem cell (hiPSC) models offer a valuable tool to study the effects of IL-6 on features relevant for human neurodevelopment in vitro. We previously reported that hiPSC-derived microglia-like cells (MGLs) respond to IL-6, but neural progenitor cells (NPCs) in monoculture do not. Therefore, we investigated whether co-culturing hiPSC-derived MGLs with NPCs would trigger a cellular response to IL-6 stimulation via secreted factors from the MGLs. Using N=4 donor lines without psychiatric diagnosis, we first confirmed that NPCs can respond to IL-6 through trans-signalling when recombinant IL-6Ra is present, and that this response is dose-dependent. MGLs secreted soluble IL-6R, but at lower levels than found in vivo and below that needed to activate trans-signalling in NPCs. Whilst transcriptomic and secretome analysis confirmed that MGLs undergo substantial transcriptomic changes after IL-6 exposure and subsequently secrete a cytokine milieu, NPCs in co-culture with MGLs exhibited a minimal transcriptional response. Furthermore, there were no significant cell fate-acquisition changes when differentiated into post-mitotic cultures, nor alterations in synaptic densities in mature neurons. These findings highlight the need to investigate if trans-IL-6 signalling to NPCs is a relevant disease mechanism linking prenatal IL-6 exposure to increased risk for psychiatric disorders. Moreover, our findings underscore the importance of establishing more complex in vitro human models with diverse cell types, which may show cell-specific responses to microglia-released cytokines to fully understand how IL-6 exposure may influence human neurodevelopment.
ABSTRACT
Maternal immune dysregulation is a prenatal risk factor for autism spectrum disorder (ASD). Importantly, a clinically relevant connection exists between inflammation and metabolic stress that can result in aberrant cytokine signaling and autoimmunity. In this study we examined the potential for maternal autoantibodies (aAbs) to disrupt metabolic signaling and induce neuroanatomical changes in the brains of exposed offspring. To accomplish this, we developed a model of maternal aAb exposure in rats based on the clinical phenomenon of maternal autoantibody-related ASD (MAR-ASD). Following confirmation of aAb production in rat dams and antigen-specific immunoglobulin G (IgG) transfer to offspring, we assessed offspring behavior and brain structure longitudinally. MAR-ASD rat offspring displayed a reduction in pup ultrasonic vocalizations and a pronounced deficit in social play behavior when allowed to freely interact with a novel partner. Additionally, longitudinal in vivo structural magnetic resonance imaging (sMRI) at postnatal day 30 (PND30) and PND70, conducted in a separate cohort of animals, revealed sex-specific differences in total and regional brain volume. Treatment-specific effects by region appeared to converge on midbrain and cerebellar structures in MAR-ASD offspring. Simultaneously, in vivo 1H magnetic resonance spectroscopy (1H-MRS) data were collected to examine brain metabolite levels in the medial prefrontal cortex. Results showed that MAR-ASD offspring displayed decreased levels of choline-containing compounds and glutathione, accompanied by increased taurine compared to control animals. Overall, we found that rats exposed to MAR-ASD aAbs present with alterations in behavior, brain structure, and neurometabolites; reminiscent of findings observed in clinical ASD.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Prenatal Exposure Delayed Effects , Humans , Male , Pregnancy , Female , Rats , Animals , Autistic Disorder/metabolism , Autism Spectrum Disorder/metabolism , Autoantibodies , Prenatal Exposure Delayed Effects/metabolism , Brain/metabolism , Maternal ExposureABSTRACT
BACKGROUND: Prenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. RESULTS: We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/ß, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3 h and 24 h. CONCLUSION: Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.
Subject(s)
Interleukin-6 , Microglia , Neural Stem Cells , Receptors, Interleukin-6 , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Interferon Regulatory Factors/metabolism , Interleukin-6/adverse effects , Interleukin-6/metabolism , Interleukin-6/pharmacology , Microglia/drug effects , Microglia/metabolism , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Receptors, Interleukin-6/metabolismABSTRACT
Maternal immune activation (MIA) during prenatal development is an environmental risk factor for psychiatric disorders including schizophrenia (SZ). Converging lines of evidence from human and animal model studies suggest that elevated cytokine levels in the maternal and fetal compartments are an important indication of the mechanisms driving this association. However, there is variability in susceptibility to the psychiatric risk conferred by MIA, likely influenced by genetic factors. How MIA interacts with a genetic profile susceptible to SZ is challenging to test in animal models. To address this gap, we examined whether differential gene expression responses occur in forebrain-lineage neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) generated from three individuals with a diagnosis of schizophrenia and three healthy controls. Following acute (24 h) treatment with either interferon-gamma (IFNγ; 25 ng/µl) or interleukin (IL)-1ß (10 ng/µl), we identified, by RNA sequencing, 3380 differentially expressed genes (DEGs) in the IFNγ-treated control lines (compared to untreated controls), and 1980 DEGs in IFNγ-treated SZ lines (compared to untreated SZ lines). Out of 4137 genes that responded significantly to IFNγ across all lines, 1223 were common to both SZ and control lines. The 2914 genes that appeared to respond differentially to IFNγ treatment in SZ lines were subjected to a further test of significance (multiple testing correction applied to the interaction effect between IFNγ treatment and SZ diagnosis), yielding 359 genes that passed the significance threshold. There were no differentially expressed genes in the IL-1ß-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1ß impacts immune function and neuronal differentiation. Overall, our data suggest that a) SZ NPCs show an attenuated transcriptional response to IFNγ treatment compared to controls; b) Due to low IL-1ß receptor expression in NPCs, NPC cultures appear to be less responsive to IL-1ß than IFNγ; and c) the genes differentially regulated in SZ lines - in the face of a cytokine challenge - are primarily associated with mitochondrial, "loss-of-function", pre- and post-synaptic gene sets. Our findings particularly highlight the role of early synaptic development in the association between maternal immune activation and schizophrenia risk.
Subject(s)
Induced Pluripotent Stem Cells , Neural Stem Cells , Schizophrenia , Animals , Cytokines/metabolism , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Pregnancy , Prosencephalon , Schizophrenia/genetics , Schizophrenia/metabolismABSTRACT
Neurogenesis, the process in which new neurons are generated, occurs throughout life in the mammalian hippocampus. Decreased adult hippocampal neurogenesis (AHN) is a common feature across psychiatric disorders, including schizophrenia, depression- and anxiety-related behaviours, and is highly regulated by environmental influences. Epidemiological studies have consistently implicated maternal immune activation (MIA) during neurodevelopment as a risk factor for psychiatric disorders in adulthood. The extent to which the reduction of hippocampal neurogenesis in adulthood may be driven by early life exposures, such as MIA, is however unclear. We therefore reviewed the literature for evidence of the involvement of MIA in disrupting AHN. Consistent with our hypothesis, data from both in vivo murine and in vitro human models of AHN provide evidence for key roles of specific cytokines induced by MIA in the foetal brain in disrupting hippocampal neural progenitor cell proliferation and differentiation early in development. The precise molecular mechanisms however remain unclear. Nonetheless, these data suggest a potential latent vulnerability mechanism, whereby MIA primes dysfunction in the unique hippocampal pool of neural stem/progenitor cells. This renders offspring potentially more susceptible to additional environmental exposures later in life, such as chronic stress, resulting in the unmasking of psychopathology. We highlight the need for studies to test this hypothesis using validated animal models of MIA, but also to test the relevance of such data for human pathology at a molecular basis through the use of patient-derived induced pluripotent stem cells (hiPSC) differentiated into hippocampal progenitor cells.
Subject(s)
Hippocampus , Neurogenesis , Adult , Animals , Anxiety Disorders , Cell Differentiation , Humans , Mice , NeuronsABSTRACT
Infectious or noninfectious maternal immune activation (MIA) is an environmental risk factor for psychiatric and neurological disorders with neurodevelopmental etiologies. Whilst there is increasing evidence for significant health consequences, the effects of MIA on the offspring appear to be variable. Here, we aimed to identify and characterize subgroups of isogenic mouse offspring exposed to identical MIA, which was induced in C57BL6/N mice by administration of the viral mimetic, poly(I:C), on gestation day 12. Cluster analysis of behavioral data obtained from a first cohort containing >150 MIA and control offspring revealed that MIA offspring could be stratified into distinct subgroups that were characterized by the presence or absence of multiple behavioral dysfunctions. The two subgroups also differed in terms of their transcriptional profiles in cortical and subcortical brain regions and brain networks of structural covariance, as measured by ex vivo structural magnetic resonance imaging (MRI). In a second, independent cohort containing 50 MIA and control offspring, we identified a subgroup of MIA offspring that displayed elevated peripheral production of innate inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, in adulthood. This subgroup also showed significant impairments in social approach behavior and sensorimotor gating, whereas MIA offspring with a low inflammatory cytokine status did not. Taken together, our results highlight the existence of subgroups of MIA-exposed offspring that show dissociable behavioral, transcriptional, brain network, and immunological profiles even under conditions of genetic homogeneity. These data have relevance for advancing our understanding of the variable neurodevelopmental effects induced by MIA and for biomarker-guided approaches in preclinical psychiatric research.
Subject(s)
Behavior, Animal , Prenatal Exposure Delayed Effects , Animals , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Pregnancy , Social BehaviorABSTRACT
Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.
Subject(s)
Induced Pluripotent Stem Cells , Neurodevelopmental Disorders , Humans , Induced Pluripotent Stem Cells/metabolism , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/metabolism , Neurons/metabolism , PhenotypeABSTRACT
Microglia, the resident tissue macrophages of the brain, are increasingly implicated in the pathophysiology of psychiatric disorders with a neurodevelopmental origin, including schizophrenia. To date, however, our understanding of the potential role for these cells in schizophrenia has been informed by studies of aged post-mortem samples, low resolution in vivo neuroimaging and rodent models. Whilst these have provided important insights, including signs of the heterogeneous nature of microglia, we currently lack a validated human in vitro system to characterize microglia in the context of brain health and disease during neurodevelopment. Primarily, this reflects a lack of access to human primary tissue during developmental stages. In this review, we first describe microglia, including their ontogeny and heterogeneity and consider their role in brain development. We then provide an evaluation of the potential for differentiating microglia from human induced pluripotent stem cells (hiPSCs) as a robust in vitro human model system to study these cells. We find the majority of protocols for hiPSC-derived microglia generate cells characteristically similar to foetal stage microglia when exposed to neuronal environment-like cues. This may represent a robust and relevant model for the study of cellular and molecular mechanisms in schizophrenia. Each protocol however, provides unique benefits as well as shortcomings, highlighting the need for context-dependent protocol choice and cross-lab collaboration and communication to identify the most robust and translatable microglia model.
ABSTRACT
Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABAB receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABAB receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties.