Δ⁹-tetrahydrocannabinol and 2-AG decreases neurite outgrowth and differentially affects ERK1/2 and Akt signaling in hiPSC-derived cortical neurons

Endocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the decrease in neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in developing human neurons

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (including scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Clusterin Is Required for β-Amyloid Toxicity in Human iPSC-Derived Neurons

Our understanding of the molecular processes underlying Alzheimer’s disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aβ25-35 peptides and Aβ1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs i. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (incl. scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Interferon-γ exposure of human iPSC-derived neurons alters major histocompatibility complex I and synapsin protein expression

Human epidemiological data links maternal immune activation (MIA) during gestation with increased risk for psychiatric disorders with a putative neurodevelopmental origin, including schizophrenia and autism. Animal models of MIA provide evidence for this association and suggest that inflammatory cytokines represent one critical link between maternal infection and any potential impact on offspring brain and behavior development. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. It is also unclear how specific cytokines may impact the development of specific cell types. Using a human cellular model, we recently demonstrated that acute exposure to interferon-γ (IFNγ) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFNγ can impact the development of immature glutamatergic neurons using an induced neuronal cellular system. We find that acute exposure to IFNγ activates a signal transducer and activator of transcription 1 (STAT1)-pathway in immature neurons, and results in significantly increased major histocompatibility complex I (MHCI) expression at the mRNA and protein level. Furthermore, acute IFNγ exposure decreased synapsin I/II protein in neurons but did not affect the expression of synaptic genes. Interestingly, complement component 4A (C4A) gene expression was significantly increased following acute IFNγ exposure. This study builds on our previous work by showing that IFNγ-mediated disruption of relevant synaptic proteins can occur at early stages of neuronal development, potentially contributing to neurodevelopmental disorder phenotypes

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory, and anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behavior. This study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomized, estradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the 3(rd) ventricle 30 minutes prior to euthanasia. Proteins from SKF-treated hypothalami were pulled-down with GST-tagged mouse PR-A or PR-B and the interactomes were analyzed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed estradiol-induced PR-expressing hypothalamic-like neurons derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin-1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised of metabolic regulators involved in glucose metabolism, lipid synthesis, and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic, and mental health disorders in women

Attenuated transcriptional response to pro-inflammatory cytokines in schizophrenia hiPSC-derived neural progenitor cells

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)-1β (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-1β-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1β 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-1β receptor expression in NPCs, NPC cultures appear to be less responsive to IL-1β 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