Morphological and transcriptomic analyses of stem cell-derived cortical neurons reveal mechanisms underlying synaptic dysfunction in schizophrenia.

BACKGROUND: Postmortem studies in schizophrenia consistently show reduced dendritic spines in the cerebral cortex but the mechanistic underpinnings of these deficits remain unknown. Recent genome-wide association studies and exome sequencing investigations implicate synaptic genes and processes in the disease biology of schizophrenia. METHODS: We generated human cortical pyramidal neurons by differentiating iPSCs of seven schizophrenia patients and seven healthy subjects, quantified dendritic spines and synapses in different cortical neuron subtypes, and carried out transcriptomic studies to identify differentially regulated genes and aberrant cellular processes in schizophrenia. RESULTS: Cortical neurons expressing layer III marker CUX1, but not those expressing layer V marker CTIP2, showed significant reduction in dendritic spine density in schizophrenia, mirroring findings in postmortem studies. Transcriptomic experiments in iPSC-derived cortical neurons showed that differentially expressed genes in schizophrenia were enriched for genes implicated in schizophrenia in genome-wide association and exome sequencing studies. Moreover, most of the differentially expressed genes implicated in schizophrenia genetic studies had lower expression levels in schizophrenia cortical neurons. Network analysis of differentially expressed genes led to identification of NRXN3 as a hub gene, and follow-up experiments showed specific reduction of the NRXN3 204 isoform in schizophrenia neurons. Furthermore, overexpression of the NRXN3 204 isoform in schizophrenia neurons rescued the spine and synapse deficits in the cortical neurons while knockdown of NRXN3 204 in healthy neurons phenocopied spine and synapse deficits seen in schizophrenia cortical neurons. The antipsychotic clozapine increased expression of the NRXN3 204 isoform in schizophrenia cortical neurons and rescued the spine and synapse density deficits. CONCLUSIONS: Taken together, our findings in iPSC-derived cortical neurons recapitulate cell type-specific findings in postmortem studies in schizophrenia and have led to the identification of a specific isoform of NRXN3 that modulates synaptic deficits in schizophrenia neurons.

Comparative Transcriptomic Analysis of Cerebral Organoids and Cortical Neuron Cultures Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) can be differentiated along various neuronal lineages to generate two-dimensional neuronal cultures as well as three-dimensional brain organoids. Such iPSC-derived cellular models are being utilized to study the basic biology of human neuronal function and to interrogate the molecular underpinnings of disease biology. The different cellular models generated from iPSCs have varying properties in terms of the diversity and organization of the cells as well as the cellular functions that are present. To understand transcriptomic differences in iPSC-derived monolayer neuronal cultures and three-dimensional brain organoids, we differentiated eight human iPSC lines from healthy control subjects to generate cerebral organoids and cortical neuron monolayer cultures from the same set of iPSC lines. We undertook RNA-seq experiments in these model systems and analyzed the gene expression data to identify genes that are differentially expressed in cerebral organoids and two-dimensional cortical neuron cultures. In cerebral organoids, gene ontology analysis showed enrichment of genes involved in tissue development, response to stimuli, and the interferon-γ pathway, while two-dimensional cortical neuron cultures showed enrichment of genes involved in nervous system development and neurogenesis. We also undertook comparative analysis of these gene expression profiles with transcriptomic data from the human fetal prefrontal cortex (PFC). This analysis showed greater overlap of the fetal PFC transcriptome with cerebral organoid gene expression profiles compared to monolayer cortical neuron culture profiles. Our studies delineate the transcriptomic differences between cortical neuron monolayer cultures and three-dimensional cerebral organoids and can help inform the appropriate use of these model systems to address specific scientific questions.

Synaptic deficits in iPSC-derived cortical interneurons in schizophrenia are mediated by NLGN2 and rescued by N-acetylcysteine.

Human postmortem studies suggest a major role for abnormalities in GABAergic interneurons in the prefrontal cortex in schizophrenia. Cortical interneurons differentiated from induced pluripotent stem cells (iPSCs) of schizophrenia subjects showed significantly lower levels of glutamate decarboxylase 67 (GAD67), replicating findings from multiple postmortem studies, as well as reduced levels of synaptic proteins gehpyrin and NLGN2. Co-cultures of the interneurons with excitatory cortical pyramidal neurons from schizophrenia iPSCs showed reduced synaptic puncta density and lower action potential frequency. NLGN2 overexpression in schizophrenia neurons rescued synaptic puncta deficits while NLGN2 knockdown in healthy neurons resulted in reduced synaptic puncta density. Schizophrenia interneurons also had significantly smaller nuclear area, suggesting an innate oxidative stressed state. The antioxidant N-acetylcysteine increased the nuclear area in schizophrenia interneurons, increased NLGN2 expression and rescued synaptic deficits. These results implicate specific deficiencies in the synaptic machinery in cortical interneurons as critical regulators of synaptic connections in schizophrenia and point to a nexus between oxidative stress and NLGN2 expression in mediating synaptic deficits in schizophrenia.

Increased synapse elimination by microglia in schizophrenia patient-derived models of synaptic pruning.

Synapse density is reduced in postmortem cortical tissue from schizophrenia patients, which is suggestive of increased synapse elimination. Using a reprogrammed in vitro model of microglia-mediated synapse engulfment, we demonstrate increased synapse elimination in patient-derived neural cultures and isolated synaptosomes. This excessive synaptic pruning reflects abnormalities in both microglia-like cells and synaptic structures. Further, we find that schizophrenia risk-associated variants within the human complement component 4 locus are associated with increased neuronal complement deposition and synapse uptake; however, they do not fully explain the observed increase in synapse uptake. Finally, we demonstrate that the antibiotic minocycline reduces microglia-mediated synapse uptake in vitro and its use is associated with a modest decrease in incident schizophrenia risk compared to other antibiotics in a cohort of young adults drawn from electronic health records. These findings point to excessive pruning as a potential target for delaying or preventing the onset of schizophrenia in high-risk individuals.

Lysine Deacetylation by HDAC6 Regulates the Kinase Activity of AKT in Human Neural Progenitor Cells.

The AKT family of serine-threonine kinases functions downstream of phosphatidylinositol 3-kinase (PI3K) to transmit signals by direct phosphorylation of a number of targets, including the mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß), and ß-catenin. AKT binds to phosphatidylinositol (3,4,5)-triphosphate (PIP3) generated by PI3K activation, which results in its membrane localization and subsequent activation through phosphorylation by phosphoinositide-dependent protein kinase 1 (PDK1). Together, the PI3K-AKT signaling pathway plays pivotal roles in many cellular systems, including in the central nervous system where it governs both neurodevelopment and neuroplasticity. Recently, lysine residues (Lys14 and Lys20) on AKT, located within its pleckstrin homology (PH) domain that binds to membrane-bound PIP3, have been found to be acetylated under certain cellular contexts in various cancer cell lines. These acetylation modifications are removed by the enzymatic action of the class III lysine deacetylases, SIRT1 and SIRT2, of the sirtuin family. The extent to which reversible acetylation regulates AKT function in other cell types remains poorly understood. We report here that AKT kinase activity is modulated by a class IIb lysine deacetylase, histone deacetylase 6 (HDAC6), in human neural progenitor cells (NPCs). We find that HDAC6 and AKT physically interact with each other in the neuronal cells, and in the presence of selective HDAC6 inhibition, AKT is acetylated at Lys163 and Lys377 located in the kinase domain, two novel sites distinct from the acetylation sites in the PH-domain modulated by the sirtuins. Measurement of the functional effect of HDAC6 inhibition on AKT revealed decreased binding to PIP3, a correlated decrease in AKT kinase activity, decreased phosphorylation of Ser552 on ß-catenin, and modulation of neuronal differentiation trajectories. Taken together, our studies implicate the deacetylase activity of HDAC6 as a novel regulator of AKT signaling and point to novel mechanisms for regulating AKT activity with small-molecule inhibitors of HDAC6 currently under clinical development.

Stem cell-derived neurons in the development of targeted treatment for schizophrenia and bipolar disorder.

The recent advent of induced pluripotent stem cells has enabled the study of patient-specific and disease-related neurons in vitro and has facilitated new directions of inquiry into disease mechanisms. With these approaches, we now have the possibility of correlating ex vivo cellular phenotypes with individual patient response to treatment and/or side effects, which makes targeted treatments for schizophrenia and bipolar disorder a distinct prospect in the coming years. Here, we briefly review the current state of stem cell-based models and explore studies that are providing new insights into the disease biology of schizophrenia and bipolar disorder, which are laying the foundations for the development of novel targeted therapies.

Unbiased Metabolite Profiling of Schizophrenia Fibroblasts under Stressful Perturbations Reveals Dysregulation of Plasmalogens and Phosphatidylcholines.

We undertook an unbiased metabolite profiling of fibroblasts from schizophrenia patients and healthy controls to identify metabolites and pathways that are dysregulated in disease, seeking to gain new insights into the disease biology of schizophrenia and to discover potential disease-related biomarkers. We measured polar and nonpolar metabolites in the fibroblasts under normal conditions and under two stressful physiological perturbations: growth in low-glucose media and exposure to the steroid hormone dexamethasone. We found that metabolites that were significantly different between schizophrenia and control subjects showed separation of the two groups by partial least-squares discriminant analysis methods. This separation between schizophrenia and healthy controls was more robust with metabolites identified under the perturbation conditions. The most significant individual metabolite differences were also found in the perturbation experiments. Metabolites that were significantly different between schizophrenia and healthy controls included a number of plasmalogens and phosphatidylcholines. We present these results in the context of previous reports of metabolic profiling of brain tissue and plasma in schizophrenia. These results show the applicability of metabolite profiling under stressful perturbations to reveal cellular pathways that may be involved in disease biology.

Perturbational Profiling of Metabolites in Patient Fibroblasts Implicates α-Aminoadipate as a Potential Biomarker for Bipolar Disorder.

Many studies suggest the presence of aberrations in cellular metabolism in bipolar disorder. We studied the metabolome in bipolar disorder to gain insight into cellular pathways that may be dysregulated in bipolar disorder and to discover evidence of novel biomarkers. We measured polar and nonpolar metabolites in fibroblasts from subjects with bipolar I disorder and matched healthy control subjects, under normal conditions and with two physiologic perturbations: low-glucose media and exposure to the stress-mediating hormone dexamethasone. Metabolites that were significantly different between bipolar and control subjects showed distinct separation by principal components analysis methods. The most statistically significant findings were observed in the perturbation experiments. The metabolite with the lowest p value in both the low-glucose and dexamethasone experiments was α-aminoadipate, whose intracellular level was consistently lower in bipolar subjects. Our study implicates α-aminoadipate as a possible biomarker in bipolar disorder that manifests under cellular stress. This is an intriguing finding given the known role of α-aminoadipate in the modulation of kynurenic acid in the brain, especially as abnormal kynurenic acid levels have been implicated in bipolar disorder.

Disease signatures for schizophrenia and bipolar disorder using patient-derived induced pluripotent stem cells.

Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances.

Human pluripotent stem cell derived midbrain PITX3(eGFP/w) neurons: a versatile tool for pharmacological screening and neurodegenerative modeling.

PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter. MPP(+) and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3(eGFP/w) neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3(eGFP/w) neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

In vitro maturation of dopaminergic neurons derived from mouse embryonic stem cells: implications for transplantation.

The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca(2+) and Cl(-) imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl(-)](i) progressively dropped in tyrosine hydroxylase positive (TH(+)) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i). As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH(+) and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.

A targeted NKX2.1 human embryonic stem cell reporter line enables identification of human basal forebrain derivatives.

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.