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Stable T98G cells expressing ShcD-WT show increased invasion but fail to exhibit ShcD-Tie2 synergy.

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Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2–ShcD signaling axis in glioma cell invasion, which may be of clinical significance.Implications:ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

BackgroundTheCYFIP1gene, located in the neurodevelopmental risk locus 15q11.2, is highly expressed in microglia, but its role in human microglial function as it relates to neurodevelopment is not well understood.MethodsWe generated multiple CRISPR knockoutsof CYFIP1in patient-derived models of microglia to characterize function and phenotype. Using microglia-like cells reprogrammed from peripheral blood mononuclear cells, we quantified phagocytosis of synaptosomes (isolated and purified synaptic vesicles) from human iPSC-derived neuronal cultures as anin vitromodel of synaptic pruning. We repeated these analyses in human iPSC-derived microglia, and characterized microglial development and function through morphology and motility.ResultsCYFIP1knockout using orthogonal CRISPR constructs in multiple patient-derived cell lines was associated with statistically significant decrease in synaptic vesicle phagocytosis in microglia models derived from both PBMCs and iPSCs (pCYFIP1knockout lines retained the ability to differentiate to functional microglia.Conclusion: The changes in microglial phenotype and function from loss ofCYFIP1may contribute to pruning abnormalities observed inCYFIP1-associated neurodevelopmental disorders. Investigating risk genes in a range of CNS cell types may be required to fully understand the way in which common and rare variants intersect to yield neuropsychiatric disorders.

Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in co-cultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.

Gene expression data can offer deep, physiological insights beyond the static coding of the genome alone. We believe that realizing this potential requires specialized, high-capacity machine learning methods capable of using underlying biological structure, but the development of such models is hampered by the lack of published benchmark tasks and well characterized baselines. In this work, we establish such benchmarks and baselines by profiling many classifiers against biologically motivated tasks on two curated views of a large, public gene expression dataset (the LINCS corpus) and one privately produced dataset. We provide these two curated views of the public LINCS dataset and our benchmark tasks to enable direct comparisons to future methodological work and help spur deep learning method development on this modality. In addition to profiling a battery of traditional classifiers, including linear models, random forests, decision trees, K nearest neighbor (KNN) classifiers, and feed-forward artificial neural networks (FF-ANNs), we also test a method novel to this data modality: graph convolugtional neural networks (GCNNs), which allow us to incorporate prior biological domain knowledge. We find that GCNNs can be highly performant, with large datasets, whereas FF-ANNs consistently perform well. Non-neural classifiers are dominated by linear models and KNN classifiers.

Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.

Multiple lines of evidence implicate dysregulated microglia-mediated synaptic pruning in the pathophysiology of schizophrenia. In vitro human cellular studies represent a promising means of pursuing this hypothesis, complementing efforts with animal models and postmortem human data while addressing their limitations. The challenges in culturing homogeneous populations of cells derived from postmortem or surgical biopsy brain material from patients, and their limited availability, has led to a focus on differentiation of induced pluripotent stem cells. These methods too have limitations, in that they disrupt the epigenome and can demonstrate line-to-line variability due in part to extended time in culture, partial reprogramming, and/or residual epigenetic memory from the cell source, yielding large technical artifacts. Yet another strategy uses direct transdifferentiation of peripheral mononuclear blood cells, or umbilical cord blood cells, to microglia-like cells. Any of these approaches can be paired with patient-derived synaptosomes from differentiated neurons as a simpler alternative to co-culture. Patient-derived microglia models may facilitate identification of novel modulators of synaptic pruning and identification of biomarkers that may allow more targeted early interventions.

Postsynaptic density is reduced in schizophrenia, and risk variants increasing complement component 4A (C4A) gene expression are linked to excessive synapse elimination. In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in patients with first-episode psychosis (FEP) who develop schizophrenia (FEP-SCZ: median 0.41 fmol/ul [CI = 0.34–0.45], FEP-non-SCZ: median 0.29 fmol/ul [CI = 0.22–0.35], healthy controls: median 0.28 [CI = 0.24–0.33]). We show that the CSF elevation of C4A in FEP-SCZ exceeds what can be expected from genetic risk variance in the C4 locus, and in patient-derived cellular models we identify a mechanism dependent on the disease-associated cytokines interleukin (IL)−1beta and IL-6 to selectively increase neuronal C4A mRNA expression. In patient-derived CSF, we confirm that IL-1beta correlates with C4A controlled for genetically predicted C4A RNA expression (r = 0.39; CI: 0.01–0.68). These results suggest a role of C4A in early schizophrenia pathophysiology.

Excessive synapse loss is a core feature of schizophrenia and is linked to the complement component 4A gene (C4A). In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in first-episode psychosis patients who develop schizophrenia and correlates with CSF measurements of synapse density. Using patient-derived cellular modeling, we find that disease-associated cytokines increase neuronal C4A expression and that IL-1β associates with C4A in patient-derived CSF.

Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection initiate neuronal cell death and cause a loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synapse engulfment. To a large extent, SARS-CoV-2 exposed microglia adopt a transcriptomic profile overlapping with neurodegenerative disorders that display an early synapse loss as well as an increased incident risk after a SARS-CoV-2 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from COVID-19.

The ability of small secretory microvesicles known as exosomes to influence neuronal and glial function via their microRNA (miRNA) cargo has positioned them as a novel and effective method of cell-to-cell communication. However, little is known about the role of exosome-secreted miRNAs in the regulation of glutamate receptor gene expression and their relevance for schizophrenia (SCZ) and bipolar disorder (BD). Using mature miRNA profiling and quantitative real-time PCR (qRT-PCR) in the orbitofrontal cortex (OFC) of SCZ (N = 29; 20 male and 9 female), BD (N = 26; 12 male and 14 female), and unaffected control (N = 25; 21 male and 4 female) subjects, we uncovered that miR-223, an exosome-secreted miRNA that targets glutamate receptors, was increased at the mature miRNA level in the OFC of SCZ and BD patients with positive history of psychosis at the time of death and was inversely associated with deficits in the expression of its targets glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B) and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2). Furthermore, changes in miR-223 levels in the OFC were positively and negatively correlated with inflammatory and GABAergic gene expression, respectively. Moreover, miR-223 was found to be enriched in astrocytes and secreted via exosomes, and antipsychotics were shown to control its cellular and exosomal localization in a cell-specific manner. Furthermore, addition of astrocytic exosomes in neuronal cultures resulted in a significant increase in miR-223 expression and a notable reduction in Grin2b and Gria2 mRNA levels, which was strongly inversely associated with miR-223 expression. Lastly, inhibition of astrocytic miR-223 abrogated the exosomal-mediated reduction in neuronal Grin2b expression. Taken together, our results demonstrate that the exosomal secretion of a psychosis-altered and glial-enriched miRNA that controls neuronal gene expression is regulated by antipsychotics.

The X-linked neurodevelopmental diseases CDKL5 deficiency disorder (CDD) and Rett syndrome (RTT) are associated with intellectual disability, infantile spasms and seizures. Although mitochondrial dysfunction has been suggested in RTT, less is understood about mitochondrial function in CDD. A comparison of bioenergetics and mitochondrial function between isogenic wild-type and mutant neural progenitor cell (NPC) lines revealed increased oxygen consumption in CDD mutant lines, which is associated with altered mitochondrial function and structure. Transcriptomic analysis revealed differential expression of genes related to mitochondrial and REDOX function in NPCs expressing the mutant CDKL5. Furthermore, a similar increase in oxygen consumption specific to RTT patient–derived isogenic mutant NPCs was observed, though the pattern of mitochondrial functional alterations was distinct from CDKL5 mutant–expressing NPCs. We propose that aberrant neural bioenergetics is a common feature between CDD and RTT disorders. The observed changes in oxidative stress and mitochondrial function may facilitate the development of therapeutic agents for CDD and related disorders.

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.

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2–ShcD signaling axis in glioma cell invasion, which may be of clinical significance. Implications: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium’s suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium’s attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Evidence from epidemiological and laboratory studies, as well as randomized placebo-controlled trials, suggests supplementation with n-3 polyunsaturated fatty acids (PUFAs) may be efficacious for treatment of major depressive disorder (MDD). The mechanisms underlying n-3 PUFAs potential therapeutic properties remain unknown. There are suggestions in the literature that glial hypofunction is associated with depressive symptoms and that antidepressants may normalize glial function. In this study, iPSC-derived neuronal stem cell lines were generated from individuals with MDD. Astrocytes differentiated from patient-derived neuronal stem cells (iNSCs) were verified by GFAP. Cells were treated with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and stearic acid (SA). During astrocyte differentiation, we found that n-3 PUFAs increased GFAP expression and GFAP positive cell formation. BDNF and GDNF production were increased in the astrocytes derived from patients subsequent to n-3 PUFA treatment. Stearic Acid (SA) treatment did not have this effect. CREB activity (phosphorylated CREB) was also increased by DHA and EPA but not by SA. Furthermore, when these astrocytes were treated with n-3 PUFAs, the cAMP antagonist, RP-cAMPs did not block n- 3 PUFA CREB activation. However, the CREB specific inhibitor (666-15) diminished BDNF and GDNF production induced by n-3 PUFA, suggesting CREB dependence. Together, these results suggested that n-3 PUFAs facilitate astrocyte differentiation, and may mimic effects of some antidepressants by increasing production of neurotrophic factors. The CREB-dependence and cAMP independence of this process suggests a manner in which n-3 PUFA could augment antidepressant effects. These data also suggest a role for astrocytes in both MDD and antidepressant action.

Comment on “A microRNA cluster in the Fragile‐X region expressed during spermatogenesis targets FMR1” by Ramaiah et al.[Image: see text]

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KImice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region ofP301Smice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENTAccumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KItransgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

Genetic variation within the transcription factor TCF4 locus can cause the intellectual disability and developmental disorder Pitt-Hopkins syndrome (PTHS), whereas single-nucleotide polymorphisms within noncoding regions are associated with schizophrenia. These genetic findings position TCF4 as a link between transcription and cognition; however, the neurobiology of TCF4 remains poorly understood. Here, we quantitated multiple distinct TCF4 transcript levels in human induced pluripotent stem cell-derived neural progenitors and differentiated neurons, and PTHS patient fibroblasts. We identify two classes of pharmacological treatments that regulate TCF4 expression: WNT pathway activation and inhibition of class I histone deacetylases. In PTHS fibroblasts, both of these perturbations upregulate a subset of TCF4 transcripts. Finally, using chromatin immunoprecipitation sequencing in conjunction with genome-wide transcriptome analysis, we identified TCF4 target genes that may mediate the effect of TCF4 loss on neuroplasticity. Our studies identify new pharmacological assays, tools, and targets for the development of therapeutics for cognitive disorders.

Engulfment of synapses and neural progenitor cells (NPCs) by microglia is critical for the development and maintenance of proper brain circuitry, and has been implicated in neurodevelopmental as well as neurodegenerative disease etiology. We have developed and validated models of these mechanisms by reprogramming microglia-like cells from peripheral blood mononuclear cells, and combining them with NPCs and neurons derived from induced pluripotent stem cells to create patient-specific cellular models of complement-dependent synaptic pruning and elimination of NPCs. The resulting microglia-like cells express appropriate markers and function as primary human microglia, while patient-matched macrophages differ markedly. As a demonstration of disease-relevant application, we studied the role of C4, recently implicated in schizophrenia, in engulfment of synaptic structures by human microglia. The ability to create complete patient-specific cellular models of critical microglial functions utilizing samples taken during a single clinical visit will extend the ability to model central nervous system disease while facilitating high-throughput screening.

Testis‐expressed X‐linked genes typically evolve rapidly. Here, we report on a testis‐expressed X‐linked microRNA (miRNA) cluster that despite rapid alterations in sequence has retained its position in the Fragile‐X region of the X chromosome in placental mammals. Surprisingly, the miRNAs encoded by this cluster (Fx‐mir) have a predilection for targeting the immediately adjacent gene, Fmr1, an unexpected finding given that miRNAs usually act in trans, not in cis. Robust repression of Fmr1 is conferred by combinations of Fx‐mir miRNAs induced in Sertoli cells (SCs) during postnatal development when they terminate proliferation. Physiological significance is suggested by the finding that FMRP, the protein product of Fmr1, is downregulated when Fx‐mir miRNAs are induced, and that FMRP loss causes SC hyperproliferation and spermatogenic defects. Fx‐mir miRNAs not only regulate the expression of FMRP, but also regulate the expression of eIF4E and CYFIP1, which together with FMRP form a translational regulatory complex. Our results support a model in which Fx‐mir family members act cooperatively to regulate the translation of batteries of mRNAs in a developmentally regulated manner in SCs.

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition., eLife, 7, ISSN:2050-084X

Schizophrenia patients display decreased synaptic density in postmortem studies, suggesting aberrant microglial synapse elimination during neurodevelopment. Here, we use cellular reprogramming to create patient-specific in vitro models of microglia-mediated synapse engulfment that demonstrate increased synapse elimination in schizophrenia-derived models compared to healthy controls. We show that excessive synaptic pruning in schizophrenia reflects abnormalities in microglia-like cells as well as synaptic structures. Further, we find that schizophrenia risk-associated variants within the complement component 4 locus contribute to the increased uptake in schizophrenia models. Finally, we demonstrate that the antibiotic minocycline reduces microglia-mediated synapse uptake and show that minocycline treatment for acne is associated with a reduction in incident schizophrenia risk compared to other treatments in a cohort of more than 9,000 young adults drawn from health records. Specific pharmacological interventions targeting excessive pruning merit further study for their capacity to delay or prevent the onset of schizophrenia in high-risk individuals.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared to their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4+ NPCs with a pharmacological inhibitor of glycogen synthase kinase 3 (GSK3), a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD-patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active non-phosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2: CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the " lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways., Funding Agencies:NIH's Library of Integrated Network-based Cellular Signatures Program Viterbi Foundation Neuroscience Initiative Stanley Medical Research Institute R21MH093958 R33MH087896 R01MH095088 Tau Consortium California Institute of Regenerative Medicine training grants University of California, San Diego T32 training grant in psychiatry California Bipolar Foundation International Bipolar Foundation Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Ministry of Education, Science, Sports and Culture in Japan 42890001 RC2MH090011

Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Wnt/β-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/β-catenin signaling in disease-relevant contexts are needed. The use of human patient–derived induced pluripotent stem cell (iPSC) models provides ideal disease-relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/β-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose-responsive stimulation by several known Wnt/β-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1500 compounds from a library of Food and Drug Administration–approved drugs and known bioactives and confirmed the HTS hits, revealing multiple chemical and biological classes of novel small-molecule probes of Wnt/β-catenin signaling. Generating these type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorders., National Institute of Mental Health (U.S.) (Grant R01MH091115), Stanley Medical Research Institute

Several genome-wide association studies (GWAS) for bipolar disorder (BD) have found a strong association of the Ankyrin3 (ANK3) gene. This association spans numerous linked single nucleotide polymorphisms (SNPs) in a ~250 kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as ankyrin-G (AnkG). Using RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites (TSSs) and coupling of specific 5’ ends with 3’ mRNA splicing events in post-mortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD–associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 may be relevant to BD pathophysiology.

Human cerebral organoids reveal deficits in neurogenesis and neuronal migration in MeCP2-deficient neural progenitors

Noncoding RNAs connect genetic risk factors to the neurodevelopmental basis of bipolar disorder

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10−8) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10−10). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.

Rett syndrome is a severe childhood onset neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein 2 (MECP2), with known disturbances in catecholamine synthesis. Here, we show that treatment with the β2-adrenergic receptor agonist clenbuterol increases survival, rescues abnormalities in respiratory function and social recognition, and improves motor coordination in young male Mecp2-null (Mecp2(-/y)) mice. Importantly, we demonstrate that short-term treatment with clenbuterol in older symptomatic female heterozygous (Mecp2(-/+)) mice rescues respiratory, cognitive, and motor coordination deficits, and induces an anxiolytic effect. In addition, we reveal abnormalities in a microRNA-mediated pathway, downstream of brain-derived neurotrophic factor that affects insulin-like growth factor 1 (IGF1) expression in Mecp2(-/y) mice, and show that treatment with clenbuterol restores the observed molecular alterations. Finally, cotreatment with clenbuterol and recombinant human IGF1 results in additional increases in survival in male null mice. Collectively, our data support a role for IGF1 and other growth factor deficits as an underlying mechanism of Rett syndrome and introduce β2-adrenergic receptor agonists as potential therapeutic agents for the treatment of the disorder.

One of the primary mechanisms through which a cell exerts control over its metabolic state is by modulating expression levels of its enzyme-coding genes. However, the changes at the level of enzyme expression allow only indirect control over metabolite levels, for two main reasons. First, at the level of individual reactions, metabolite levels are non-linearly dependent on enzyme abundances as per the reaction kinetics mechanisms. Secondly, specific metabolite pools are tightly interlinked with the rest of the metabolic network through their production and consumption reactions. While the role of reaction kinetics in metabolite concentration control is well studied at the level of individual reactions, the contribution of network connectivity has remained relatively unclear. Here we report a modeling framework that integrates both reaction kinetics and network connectivity constraints for describing the interplay between metabolite concentrations and mRNA levels. We used this framework to investigate correlations between the gene expression and the metabolite concentration changes in Saccharomyces cerevisiae during its metabolic cycle, as well as in response to three fundamentally different biological perturbations, namely gene knockout, nutrient shock and nutrient change. While the kinetic constraints applied at the level of individual reactions were found to be poor descriptors of the mRNA-metabolite relationship, their use in the context of the network enabled us to correlate changes in the expression of enzyme-coding genes to the alterations in metabolite levels. Our results highlight the key contribution of metabolic network connectivity in mediating cellular control over metabolite levels, and have implications towards bridging the gap between genotype and metabolic phenotype., Author Summary Regulation of metabolic activity in response to environmental and genetic perturbations is fundamental to the growth and maintenance of all cells. A primary regulatory process used by cells to control the activity of their metabolic network is the alteration in the expression of enzyme-coding genes. How these alterations regulate metabolite concentrations is an important question in the quest towards unraveling the genotype-phenotype relationship. The link between the expression levels of enzymes and metabolite concentrations is governed by the kinetics of individual reactions, which in turn are interlinked with each other due to the complex connectivity structure of metabolic networks. Although the enzyme-metabolite relationship is relatively well studied at the level of individual reactions, our understanding of the regulation of metabolite levels in complex networks has remained incomplete. In this study, we show that the constraints imposed by the network connectivity are key determinants of the relationship between gene expression and metabolite concentration changes. Our results provide mechanistic insight into the function of complex metabolic networks and have implications for health and biotechnological applications.

Negative superhelical tension can drive local transitions to alternative DNA structures. Long regions of DNA may contain several sites that are susceptible to forming alternative structures. Their relative propensities to undergo transition are ordered according to the energies required for their formation. These energies have two components - the energy needed to drive the transition and the energy relieved by the partial relaxation of superhelicity that the transition provides. This coupling can cause a complex competition among the possible transitions, in which the formation of one energetically favourable alternative structure may inhibit the formation of another within the same domain. In principle, DNA structural competitions can affect the structural and energetic requirements for the initiation of transcription at distant promoter sites. We have tested this possibility by examining the effects of structural transitions on transcription initiation from promoter sites in the same superhelical domain. Specifically, we describe the effects of the presence of a Z-DNA-forming DNA sequence on the basal levels of expression of two supercoiling-sensitive promoters of Escherichia coli, ilvPG and gyrA. We demonstrate transcriptional repression of the ilvPG promoter and activation of the gyrA promoter. We present evidence that this regulation is effected by the superhelically induced B- to Z-DNA transition in a manner that is both orientation and distance independent. We discuss the mechanism of topological coupling between left-handed Z-DNA and the regulation of promoter activity. We also discuss the possibility that the coupling of DNA structural transitions and transcriptional activity might be used as a general regulatory mechanism for gene expression.