Evaluation of Nav1.8 as a therapeutic target for Pitt Hopkins Syndrome.

Pitt Hopkins Syndrome (PTHS) is a rare syndromic form of autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 (TCF4) gene. TCF4 is a basic helix-loop-helix transcription factor that is critical for neurodevelopment and brain function through its binding to cis-regulatory elements of target genes. One potential therapeutic strategy for PTHS is to identify dysregulated target genes and normalize their dysfunction. Here, we propose that SCN10A is an important target gene of TCF4 that is an applicable therapeutic approach for PTHS. Scn10a encodes the voltage-gated sodium channel Nav1.8 and is consistently shown to be upregulated in PTHS mouse models. In this perspective, we review prior literature and present novel data that suggests inhibiting Nav1.8 in PTHS mouse models is effective at normalizing neuron function, brain circuit activity and behavioral abnormalities and posit this therapeutic approach as a treatment for PTHS.

Promyelinating drugs promote functional recovery in an autism spectrum disorder mouse model of Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is an autism spectrum disorder caused by autosomal dominant mutations in the human transcription factor 4 gene (TCF4). One pathobiological process caused by murine Tcf4 mutation is a cell autonomous reduction in oligodendrocytes and myelination. In this study, we show that the promyelinating compounds, clemastine, sobetirome and Sob-AM2 are effective at restoring myelination defects in a Pitt-Hopkins syndrome mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells and normalized oligodendrocyte density. In vivo, 2-week intraperitoneal administration of clemastine also normalized oligodendrocyte precursor cell and oligodendrocyte density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in the proportion of uncompacted myelin and an overall reduction in the g-ratio. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behaviour. To confirm behavioural rescue was achieved via enhancing myelination, we show that treatment with the thyroid hormone receptor agonist sobetirome or its brain penetrating prodrug Sob-AM2, was also effective at normalizing oligodendrocyte precursor cell and oligodendrocyte densities and behaviour in the Pitt-Hopkins syndrome mouse model. Together, these results provide preclinical evidence that promyelinating therapies may be beneficial in Pitt-Hopkins syndrome and potentially other neurodevelopmental disorders characterized by dysmyelination.

Modeling Psychiatric Disorder Biology with Stem Cells.

PURPOSE OF REVIEW: We review the ways in which stem cells are used in psychiatric disease research, including the related advances in gene editing and directed cell differentiation. RECENT FINDINGS: The recent development of induced pluripotent stem cell (iPSC) technologies has created new possibilities for the study of psychiatric disease. iPSCs can be derived from patients or controls and differentiated to an array of neuronal and non-neuronal cell types. Their genomes can be edited as desired, and they can be assessed for a variety of phenotypes. This makes them especially interesting for studying genetic variation, which is particularly useful today now that our knowledge on the genetics of psychiatric disease is quickly expanding. The recent advances in cell engineering have led to powerful new methods for studying psychiatric illness including schizophrenia, bipolar disorder, and autism. There is a wide array of possible applications as illustrated by the many examples from the literature, most of which are cited here.

The Role for the DSB Response Pathway in Regulating Chromosome Translocations.

In response to DNA double strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that coordinate their detection, signaling and repair. Central to this network is the ATM kinase and its substrates at chromatin surrounding DSBs H2AX, MDC1 and 53BP1. In humans, germline inactivation of ATM causes Ataxia Telangiectasia (A-T), an autosomal recessive syndrome of increased proneness to hematological malignancies driven by clonal chromosomal translocations. Studies of cancers arising in A-T patients and in genetically engineered mouse models (GEMM) deficient for ATM and its substrates have revealed complex, multilayered roles for ATM in translocation suppression and identified functional redundancies between ATM and its substrates in this context. "Programmed" DSBs at antigen receptor loci in developing lymphocytes employ ubiquitous DDR factors for signaling and repair and have been particularly useful for mechanistic studies because they are region-specific and can be monitored in vitro and in vivo. In this context, murine thymocytes deficient for ATM recapitulate the molecular events that lead to transformation in T cells from A-T patients and provide a widely used model to study the mechanisms that suppress RAG recombinase-dependent translocations. Similarly, analyses of the fate of Activation induced Cytidine Deaminase (AID)-dependent DSBs during mature B cell Class Switch Recombination (CSR) have defined the genetic requirements for end-joining and translocation suppression in this setting. Moreover, a unique role for 53BP1 in the promotion of synapsis of distant DSBs has emerged from these studies.

TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons.

BACKGROUND: Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. METHODS: To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. RESULTS: Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. CONCLUSIONS: Taken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons.

Genetic variation in the transcription factor 4 ( TCF4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of ASD called Pitt Hopkins Syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models is shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. Here we show that cortical neurons derived from patients with TCF4 mutations have deficits in spontaneous synaptic transmission, network excitability and homeostatic plasticity. Transcriptomic analysis indicates these phenotypes result from altered expression of genes involved in presynaptic neurotransmission and identifies the presynaptic binding protein, RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. Together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

Transcriptomic data of Clozapine-treated Ngn2-induced Human Excitatory Neurons.

We generated human excitatory neurons using a protocol for rapid 21-day induction using neurogenin-2 overexpression (Zhang et al., 2013) in a publicly available control iPSC line. We validated the glutamatergic neuronal identity of the neurons by immunofluorescence and transcriptomics. We exposed 6 of the 12 replicate neuron cultures to therapeutic plasma levels of clozapine (300 ng/mL) for the last 3 days of culture, and the remaining 6 to replicates to the clozapine solvent alone (methanol) to be used as controls. We harvested the cultures and extracted total RNA, depleted ribosomal RNA and subjected them to RNA sequencing. Of the 6 control replicates 2 failed RNA quality control, and thus a total of 6 exposed and 4 control cultures were used for further analysis. Here, we provide that raw sequencing data as well as a list of all of the genes and their expression levels resulting from the RNA-sequencing. This dataset can be used as a reference data for future studies that access additional neuronal cell types, clozapine exposure conditions, and other antipsychotic medication. Related Research Article: Das, D., Peng, X., Lam, A.N., Bader, J.S., Avramopoulos, D., 2021. Transcriptome analysis of human induced excitatory neurons supports a strong effect of clozapine on cholesterol biosynthesis. Schizophr Res 228, 324-326. (Das et al., 2021).

Transcriptome analysis of human induced excitatory neurons supports a strong effect of clozapine on cholesterol biosynthesis.

Antipsychotics are known to modulate dopamine and other neurotransmitters which is often thought to be the mechanism underlying their therapeutic effects. Nevertheless, other less studied consequences of antipsychotics on neuronal function may contribute to their efficacy. Revealing the complete picture behind their action is of paramount importance for precision medicine and accurate drug selection. Progress in cell engineering allows the generation of induced pluripotent stem cells (iPSCs) and their differentiation to a variety of neuronal types, providing new tools to study antipsychotics. Here we use excitatory cortical neurons derived from iPSCs to explore their response to therapeutic levels of Clozapine as measured by their transcriptomic output, a proxy for neuronal homeostasis. To our surprise, but in agreement with the results of many investigators studying glial-like cells, Clozapine had a very strong effect on cholesterol metabolism. More than a quarter (12) of all annotated cholesterol genes (46) in the genome were significantly changed at FDR < 0.1, all upregulated. This is a 35-fold enrichment with an adjusted p = 8 × 10-11. Notably no other functional category showed evidence of enrichment. Cholesterol is a major component of the neuronal membrane and myelin but it does not cross the blood brain barrier, it is produced locally mostly by glia but also by neurons. By singling out increased expression of cholesterol metabolism genes as the main response of cortical excitatory neurons to antipsychotics, our work supports the hypothesis that cholesterol metabolism may be a contributing mechanism to the beneficial effects of Clozapine and possibly other antipsychotics.

Publicly Available hiPSC Lines with Extreme Polygenic Risk Scores for Modeling Schizophrenia.

Schizophrenia (SZ) is a common and debilitating psychiatric disorder with limited effective treatment options. Although highly heritable, risk for this polygenic disorder depends on the complex interplay of hundreds of common and rare variants. Translating the growing list of genetic loci significantly associated with disease into medically actionable information remains an important challenge. Thus, establishing platforms with which to validate the impact of risk variants in cell-type-specific and donor-dependent contexts is critical. Towards this, we selected and characterized a collection of 12 human induced pluripotent stem cell (hiPSC) lines derived from control donors with extremely low and high SZ polygenic risk scores (PRS). These hiPSC lines are publicly available at the California Institute for Regenerative Medicine (CIRM). The suitability of these extreme PRS hiPSCs for CRISPR-based isogenic comparisons of neurons and glia was evaluated across 3 independent laboratories, identifying 9 out of 12 meeting our criteria. We report a standardized resource of publicly available hiPSCs on which we hope to perform genome engineering and generate diverse kinds of functional data, with comparisons across studies facilitated by the use of a common set of genetic backgrounds.

Human Forebrain Organoids from Induced Pluripotent Stem Cells: A Novel Approach to Model Repair of Ionizing Radiation-Induced DNA Damage in Human Neurons.

Human induced pluripotent stem cells (iPSCs) can generate virtually any cell type and therefore are applied to studies of organ development, disease modeling, drug screening and cell replacement therapy. Under proper culture conditions in vitro induced pluripotent stem cells (iPSCs) can be differentiated to form organ-like tissues, also known as "organoids", which resemble organs more closely than cells, in vivo. We hypothesized that human brain organoids can be used as an experimental model to study mechanisms underlying DNA repair in human neurons and their progenitors after radiation-induced DNA double-strand breaks (DSBs), the most severe form of DNA damage. To this end, we customized a protocol for brain organoid generation that is time efficient. These organoids recapitulate key features of human cortical neuron development, including a subventricular zone containing neural progenitors that mature to postmitotic cortical neurons. Using immunofluorescence to measure DNA DSB markers, such as γ-H2AX and 53BP1, we quantified the kinetics of DSB repair in neural progenitors within the subventricular zone for up to 24 h after a single 2 Gy dose of ionizing radiation. Our data on DNA repair in progenitor versus mature neurons indicate a similar timeline: both repair DNA DSBs which is mostly resolved by 18 h postirradiation. However, repair kinetics are more acute in progenitors than mature neurons in the mature organoid. Overall, this study supports the use of 3D organoid culture technology as a novel platform to study DNA damage responses in developing or mature neurons, which has been previously difficult to study.

Generation and characterization of a novel human iPSC line from a resilient Alzheimer's disease patient.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is the major cause of dementia in older people. Here, we report the derivation of human induced pluripotent stem cells (iPSCs) from an AD patient at age of 80 who has the APOE ε4/ε4 genotype and is resilient to cognitive decline for 10 years. The iPSCs reprogrammed from the blood cells of this patient by transient expression of pluripotency genes maintain the ε4/ε4 genotype, are karyotypically normal and display typical iPSC characteristics. Upon differentiation, the iPSCs are able to differentiate into cells of the three germ layers, confirming their pluripotency.

Astrocyte- and Neuron-Derived Extracellular Vesicles from Alzheimer's Disease Patients Effect Complement-Mediated Neurotoxicity.

We have previously shown that blood astrocytic-origin extracellular vesicles (AEVs) from Alzheimer's disease (AD) patients contain high complement levels. To test the hypothesis that circulating EVs from AD patients can induce complement-mediated neurotoxicity involving Membrane Attack Complex (MAC) formation, we assessed the effects of immunocaptured AEVs (using anti-GLAST antibody), in comparison with neuronal-origin (N)EVs (using anti-L1CAM antibody), and nonspecific CD81+ EVs (using anti-CD81 antibody), from the plasma of AD, frontotemporal lobar degeneration (FTLD), and control participants. AEVs (and, less effectively, NEVs) of AD participants induced Membrane Attack Complex (MAC) expression on recipient neurons (by immunohistochemistry), membrane disruption (by EthD-1 assay), reduced neurite density (by Tuj-1 immunohistochemistry), and decreased cell viability (by MTT assay) in rat cortical neurons and human iPSC-derived neurons. Demonstration of decreased cell viability was replicated in a separate cohort of autopsy-confirmed AD patients. These effects were not produced by CD81+ EVs from AD participants or AEVs/NEVs from FTLD or control participants, and were suppressed by the MAC inhibitor CD59 and other complement inhibitors. Our results support the stated hypothesis and should motivate future studies on the roles of neuronal MAC deposition and AEV/NEV uptake, as effectors of neurodegeneration in AD.

The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice.

Shroom3 is an actin-associated regulator of cell morphology that is required for neural tube closure, formation of the lens placode, and gut morphogenesis in mice and has been linked to chronic kidney disease and directional heart looping in humans. Numerous studies have shown that Shroom3 likely regulates these developmental processes by directly binding to Rho-kinase and facilitating the assembly of apically positioned contractile actomyosin networks. We have characterized the molecular basis for the neural tube defects caused by an ENU-induced mutation that results in an arginine-to-cysteine amino acid substitution at position 1838 of mouse Shroom3. We show that this substitution has no effect on Shroom3 expression or localization but ablates Rock binding and renders Shroom3 non-functional for the ability to regulate cell morphology. Our results indicate that Rock is the major downstream effector of Shroom3 in the process of neural tube morphogenesis. Based on sequence conservation and biochemical analysis, we predict that the Shroom-Rock interaction is highly conserved across animal evolution and represents a signaling module that is utilized in a variety of biological processes.

Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.

Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction.

Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock-Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.