Patterning ganglionic eminences in developing human brain organoids using a morphogen-gradient-inducing device.

In early neurodevelopment, the central nervous system is established through the coordination of various neural organizers directing tissue patterning and cell differentiation. Better recapitulation of morphogen gradient production and signaling will be crucial for establishing improved developmental models of the brain in vitro. Here, we developed a method by assembling polydimethylsiloxane devices capable of generating a sustained chemical gradient to produce patterned brain organoids, which we termed morphogen-gradient-induced brain organoids (MIBOs). At 3.5 weeks, MIBOs replicated dorsal-ventral patterning observed in the ganglionic eminences (GE). Analysis of mature MIBOs through single-cell RNA sequencing revealed distinct dorsal GE-derived CALB2+ interneurons, medium spiny neurons, and medial GE-derived cell types. Finally, we demonstrate long-term culturing capabilities with MIBOs maintaining stable neural activity in cultures grown up to 5.5 months. MIBOs demonstrate a versatile approach for generating spatially patterned brain organoids for embryonic development and disease modeling.

Schizophrenia-associated NRXN1 deletions induce developmental-timing- and cell-type-specific vulnerabilities in human brain organoids.

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

Generation and Co-culture of Cortical Glutamatergic and GABAergic-Induced Neuronal Cells.

The study of neurological disorders requires experimentation on human neurons throughout their development. Primary neurons can be difficult to obtain, and animal models may not fully recapitulate phenotypes observed in human neurons. Human neuronal culturing schemes which contain a balanced mixture of excitatory and inhibitory neurons that resemble physiological ratios seen in vivo will be useful to probe the neurological basis of excitation-inhibition (E-I) balance. Here, we describe a method for directly inducing a homogenous population of cortical excitatory neurons and cortical interneurons from human pluripotent stem cells, as well as the generation of mixed cultures using these induced neurons. The obtained cells display robust neuronal synchronous network activity as well as complex morphologies that are amenable to studies probing the molecular and cellular basis of disease mutations or other aspects of neuronal and synaptic development.

Method to Generate Dorsal Forebrain Brain Organoids from Human Pluripotent Stem Cells.

Region-specific brain organoids, such as dorsal forebrain brain organoid, have become increasingly useful to model early brain development. Importantly, these organoids provide an avenue to investigate mechanisms underlying neurodevelopmental disorders, as they undergo developmental milestones resembling early neocortical formation. These milestones include the generation of neural precursors which transition into intermediate cell types and subsequently to neurons and astrocytes, as well as the fulfillment of key neuronal maturation events such as synapse formation and pruning. Here we describe how to generate free-floating dorsal forebrain brain organoids from human pluripotent stem cells (hPSCs). We also describe validation of the organoids via cryosectioning and immunostaining. Additionally, we include an optimized protocol that allows high-quality dissociation of the brain organoids to live single cells, a critical step for downstream single-cell assays.

Measuring Neuronal Network Activity Using Human Induced Neuronal Cells.

Synchronous firing of neurons, often referred to as "network activity" or "network bursting," is an indication of a mature and synaptically connected network of neurons. We previously reported this phenomenon in 2D human neuronal in vitro models (McSweeney et al. iScience 25:105187, 2022). Using induced neurons (iNs) differentiated from human pluripotent stem cells (hPSCs) coupled with high-density microelectrodes arrays (HD-MEAs), we probed the underlying patterns of neuronal activity and found irregularities in network signaling across mutant states (McSweeney et al. iScience 25:105187, 2022). Here, we describe methods for plating cortical excitatory iNs differentiated from hPSCs on top of HD-MEAs and culturing iNs to maturity, examples of representative human wild-type Ngn2-iN data, and troubleshooting tips and tricks for the experimenter interested in integrating HD-MEAs into one's research approach.

oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.

Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1-60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.

Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism.

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

CASK loss of function differentially regulates neuronal maturation and synaptic function in human induced cortical excitatory neurons.

Loss-of-function (LOF) mutations in CASK cause severe developmental phenotypes, including microcephaly with pontine and cerebellar hypoplasia, X-linked intellectual disability, and autism. Unraveling the pathological mechanisms of CASK-related disorders has been challenging owing to limited human cellular models to study the dynamic roles of this molecule during neuronal maturation and synapse development. Here, we investigate cell-autonomous functions of CASK in cortical excitatory induced neurons (iNs) generated from CASK knockout (KO) isogenic human embryonic stem cells (hESCs) using gene expression, morphometrics, and electrophysiology. While immature CASK KO iNs show robust neuronal outgrowth, mature CASK KO iNs display severe defects in synaptic transmission and synchronized network activity without compromising neuronal morphology and synapse numbers. In the developing human cortical excitatory neurons, CASK functions to promote both structural integrity and establishment of cortical excitatory neuronal networks. These results lay the foundation for future studies identifying suppressors of such phenotypes relevant to human patients.

Probing the molecular and cellular pathological mechanisms of schizophrenia using human induced pluripotent stem cell models.

With recent advancements in psychiatric genomics, as a field, "stem cell-based disease modelers" were given the exciting yet daunting task of translating the extensive list of disease-associated risks into biologically and clinically relevant information in order to deliver therapeutically meaningful leads and insights. Despite their limitations, human induced pluripotent stem cell (iPSCs) based models have greatly aided our understanding of the molecular and cellular mechanisms underlying the complex etiology of brain disorders including schizophrenia (SCZ). In this review, we summarize the major findings from studies in the past decade which utilized iPSC models to investigate cell type-specific phenotypes relevant to idiopathic SCZ and disease penetrant alleles. Across cell type differences, several biological themes emerged, serving as potential neurodevelopmental mechanisms of SCZ, including oxidative stress and mitochondrial dysfunction, depletion of progenitor pools and insufficient differentiation potential of these progenitors, and structural and functional deficits of neurons and other supporting cells. Here, we discuss both the recent progress as well as challenges and improvements needed for future studies utilizing iPSCs as a model for SCZ and other neuropsychiatric disorders.

Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Patterning Neuroepithelial Cell Sheet via a Sustained Chemical Gradient Generated by Localized Passive Diffusion Devices.

Recent advances in human pluripotent stem cells (hPSCs)-derived in vitro models open a new avenue for studying early stage human development. While current approaches leverage the self-organizing capability of hPSCs, it remains unclear whether extrinsic morphogen gradients are sufficient to pattern neuroectoderm tissues in vitro. While microfluidics or hydrogel-based approaches to generate chemical gradients are well-established, these systems either require continuous pumping or encapsulating cells in gels, making it difficult for adaptation in standard biology laboratories and downstream analysis. In this work, we report a new device design that leverages localized passive diffusion, or LPaD for short, to generate a stable chemical gradient in an open environment. As LPaD is operated simply by media changing, common issues for microfluidic systems such as leakage, bubble formation, and contamination can be avoided. The device contains a slit carved in a film filled with solid gelatin and connected to a static aqueous morphogen reservoir. Concentration gradients generated by the device were visualized via DAPI fluorescent intensity and were found to be stable for up to 168 h. Using this device, we successfully induced cellular response of Madin-Darby canine kidney (MDCK) cells to the concentration gradient of a small-molecule drug, cytochalasin D. Furthermore, we efficiently patterned the dorsal-ventral axis of hPSC-derived forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal gradient generated by the LPaD devices. Together, LPaD devices are powerful tools to control the local chemical microenvironment for engineering organotypic structures in vitro.

Copy number variants in neurexin genes: phenotypes and mechanisms.

Neurexins are central to trans-synaptic cell adhesion and signaling during synapse specification and maintenance. The past two decades of human genetics research have identified structural variations in the neurexin gene family, in particular NRXN1 copy number variants (CNVs), implicated in multiple neuropsychiatric and developmental disorders. The heterogeneity and reduced penetrance of NRXN1 deletions, in addition to the pleiotropic, circuit-specific functions of NRXN1, present substantial obstacles to understanding how compromised NRXN1 function predisposes individuals to neuropsychiatric disorders. Here, we provide an updated review of NRXN1 genetics in disease, followed by recently published work using both human induced pluripotent stem cell (iPSC) derived systems and animal models to understand the mechanisms of disease pathophysiology. Finally, we suggest our outlook on how the field should progress to improve our understanding of neurexin mediated disease pathogenesis. We believe that understanding how structural genetic variants in NRXN1 contribute to disease pathophysiology requires parallel approaches in iPSC and mouse model systems, each leveraging their unique strengths - analysis of genetic interactions and background effects in iPSCs and neural circuit and behavioral analysis in mice.

Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel.

Bioengineers have designed numerous instructive brain extracellular matrix (ECM) environments with tailored and tunable protein compositions and biomechanical properties in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and synthetic model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under "normal" culture conditions. Here, a synthetic hydrogel is introduced, which for the first time demonstrates maintenance of astrocyte quiescence and activation on demand. With this synthetic brain hydrogel, the brain-specific integrin-binding and matrix metalloprotease-degradable domains of proteins are shown to control astrocyte star-shaped morphologies, and an ECM condition that maintains astrocyte quiescence with minimal activation can be achieved. In addition, activation can be induced in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. This synthetic brain hydrogel is envisioned as a new tool to study the physiological role of astrocytes in health and disease.

The Conserved, Disease-Associated RNA Binding Protein dNab2 Interacts with the Fragile X Protein Ortholog in Drosophila Neurons.

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.

Presynaptic Neuronal Pentraxin Receptor Organizes Excitatory and Inhibitory Synapses.

Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1ß-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses. SIGNIFICANCE STATEMENT: Neuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.

Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons.

Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired hyperpolarization-activated cation (Ih) channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) that form Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest that SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1.

Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

Generation of induced neuronal cells by the single reprogramming factor ASCL1.

Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length.

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.