Elisa A. Waxman

Objective: STXBP1 ‐related disorders ( STXBP1 ‐RD) are among the most common genetic neurodevelopmental disorders, marked by early onset epilepsy, global developmental delay, and motor impairments. Many missense variants remain uncharacterized, limiting accurate variant interpretation and hindering development of precision therapies. Current American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) variant curation frameworks provide limited discriminatory power for STXBP1 missense variants due to limited functional data and frequently incomplete parental testing, leaving a substantial proportion of variants classified as variants of uncertain significance. Methods: We developed a fluorometric assay that leverages split fluorescence complementation to simultaneously measure STXBP1 protein stability and its binding affinity to a primary interaction partner, syntaxin‐1A (STX1A), including genetically engineered closed and open STX1A conformations, which are essential for SNARE complex function. Using this platform, we evaluated five benign variants, six pathogenic variants, and four variants that result in an atypically mild clinical presentation. Results: Benign population variants showed minimal effects on stability or binding, whereas known pathogenic variants consistently demonstrated reduced protein stability and diminished binding to STX1A, with some exhibiting STX1A conformation‐specific binding deficits. Variants associated with atypically mild phenotypes displayed intermediate, variant‐specific profiles. To quantify discriminatory performance, we developed and trained an exploratory support vector machine classifier using stability and binding metrics. With the exception of a single STXBP1 variant in an individual with a mild clinical presentation, the model correctly classified benign and pathogenic variants using leave‐one‐out cross‐validation. For variants with available clinical data, classifier scores showed a statistically significant correlation with clinical severity. Significance: Together, these findings demonstrate that a scalable, quantitative assay capturing STXBP1 stability and syntaxin‐binding behavior can differentiate benign from pathogenic variants and provide functional insights into mechanisms of pathogenicity. This platform offers a path toward improved variant interpretation, supports refinement of ACMG/AMP criteria for synaptic disorders, and may inform future therapeutic targeting strategies for STXBP1 ‐RD.

Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1 -related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1 , normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR‐modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two‐dimensional cultures of hPSC‐derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC.Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neuronsSupport Protocol 1: Harvesting and fixing cells for flow cytometry analysesSupport Protocol 2: Performing flow cytometry analysesSupport Protocol 3: Thawing NPCs from a cryopreserved stockAlternate Protocol 1: Continuing Expansion of NPCsAlternate Protocol 2: Treatment of neurons with Ara‐C to ablate radial gliaSupport Protocol 4: Experimental methods for validation of excitatory cortical neurons

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifierEZH1as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals.EZH1encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impairEZH1expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of twoEZH1missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate thatEZH1variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine‐generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA‐approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose‐ and time‐dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients.

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcriptional activator of antioxidant response elements (ARE), which function to increase expression of antioxidant enzymes. Recent works suggests Nrf2 activation is a promising protective mechanism against the progressive neurodegeneration in Parkinson’s disease (PD). While Nrf2 inducers show some promising results in animal models of PD, the response is limit in dopaminergic neurons with the protection mostly conferred by surrounding glia. The present study characterizes ARE transcriptional repressors Bach1 and Bach 2 (Broad complex– Tramtrack–Bric-a-brac and Cap n’ Collar homology, basic leucine zipper transcription factors 1 and 2) as a potential explanation for limited Nrf2 activity in these neurons. The current work identified Bach1 and Bach2 in dopaminergic neurons of the human substantia nigra by immunocytochemical analyses. We further identified Bach2 as a more robust inhibitor of Nrf2 responses. The effects of both Bach1 and Bach2 were dependent on their DNA-binding domains, but the DNA-binding domains did not entirely explain the differences in their relative repressor activities. Using IMR-32 neuroblastoma cells, we found differentiation into a dopaminergic neuronal phenotype resulted in increased Bach2 expression, decreased full-length Bach1 expression, increased truncated Bach1, and blunted Nrf2-mediated responses. Bach inhibitors cobalt protoporphyrin or cadmium chloride, which were more effective against Bach1 than Bach2, did not rescue Nrf2 responses. These results provide a novel mechanism for the lack of antioxidant responses and sensitivity of dopaminergic neurons to oxidative damage, in addition to proposing Bach2 as a novel drug target for the treatment of PD. Significance: Oxidative stress and damage are unifying contributors to explain the progressive loss of dopaminergic neurons in Parkinson’s disease (PD). Although increased transcription of antioxidant enzymes by nuclear factor erythroid 2-related factor 2 (Nrf2) shows potential in combating oxidative damage, Nrf2 protection is mostly conferred by surrounding glia with a relative inability of neurons to mount a protective response. This work characterizes transcriptional repressors Bach1 and Bach2 that inhibit Nrf2 responses and prevent antioxidant enzyme production. This study supports Bach2 as a major factor in preventing antioxidant production in neurons and as a novel drug target to protect against PD.