Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

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 modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes 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 impair EZH1 expression 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 two EZH1 missense 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 that EZH1 variants 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

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders

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 modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes 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 impair EZH1 expression 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 two EZH1 missense 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 that EZH1 variants 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

NMDA receptor biochemical and activity-dependent regulation of p38 MAPK and glutamate transporter EAAC1

N-methyl-D-aspartate (NMDA) receptors have a high affinity for glutamate and are important for control of dendritic growth, gene transcription, and synaptic plasticity. However, high levels of NMDA receptor stimulation cause excitotoxicity. In the present work I have explored NMDA receptor-mediated regulation of two independent pathways: p38 mitogen-activated protein kinase (MAPK) activation and excitatory amino acid carrier 1 (EAAC1) cell surface expression. In the current research, using NMDA as a specific agonist, the pharmacological requirements for NMDA receptor-mediated p38 MAPK phosphorylation and dephosphorylation were examined. Low concentrations of NMDA resulted in robust, sustained phosphorylation, and high concentrations of NMDA produced only transient phosphorylation of p38 MAPK. Antagonists of NR2B-containing NMDA receptors and calcineurin each prevented NMDA-induced p38 MAPK phosphorylation. Alternatively, antagonists of NR1/2B subtype NMDA receptors and phosphoinositide-3 kinase (PI3K) inhibitors prevented the rapid loss of p38 MAPK phosphorylation with high concentrations of NMDA. The p85 (regulatory) subunit of PI3K was coimmunoprecipitated with the NR2B, but not the NR2A subunit, and increased phosphorylation of NR2B-Y1336 was observed after treatment with high concentrations of NMDA. These data suggest that p38 MAPK phosphorylation (and thereby activation) occurs through NR1/2A/2B subtype NMDA receptors through calcineurin, and dephosphorylation occurs through phosphorylation of NR2B-Y1336, resulting in PI3K activation, in conjunction with NR1/2B subtype NMDA receptors. Independently, an association and regulation of EAAC1 by the NMDA receptor was identified. Antibodies that pull-down EAAC1 coimmunoprecipitated NMDA receptor subunits NR1, NR2A, and NR2B in hippocampal cultures and in C6 glioma cotransfected with myc-EAAC1, NR1, NR2A, and NR2B. C6 glioma, transfected with both NR1 and NR2 subunits, increased levels of cell surface EAAC1. Furthermore, high concentrations of NMDA produced a substantial loss of cell surface EAAC1 in hippocampal cultures. These effects of NMDA were inhibited by NMDA receptor antagonists, Ca2+ chelators, and hypertonic sucrose. These data suggest that NMDA receptors interact with EAAC1, facilitating its cell surface expression, and that NMDA receptor activation induces internalization of EAAC1 through overall increases in intracellular calcium and clathrin-mediated endocytosis. This work has identified novel mechanisms by which NMDA receptors may affect synaptic plasticity and/or excitotoxicity

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders

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 modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes 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 impair EZH1 expression 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 two EZH1 missense 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 that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorder

Generation of a human Tropomyosin 1 knockout iPSC line

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line

Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching

Abstract 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

Screening of approved drugs identifies 3rd generation retinoids as in vitro therapeutic agents in multiple sulfatase deficiency

Schlotawa L, Kettwig M, Ahrens-Nicklas R, et al. Screening of approved drugs identifies 3rd generation retinoids as in vitro therapeutic agents in multiple sulfatase deficiency. In: Program and Abstracts WORLDSymposiumTM 2023* 19th Annual Research Meeting & Scientific Sessions. Molecular Genetics and Metabolism. Vol 138. San Diego: Elsevier; 2023: 117-118

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency

Schlotawa L, Tyka K, Kettwig M, et al. Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency. EMBO Molecular Medicine. 2023: e14837.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. © 2023 The Authors. Published under the terms of the CC BY 4.0 license