RESUMO
Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase function. The OTUD7A protein-protein interaction network included synaptic, axonal, and cytoskeletal proteins and was enriched for ASD and epilepsy risk genes (Ank3, Ank2, SPTAN1, SPTBN1). The interactions between OTUD7A and Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment, while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Furthermore, our study highlights the utility of targeting CNV genes using cell type-specific proteomics to identify shared and unexplored disease mechanisms across NDDs.
Assuntos
Anquirinas , Epilepsia , Humanos , Camundongos , Animais , Anquirinas/genética , Variações do Número de Cópias de DNA , Epilepsia/genética , NeurôniosRESUMO
Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.
Assuntos
Transtornos Cromossômicos/enzimologia , Transtornos Cromossômicos/genética , Enzimas Desubiquitinantes/fisiologia , Endopeptidases/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Convulsões/enzimologia , Convulsões/genética , Animais , Transtorno do Espectro Autista/genética , Deleção Cromossômica , Cromossomos Humanos Par 15/enzimologia , Cromossomos Humanos Par 15/genética , Espinhas Dendríticas/metabolismo , Enzimas Desubiquitinantes/genética , Endopeptidases/metabolismo , Feminino , Deleção de Genes , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Fenótipo , Prosencéfalo/patologiaRESUMO
OBJECTIVE: Added transforming growth factor beta (TGFbeta) inhibits the proliferation of immature cardiomyocytes. We have now examined the hypothesis that suppression of endogenous TGFbeta signaling will boost the proliferative response (DNA synthesis) of cardiac myocytes to serum and/or to the mitogenic factor fibroblast growth factor-2 (FGF-2). METHODS AND RESULTS: Overexpression of a kinase-deficient TGFbeta type II receptor (TGFbetaRIIDeltaKD) resulted in a 2.8-fold increase in cardiomyocyte DNA synthesis in serum-rich cultures, an effect requiring active FGFR-1 since it was not observed in the presence of excess kinase-deficient FGFR-1. This finding suggested that endogenous TGFbeta-TGFbetaRII suppressed endogenous FGFR-1-mediated signals that stimulate or are permissive for DNA synthesis. TGFbeta had no effect, however, on the FGF-2-induced acute stimulation of extracellular signal regulated kinase1/2. FGF-2, added in the absence or presence of TGFbeta inhibition, elicited a 3- or a 13-fold stimulation of DNA synthesis, respectively, pointing to a synergistic effect. CONCLUSION: Inhibition of TGFbetaRII-transduced signaling upregulates the proliferative response of cardiomyocytes to serum, and greatly potentiates the stimulatory effect of FGF-2. A combinatorial strategy including activation of FGF-2 and inhibition of TGFbeta-triggered signal transduction may be required for maximal stimulation of immature cardiomyocyte DNA synthesis.