Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub.

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional 'tug-of-war' on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

ngn-1/neurogenin Activates Transcription of Multiple Terminal Selector Transcription Factors in the Caenorhabditis elegans Nervous System.

Proper nervous system development is required for an organism's survival and function. Defects in neurogenesis have been linked to neurodevelopmental disorders such as schizophrenia and autism. Understanding the gene regulatory networks that orchestrate neural development, specifically cascades of proneural transcription factors, can better elucidate which genes are most important during early neurogenesis. Neurogenins are a family of deeply conserved factors shown to be both necessary and sufficient for the development of neural subtypes. However, the immediate downstream targets of neurogenin are not well characterized. The objective of this study was to further elucidate the role of ngn-1/neurogenin in nervous system development and to identify its downstream transcriptional targets, using the nematode Caenorhabditis elegans as a model for this work. We found that ngn-1 is required for axon outgrowth, nerve ring architecture, and neuronal cell fate specification. We also showed that ngn-1 may have roles in neuroblast migration and epithelial integrity during embryonic development. Using RNA sequencing and comparative transcriptome analysis, we identified eight transcription factors (hlh-34/NPAS1, unc-42/PROP1, ceh-17/PHOX2A, lim-4/LHX6, fax-1/NR2E3, lin-11/LHX1, tlp-1/ZNF503, and nhr-23/RORB) whose transcription is activated, either directly or indirectly, by ngn-1 Our results show that ngn-1 has a role in transcribing known terminal regulators that establish and maintain cell fate of differentiated neural subtypes and confirms that ngn-1 functions as a proneural transcription factor in C. elegans neurogenesis.