CLP1 Founder Mutation Links tRNA Splicing and Maturation to Cerebellar Development and Neurodegeneration

SummaryNeurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans

Transgenic overexpression of the transcription factor Nkx6.1 in β-cells of mice does not increase β-cell proliferation, β-cell mass, or improve glucose clearance

The loss or dysfunction of the pancreatic endocrine β-cell results in diabetes. Recent innovative therapeutic approaches for diabetes aim to induce β-cell proliferation in vivo by pharmacological intervention. Based on the finding that overexpression of the transcription factor Nkx6.1 in islets in vitro increases β-cell proliferation while maintaining β-cell function, Nkx6.1 has been proposed as a potential target for diabetes therapy. However, it is unknown whether elevated Nkx6.1 levels in β-cells in vivo have similar effects as observed in isolated islets. To this end, we sought to investigate whether overexpression of Nkx6.1 in β-cells in vivo could increase β-cell mass and/or improve β-cell function in normal or β-cell-depleted mice. Using a bigenic inducible Cre-recombinase-based transgenic model, we analyzed the effects of Nkx6.1 overexpression on β-cell proliferation, β-cell mass, and glucose metabolism. We found that mice overexpressing Nkx6.1 in β-cells displayed similar β-cell proliferation rates and β-cell mass as control mice. Furthermore, after partial β-cell ablation, Nkx6.1 overexpression was not sufficient to induce β-cell regeneration under either nondiabetic or diabetic conditions. Together these results demonstrate that sustained Nkx6.1 overexpression in vivo does not stimulate β-cell proliferation, expand β-cell mass, or improve glucose metabolism in either normal or β-cell-depleted pancreata. Thus, raising cellular Nkx6.1 levels in β-cells in vivo is unlikely to have a positive impact on type 2 diabetes

Biallelic loss of human CTNNA2, encoding alpha N-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migration

WOS: 000440423400008PubMed ID: 30013181Neuronal migration defects, including pachygyria, are among the most severe developmental brain defects in humans. Here, we identify biallelic truncating mutations in CTNNA2, encoding alpha N-catenin, in patients with a distinct recessive form of pachygyria. CTNNA2 was expressed in human cerebral cortex, and its loss in neurons led to defects in neurite stability and migration. The alpha N-catenin paralog, alpha E-catenin, acts as a switch regulating the balance between beta-catenin and Arp2/3 actin filament activities(1). Loss of alpha N-catenin did not affect beta-catenin signaling, but recombinant alpha N-catenin interacted with purified actin and repressed ARP2/3 actin-branching activity. The actin-binding domain of alpha N-catenin or ARP2/3 inhibitors rescued the neuronal phenotype associated with CTNNA2 loss, suggesting ARP2/3 de-repression as a potential disease mechanism. Our findings identify CTNNA2 as the first catenin family member with biallelic mutations in humans, causing a new pachygyria syndrome linked to actin regulation, and uncover a key factor involved in ARP2/3 repression in neurons.NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01NS041537, R01NS048453, R01NS052455, P01HD070494, P30NS047101]; Qatar National Research Fund [6-1463-351]; Simons Foundation Autism Research Initiative; Howard Hughes Medical InstituteHoward Hughes Medical Institute; A.P. Giannini Fellowship; NIH Pathway to Independence AwardUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R00HD082337]; 2014 NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation; Yale Center for Mendelian Disorders [UMIHG008900, UMIHG006504]We thank the patients and their families for participation. We thank A. Wynshaw-Boris for generous scientific and editorial input. The research was supported by NIH R01NS041537, R01NS048453, R01NS052455, P01HD070494, P30NS047101, Qatar National Research Fund number 6-1463-351, the Simons Foundation Autism Research Initiative, and the Howard Hughes Medical Institute (to J.G.G). A.E.S. is a recipient of an A.P. Giannini Fellowship and an NIH Pathway to Independence Award, R00HD082337. S.T.B. is supported by a 2014 NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation. We thank the Broad Institute and Yale Center for Mendelian Disorders (UMIHG008900 to D. MacArthur and H. Rehm, and UMIHG006504 to R. Lifton and M.G.), and the Gregory M. Kiez and Mehmet Kutman Foundation (to M.G). We acknowledge M. Gerstein, S. Mane, A. B. Ekici, and S. Uebe for sequencing support and analysis, the Yale Biomedical High Performance Computing Center for data analysis and storage, the Yale Program on Neurogenetics, and the Yale Center for Human Genetics and Genomics. Exome data have been deposited into the database of Genotypes and Phenotypes (phs000288)

An AKT3-FOXG1-reelin network underlies defective migration in human focal malformations of cortical development.

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non-cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy

Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies