ADAM10-Initiated Release of Notch Intracellular Domain Regulates Microtubule Stability and Radial Migration of Cortical Neurons.

Proper neuronal migration is orchestrated by combined membrane signal paradigms, whereas the role and mechanism of regulated intramembrane proteolysis (RIP) remain to be illustrated. We show here that the disintegrin and metalloprotease-domain containing protein 10 (ADAM10) regulates cortical neurons migration by initiating the RIP of Notch. We found that Notch intracellular domain (NICD) significantly rescued the migration defect of ADAM10-deficient neurons. Moreover, ADAM10 deficiency led to reduced neuronal motility and disrupted microtubule (MT) structure, which were associated with downregulated expression of acetylated tubulin and MT-associated proteins. Specifically, the NICD/RBPJ complex bound directly to the promoter, and regulated the neuronal expression level of doublecortin (DCX), a modulator of the MT cytoskeleton. Functionally, DCX overexpression largely restored neuron motility and reversed migration defect caused by ADAM10 knockout. Taken together, these findings demonstrate the direct requirement of ADAM10 in cortical radial migration and reveal the underlying mechanism by linking ADAM10-initiated RIP of Notch to the regulation of MT cytoskeleton through transcriptional control of Dcx expression.

PRRT2 deficiency induces paroxysmal kinesigenic dyskinesia by regulating synaptic transmission in cerebellum.

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.