Nwd1 regulates neuronal differentiation and migration through purinosome formation in the developing cerebral cortex

Abstract

<jats:title>SUMMARY</jats:title><jats:p>Engagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze <jats:italic>de novo</jats:italic> purine synthesis are assembled as a huge multienzyme complex called “purinosome”. However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, <jats:underline>N</jats:underline>ACHT and <jats:underline>W</jats:underline>D repeat <jats:underline>d</jats:underline>omain-containing <jats:underline>1</jats:underline> (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.</jats:p>

Inka2, a novel Pak4 inhibitor, regulates actin dynamics in neuronal development

“…Cell transfection was performed as previously described (Yamada et al, 2020). Cultured cells were transfected with plasmid DNA and PEI MAX (Polysciences, Warrington, PA, USA) complexes (ratio of DNA to PEI MAX, 1:3, w/w) formed in Opti-MEM I by incubating for 15 min at 25°C.…”

“…cDNA fragments corresponding to Inka1 and Inka2 ORFs were isolated using RT-PCR of the RNAs of E12 embryonic and adult mice brains and subcloned in-frame into a pEGFP-C2 expression vector (Takara Bio Inc., Shiga, Japan). To construct the CAG promoter-driven Inka2 in mammalian cells, the EGFP-Inka2 ORF fragment was subcloned into the pCAG-GS vector (Yamada et al, 2020). Deletion mutant EGFP- Inka2ΔiBox and EGFP-Inka2ΔCC were constructed from pEGFP-Inka2 using Q5 Site- Directed Mutagenesis Kit (New England BioLabs, Ipswich, MA, USA).…”

Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

“…Primary NSPCs were isolated from the E12 telencephalon (Yamada and Sakakibara, 2018). PCNs were prepared from the cerebral cortex of E16-17 mice (Yamada et al, 2020). Cortices were dissociated using 0.25% trypsin, followed by trituration.…”

“…Cultured cell lines were transfected with plasmids using PEI MAX (Polysciences) complexes with a DNA to PEI MAX ratio of 1:3 w/w (Akiyama et al, 2020). Mouse NSPCs and primary cortical neurons were electroporated using the NEPA21 Electroporator (Nepagene) (Yamada et al, 2020).…”

“…Pregnant mice were anesthetized with medetomidine, midazolam, and butorphanol (Yamada et al, 2020). Plasmid DNA solution was injected into the lateral ventricle of E14.5 mice through the uterus wall and electroporated using the NEPA21 electric-pulse generator (Nepagene) according to the manufacturer's instructions.…”