Zika-Virus-Encoded NS2A Disrupts Mammalian Cortical Neurogenesis by Degrading Adherens Junction Proteins

Abstract

SUMMARY Zika virus (ZIKV) directly infects neural progenitors and impairs their proliferation. How ZIKV interacts with the host molecular machinery to impact neurogenesis in vivo is not well understood. Here, by systematically introducing individual proteins encoded by ZIKV into the embryonic mouse cortex, we show that expression of ZIKV-NS2A, but not Dengue virus (DENV)-NS2A, leads to reduced proliferation and premature differentiation of radial glial cells, and aberrant positioning of newborn neurons. Mechanistically, in vitro mapping of protein-interactomes and biochemical analysis suggest interactions between ZIKA-NS2A and multiple adherens junction complex (AJ) components. Functionally, ZIKV-NS2A, but not DENV-NS2A, destabilizes the AJ complex, resulting in impaired AJ formation and aberrant radial glial fiber scaffolding in the embryonic mouse cortex. Similarly, ZIKA-NS2A, but not DENV-NS2A, reduces radial glial cell proliferation and causes AJ deficits in human forebrain organoids. Together, our results reveal pathogenic mechanisms underlying ZIKV infection in the developing mammalian brain.

DISC1 Regulates Neurogenesis via Modulating Kinetochore Attachment of Ndel1/Nde1 during Mitosis

“…At day 45–47, NESTIN + RGCs in forebrain organoids proliferate robustly as revealed by mitotic NSC marker p-VIMENTINE (Figure S6A). To genetically manipulate and label RGCs in organoids, we delivered plasmids expressing GFP, GFP-DISC1 765–835, or GFP-DISC1 765–835 L822Q into organoids by electroporation (Figure S6B) (Yoon et al, 2017b). At 5 days post electroporation (dpe), we observed GFP-labeled Ki67 + PAX6 + proliferating RGCs in the ventricular zone-like structure (VZ, defined by PAX6 labeling; Figure S6C).…”

“…Plasmids expressing GFP, or the DISC1 765–835 peptide or the DISC1 765–835 L822Q peptide mixed with EGFP expressing plasmid pSUbGW) (~ 2μg/μl) were delivered to ventricular zone of embryo brain by in utero electroporation at E13.5 as previously described (Yoon et al, 2014; Yoon et al, 2017b). Briefly, DNA was injected using a beveled and calibrated micropipette with a ~10 μm opening at 15 psi, then five pulses (43 V, 50 ms in duration with a 950 ms interval) were delivered with tweezer electrodes (CUY650-5, Nepa Gene) by a CUY21SC electroporator (Nepa Gene).…”

“…Five pulses (43 V, 50 ms in duration with a 950 ms interval) were delivered with tweezer electrodes (CUY650–5, Nepa Gene) by a CUY21SC electroporator (Nepa Gene) as previously described (Yoon et al, 2017a; Yoon et al, 2017b). Electroporated organoids were transferred back to Spin 3 bioreactor and cultured until fixation.…”

Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation

“…Protocols for generation of forebrain organoids were detailed previously (Qian et al, 2016; Xu et al, 2016; Yoon et al, 2017). Briefly, human iPSCs were cultured in stem cell medium, consisting of DMEM:F12 (Invitrogen) supplemented with 20% Knockout Serum Replacer (Gibco), 1X Non-essential Amino Acids (Invitrogen), 1X Penicillin/Streptomycin (Invitrogen), 1X 2-Mercaptoenthanol (Millipore), 1X Glutamax (Invitrogen), and 10 ng/ml FGF-2 (Peprotech) on irradiated CF1 mouse embryonic fibroblasts (Charles River).…”

“…A mixture of 0.5 μl of plasmid DNA and 0.05% Fast green was injected into the lumen of neural tube structures in forebrain organoids using a calibrated micropipette (Yoon et al, 2017). About 3–4 locations on one side of each forebrain organoid were targeted by the injection.…”