Extended Data Fig. 4: Immature Neurons recDEG Atpif1 knock-down disrupts neuronal proliferation and differentiation in vitro.
a-d, Primary cortical neurons were transduced with LV-shRNA for five days. PrestoBlue HS normalized cell viability (a, one-way ANOVA followed by Dunnett’s against shCtrl: shSlc25a4 n.s. P = 0.9967, shAtp6v0c n.s. P = 0.0573, shAtpif1 *P = 0.013), confirmation of the gene knock-down (KD) by qPCR (b, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl ****P < 0.0001), and gene expression of neuronal markers in response to Atp6v0c (c, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl: Neurod1 ****P < 0.0001, Dcx **P = 0.0017, Tubb3 n.s. P = 0.8694, Map2 ***P = 0.0003, Dlg4 **P = 0.0071, Syn1 **P = 0.0037, Bax *P = 0.028) and Atpif1 knock-down (d, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl: Neurod1, Dcx, Map2, Dlg4, and Syn1 ****P < 0.0001, Tubb3 **P = 0.0046, Bax **P = 0.0022). shAtp6v0c and shSlc25a4 n = 6, shAtpif1 n = 4 (a), shAtp6v0c and shAtpif1 n = 6, shSlc25a4 n = 5 (b-d). e, Representative confocal images of EdU (red) and Nestin (green) staining of embryonic neural stem and progenitor cells transduced with LV-shRNA and maintained in proliferating media for 5 days. Scale bar, 50 µm. f-h, Neurospheres were transduced with LV-shRNA and maintained in differentiation media for five days. Confirmation of the gene knock-down by qPCR (f, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl ****P < 0.0001), and gene expression of neuronal markers in response to Atp6v0c (g, two-way ANOVA, KD n.s. P = 0.0877, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl: Neurod1 and Tubb3 ****P < 0.0001, Dcx n.s. P = 0.3704, Map2 n.s. P = 0.0826, Dlg4 n.s. P = 0.1893, Syn1 n.s. P = 0.1901) and Atpif1 knock-down (h, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl: Neurod1, Dcx, Tubb3, and Map2 ****P < 0.0001, Dlg4 **P = 0.0015, Syn1***P = 0.0004). n = 11 per group. i-l, Primary cortical neurons were transduced with LV-shRNA for five days and treated with 20 µM recombinant amyloid-beta 42 for the last 16 h (i and j), or Abeta-enriched Tg2576 conditioned-media for the last 3 h (k and l). Normalized calcein fluorescent signal indicative of live cells after 16 h amyloid-beta 42 treatment (i, Welch’s ANOVA followed by Dunnett’s T3 against shCtrl ****P < 0.0001), normalized EthD1 fluorescent signal indicative of dead cells after 16 h amyloid-beta 42 (j, Welch’s ANOVA followed by Dunnett’s T3 against shCtrl, shAtp6v0c **P = 0.0055, shAtpif1 ***P = 0.0002), normalized calcein fluorescent signal after 3 h Tg2576 conditioned-media (k, Welch’s ANOVA followed by Dunnett’s T3 against shCtrl ****P < 0.0001), and normalized EthD1 fluorescent signal after 3 h Tg2576 conditioned-media (l, one-way ANOVA followed by Dunnett’s against shCtrl, shAtp6v0c n.s. P = 0.0726, shAtpif1 n.s. P = 0.0754). n = 6 per group. m-o, Adult hippocampus derived neurospheres were transduced with LV-shRNA and maintained in differentiation media for three days. Confirmation of the gene knock-down by qPCR (m, two-tailed unpaired t-test ****P < 0.0001), PrestoBlue HS normalized cell viability (n, two-tailed unpaired t-test **P = 0.0065), and gene expression of neuronal markers in response to Atpif1 knock-down (o, two-way ANOVA, KD ****P < 0.0001, KD x gene ****P < 0.0001, followed by Fisher’s LSD compared to shCtrl: Neurod1, Dcx, and Map2 ****P < 0.0001, Tubb3 n.s. P = 0.681, Dlg4 *P = 0.0152, Syn1 n.s. P = 0.7519, Bax n.s. P = 0.9609). n = 4 (n) and 12 per group (m, o). Data represent the mean ± s.e.m. of biologically independent samples.
