Fig. 1: Postnatal lentiviral NEXMIF restoration increases expression of NEXMIF in adolescent KO mice. | Translational Psychiatry

Fig. 1: Postnatal lentiviral NEXMIF restoration increases expression of NEXMIF in adolescent KO mice.

From: Restoration of NEXMIF expression rescues abnormalities in gene transcription, neuron maturation and autistic-like behaviors in Nexmif knockout mice

Fig. 1

A Validation of the human FUW-hUbC-NEXMIF lentivirus (LV-NEXMIF). Cultured wildtype (WT) rat cortical neurons were infected with LV-NEXMIF or FUW control lentivirus (LV-Control) at 7 days in vitro (DIV) and immunostained for NEXMIF at DIV 14. Scale bar = 5 µm. B Quantification of (A) revealed that NEXMIF nuclear intensity was significantly increased in LV-NEXMIF-treated neurons (n = 97), relative to neurons infected with LV-Control (n = 84). C Western blot from DIV 14 neuronal lysates (left) and quantification (right) showing that NEXMIF protein was also significantly increased in LV-NEXMIF-treated neurons (n = 3 wells), relative to control (n = 3 wells). D Schematic illustration of the experimental timeline. LV-Control or LV-NEXMIF virus was bilaterally injected into the ventricles of WT or Nexmif knockout (KO) mouse brains at postnatal (P) days 0–1. The three injected groups were: WT + LV-Control (WT + CTRL), KO + LV-Control (KO + CTRL), and KO + LV-NEXMIF (KO + NEX) mice. From P30-P70, injected mice were subjected to a series of behavioral tests to examine rescue of ASD-like phenotypes. Following behavioral testing, mice were sacrificed and perfused for brain slice immunostaining, or brain tissue was collected for protein quantification and Golgi staining for spine morphology. E Western blot of NEXMIF and β-tubulin protein confirming successful restoration of NEXMIF expression in the medial prefrontal cortex (mPFC, left panel) and hippocampus (HPC, right panel) of KO + NEX mice. F Quantification of the western blots in (D) normalized to β-tubulin loading control revealed significantly increased expression of NEXMIF protein in the mPFC and HPC of KO + NEX mice, relative to KO + CTRL mice (n = 4 WT + CTRL, 3 KO + CTRL, 4 KO + NEX mice). G Representative brain slice images showing NEXMIF immunostaining (top row) merged with DAPI nuclear counterstain (bottom row) from layer 2/3 neurons of the mPFC in WT + CTRL (left panel), KO + CTRL (middle panel), and KO + NEX (right panel) mice. Scale bar = 100 µm. H Quantification of brain slice immunostaining of NEXMIF in WT + CTRL, KO + CTRL, and KO + NEX mice revealed significantly increased NEXMIF immunointensity in Layer 2/3 neurons of the mPFC in KO + NEX mice, relative to that in KO + CTRL mice (WT + CTRL: n = 91 cells, KO + NEX: n = 63 cells). Data are represented as average ± SEM. Two-tailed student’s t test (B-C) or One-way ANOVA (F, H) with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant. Figure 1D created with Biorender.com.

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