Gag-like proteins: Novel mediators of prenatal alcohol exposure in neural development

Abstract

Background: We previously showed that ethanol did not kill fetal neural stem cells (NSCs), but that their numbers nevertheless are decreased due to aberrant maturation and loss of self-renewal. To identify mechanisms that mediate this loss of NSCs, we focused on a family of Gag-like proteins (GLPs), derived from retroviral gene remnants within mammalian genomes. GLPs are important for fetal development, though their role in brain development is virtually unexplored. Moreover, GLPs may be transferred between cells in extracellular vesicles (EVs) and thereby transfer environmental adaptations between cells. We hypothesized that GLPs may mediate some effects of ethanol in NSCs.

Methods: Sex-segregated male and female fetal murine cortical NSCs, cultured ex vivo as nonadherent neurospheres, were exposed to a dose range of ethanol and to mitogen-withdrawal-induced differentiation. We used siRNAs to assess the effects of NSC-expressed GLP knockdown on growth, survival, and maturation and in silico GLP knockout, in an in vivo single-cell RNA-sequencing dataset, to identify GLP-mediated developmental pathways that were also ethanol-sensitive.

Results: PEG10 isoform-1, isoform-2, and PNMA2 were identified as dominant GLP species in both NSCs and their EVs. Ethanol-exposed NSCs exhibited significantly elevated PEG10 isoform-2 and PNMA2 protein during differentiation. Both PEG10 and PNMA2 were mediated apoptosis resistance and additionally, PEG10 promoted neuronal and astrocyte lineage maturation. Neither GLP influenced metabolism nor cell cycle in NSCs. Virtual PEG10 and PNMA2 knockout identified gene transcription regulation and ubiquitin-ligation processes as candidate mediators of GLP-linked prenatal alcohol effects.

Conclusions: Collectively, GLPs present in NSCs and their EVs may confer apoptosis resistance within the NSC niche and contribute to the abnormal maturation induced by ethanol.

Keywords: extracellular vesicles; gag-like proteins; neural stem cells; prenatal alcohol exposure.

FIGURE 1

Previous EtOH exposure increases PEG10 and PNMA2 protein expression during early differentiation of NSCs. Representative western immunoblots (A) stained for total protein and (B) with antibodies (green fluorescence) to PEG10 isoforms 1 and 2 and antibodies (red fluorescence) to PNMA2. Quantitative analysis of relative protein levels of (C) PEG10 isoform 1, (D) PEG10 isoform 2, and (E) PNMA2 on Days 0, 1, and 3 of early differentiation of NSCs. The same composite sample (see methods) was included on each immunoblot to allow for comparisons between blots. n = 5 samples per group. # = main effect of Treatment, * = main effect of Day, + = interaction effect, “Treatment by Day,” $ = interaction effect, “Sex by Treatment by Day.” Significance was determined using 3‐way ANOVA. ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001

FIGURE 2

PEG10 and PNMA2 are present in EVs. (A) Total protein stain of male and female NSCs and EVs isolated from culture media of the NSCs. (B) Immunoblot showing presence of GLPs in red and presence of cell‐enriched proteins (anti‐DREBRIN, NDUFS1, and ATPB antibodies) in green

FIGURE 3

PEG10 and PNMA2 are anti‐apoptotic and do not affect cellular metabolism. (A) Quantification of caspase 3/7 activity by luciferase assay following PEG10 and PNMA2 knockdown. (B) MTT absorbance values following knockdown of PEG10 and PNMA2 in NSCs. n = 5 samples per group. * = main effect of Treatment by 2‐way ANOVA. *p < 0.05, ****p < 0.0001

FIGURE 4

PEG10 and PNMA2 do not influence cell cycle dynamics in NSCs. Proportion of cells in (A) G0/G1, (B) G2/M, or (C) S phase of the cell cycle following PEG10 and PNMA2 siRNA‐mediated knockdown. (D) Representative flow cytometry image. For panel (A), mean ± SEM for each group is as follows: Scrambled Male = 48.00% ± 0.45, Scrambled Female = 49.75% ± 0.26, KD PEG10 Male = 49.11% ± 0.46, KD PEG10 Female = 50.37% ± 0.48, KD PNMA2 Male = 48.41% ± 0.45, KD PNMA2 Female = 51.27% ± 1.09. n = 5 samples per group

FIGURE 5

PEG10 has a role in neuronal and astrocytic lineage differentiation of NSCs. Expression of (A) Rbfox3/NeuN, (B) Nes, (C) Olig2, (D) Pdgfra, (E) Gfap, and (F) Glast in NSCs following siRNA‐mediated knockdown of PEG10 and PNMA2. n = 5 samples per group, * = main effect of Treatment by 2‐way ANOVA. *p < 0.05

FIGURE 6

Virtual KO reveals genes linked to PEG10 and PNMA2. Genes predicted to have altered expression by virtual KO of (A) Peg10 and (B) Pnma2. (C) PEG10 and PNMA2 transcript expression in GD14.5 fetal mouse VZ, SVZ, and TPC as identified in scRNA‐seq