294 - Differential Impact of Gene-Environment Interaction on Neural Cellular Composition During Neurodevelopment in Males versus Females

Publication Number: 4289.294

Christiana Santiago, UCLA Mattel Childrens Hospital, Los angeles, CA, United States; Bo-Chul Shin, UCLA Mattel Childrens Hospital, Los Angeles, CA, United States; Yasemin Ozay, UCLA Mattel Childrens Hospital, Los Angeles, CA, United States; Shubhamoy Ghosh, University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA, United States; Tomoko Daida, Tokyo Medical Univesity, Shinjuku, Tokyo, Japan; Sherin U.. Devaskar, University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA, United States

Background: Maternal malnutrition contributes to intrauterine growth restriction (IUGR) and long-term neurocognitive deficits. When combined with genetic susceptibility ("primary hit")-such as a neuronal GLUT3 mutation linked to seizures and microcephaly-maternal dietary restriction may serve as a "second hit," worsening neurodevelopmental outcomes. We studied the combined effects of prenatal maternal calorie restriction (50%CR, 25%CR, or ad libitum [AD]) and a neural-specific GLUT3 mutation on embryonic brain development.

Objective: We determined if calorie restriction would impair neurodevelopment in a dose-dependent manner and exacerbate deficits in the GLUT3 knockout (KO) condition.

Design/Methods: Pregnant dams carrying Emx1-GLUT3 KO or wild-type (CON) fetuses underwent 50%CR, 25%CR, or AD feeding from embryonic day (E)10-19. Fetal cortices were collected at E19 for body and head weight measurements (Figure 1; n per group shown). Single-nucleus RNA sequencing (10X Genomics) was performed on E19 cortices (n=3/group), followed by differential expression (FDR=5%) and cell-type annotation (Figure 2). Immunohistochemistry (IHC) and Western blot (WB) analyses are ongoing. Statistics included ANOVA with Tukey's post hoc or Student's t-test (P < 0.05).

Results: Body and head weights were significantly reduced in IUGR (25%CR, 50%CR) and "second-hit" (25%CR KO, 50%CR KO) groups versus the wildtype AD CON (P < 0.0001), with no change in litter size. SnRNA-seq showed that in males, 25%CR reduced mature neuronal and glial populations-including upper-layer callosal projection and corticothalamic neurons, oligodendrocytes, Cajal-Retzius cells, and astrocytes-which were more pronounced when combined with the KO genotype, while concurrently expanding atypical and intermediate progenitor pools. These effects were further amplified in 50%CR and 50%CR KO groups. In females, similar but more mild reductions occurred in 50%CR and 50%CR KO, while 25%CR and 25% KO females showed no changes.

Conclusion(s): Both 25% and 50% maternal calorie restriction reduced fetal growth in both sexes of KO and CON mice. Neurodevelopmental disruption was dose-dependent and more severe in males under 25% and 50%CR conditions, indicating sex-specific vulnerability to caloric restriction. These changes were more pronounced in the "second hit" (KO) conditions. These findings highlight male-biased susceptibility to IUGR combined with a GLUT3 mutation. Ongoing WB and IHC analyses will further validate these results. Supported by NICHD HD81206, R25 HD109116, UCLA Broad Stem Cell Center, AAP Marshall Klaus, and UCLA CDI Fellows Awards.

Bar graphs showing mean body and head weights of male and female pups at E19.
Group sizes (AD, AD KO, 25% CR, 25% CR KO, 50% CR, 50% CR KO) are shown below each bar. CON pups (no GLUT3 mutation) are shown in blue; KO pups (GLUT3 mutation) in purple. Error bars denote SEM. N per group is shown within each bar. Statistical analysis: ANOVA with Tukey's post hoc test; *P < 0.05, ns = not significant.

Figure 2. Stacked bar graph showing cell-type proportions across groups by sex.
Colors (key at left) denote experimental groups (n = 3 pups each). Each bar represents the percentage of total cells (n = shown above bars) for each cell type. Abbreviations: CThPN, corticothalamic projection neurons; DL CPN, deep-layer callosal projection neurons; NP, near-projecting neurons; SCPN, subcerebral projection neurons; UL CPN, upper-layer callosal projection neurons; VLMC, vascular leptomeningeal cells.