Sex Differences in CASA-Mediated Autophagy in ALS Glial Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that affects men and women differently, both clinically and at the cellular level. Glial cells—astrocytes and microglia—are key players in disease progression. One protective mechanism in these cells is chaperone-assisted selective autophagy (CASA), which helps clear misfolded proteins. However, whether CASA is regulated differently by sex in ALS remains unclear. To explore this, we analyzed spinal cord astrocytes from adult SOD1-G93A ALS mice. RNA sequencing revealed 620 differentially expressed genes (DEGs) in females and 187 in males, including a reduction in HSPB8 and HSPB6 in female ALS astrocytes. Conversely, qPCR showed increased expression of HSPB8 and BAG3 in male ALS astrocytes, while females showed a decrease. No differences were observed in astrocytes isolated at postnatal day 2 (P2), suggesting age-related changes. Autophagy markers confirmed these trends: p62 body counts were higher in wild-type (WT) males than in G93A, and NH4Cl treatment (blocking autophagy) enhanced this difference. In females, G93A astrocytes showed significantly more p62 bodies than WT, indicating impaired protein clearance. Female WT astrocytes also had fewer p62 bodies than male WT, hinting at more efficient baseline autophagy. LC3 puncta, representing autophagosomes, were reduced in G93A versus WT astrocytes in males, but unchanged in females. In microglial cultures activated with cytokines, LC3 mRNA decreased in both sexes in G93A samples, while BAG3 protein expression dropped specifically in females, regardless of genotype. Overall, male astrocytes appear to compensate for ALS-related stress by upregulating CASA components. Female astrocytes, although more autophagy-competent at baseline, seem more susceptible to ALS-induced dysfunction. These findings highlight the importance of considering sex as a biological variable in ALS research and therapeutic design.