Aquaporin-4 deficiency induced white matter injury via upregulated complement component 3 in cerebral small vessel disease

Cerebral small vessel disease (CSVD) is the most common neurological disorder associated with a high incidence of stroke and dementia. For the heterogeneity of the pathogenesis, effective preventive and therapeutic strategies remain limited. Reduced aquaporin-4 (AQP4) was reported in the development of CSVD, while its role and mechanisms have not been fully elucidated. We employed logistic regression analysis to assess the association between AQP4 gene single-nucleotide polymorphisms (SNPs) and CSVD presence. The functional impact of SNP mutations on AQP4 gene was evaluated using a Luciferase assay. Subsequently, Aqp4−/− mice were subjected to bilateral carotid artery stenosis (BCAS) surgery to detect the CSVD phenotype of Aqp4 reduction. Aqp4+/+ and Aqp4−/− mice in the Sham and BCAS groups were subjected to MRI, histological examinations, and behavioural tests. AAV2/9-Gfap-AQP4M1 and AAV2/9-Gfap-AQP47×cMyc−Kras were used to investigate Aqp4 expression and translocation ability after BCAS. Furthermore, RNA-sequencing was performed on the corpus callosum (CC) and Aqp4−/− astrocytes to identify transcriptional changes associated with Aqp4 deficiency. We identified four SNPs (rs335929, rs335930, rs335931 and rs455671) were significantly associated with CSVD presence. Among these, rs335929 variant was correlated with reduced AQP4 mRNA expression. Compared with Aqp4+/+ mice, fractional anisotropy values were decreased in the Sham and BCAS groups in Aqp4−/− mice. Under BCAS conditions, Aqp4−/− mice exhibited more severe demyelination and myelin density in the CC. Intraventricular delivery of AAV2/9-Gfap-AQP4M1 and AAV2/9-Gfap-AQP47×cMyc−Kras attenuated BCAS-induced white matter injury and cognitive function, no significant differences were observed between the two AAV treatment groups. RNA sequencing analysis indicated upregulation of inflammatory responses and complement cascades in the CC of Aqp4−/− mice. Complement component 3 (C3) mRNA was notably elevated in astrocytes isolated from Aqp4−/− mice. Treatment with a C3a receptor antagonist in Aqp4−/− mice improved myelin integrity and reduced MBP loss following BCAS. Reduced AQP4 expression was associated with CSVD presence in clinical studies. Experimentally, Aqp4 deficiency has been shown to exacerbate white matter injury, that effect may be mediated by upregulation of C3 mRNA in Aqp4-deficient astrocytes. Targeting C3 activation may represent a promising strategy to mitigate AQP4 loss-induced white matter injury in CSVD.