Developing Zebrafish Models to Study COL4A1-Related Disease ^†

Cerebral small vessel disease (cSVD) is a leading contributor to both stroke and vascular dementia, yet its underlying mechanisms remain poorly understood, and treatment options are limited [1]. Variants in the COL4A1 gene are known to cause a monogenic form of cSVD by disrupting the integrity of the cerebrovascular basement membrane, causing variable symptoms that are partially dependent on the age at which cerebrovascular events occur [2]. Zebrafish larvae offer a powerful model for studying cerebrovascular disorders, thanks to several experimental advantages, including optical transparency and suitability for live imaging [3]. In this study, we developed and characterised a zebrafish crispant model carrying mosaic deleterious mutations in col4a1, which successfully replicates key features of COL4A1-related cSVD, including spontaneous brain haemorrhages and cerebrovascular abnormalities. We also observed basement membrane defects and increased mmp9 (encoding matrix metalloprotease 9) expression associated with loss of col4a1. Furthermore, we generated a stable mutant zebrafish line carrying a 20-base pair deletion in col4a1, which exhibited cerebrovascular defects, impaired swimming behaviour, and heightened sensitivity to drug-induced brain haemorrhage during larval development. In adulthood, mutant animals developed spontaneous brain haemorrhages that were visible in freely swimming fish. Altogether, our findings support zebrafish as a new valid preclinical model system for COL4A1-associated cSVD, offering valuable insights into disease mechanisms and opportunities for translational research.