Loss of col4a1 in zebrafish recapitulates the cerebrovascular phenotypes associated with monogenic cerebral small vessel disease

Cerebral small vessel disease (cSVD) is a major cause of vascular dementia and stroke. Our understanding of cSVD pathophysiology is incomplete and our ability to treat patients is limited. Pathogenic variants in type IV collagen alpha 1 ( COL4A1 ) cause a monogenic form of cSVD with variable age of onset, via disturbance of cerebrovascular basement membranes. Zebrafish larvae are a powerful model organism for studying cerebrovascular disease due to their optical clarity and applicability for live imaging. In this study, we characterised a zebrafish crispant model for loss-of-function COL4A1 -associated cSVD that successfully recapitulates key disease features, including spontaneous intracerebral haemorrhage and cerebrovascular abnormalities. We also identified evidence for abnormal cerebrovascular basement membranes and elevated matrix metalloproteinase 9 ( mmp9) transcription associated with loss of col4a1 . Pharmacological inhibition of mmp9 was sufficient to ameliorate some cerebrovascular phenotypes. Finally, we describe the generation of a mutant line carrying a germline-transmissible 20bp deletion in zebrafish col4a1 ( col4a1 Δ20 ) which is associated with cerebrovascular abnormalities, swimming defects and increased susceptibility to pharmacologically induced brain haemorrhages during larval stages. In adulthood, mutant col4a1 Δ20 animals developed spontaneous brain haemorrhages that were observable in free-swimming fish. Overall, this study validates the use of zebrafish disease modelling for preclinical COL4A1 -associated cSVD research and highlights its potential for further understanding disease pathophysiology and future drug discovery projects.