HTRA1 deficiency in COL4A1 mutant hiPSC-derived astrocytes: a convergent mechanism of cerebral small vessel disease

Cerebral small vessel disease (cSVD) is a major contributor to stroke and cognitive decline, ultimately leading to vascular dementia (VaD). Genetic factors play a key role in disease susceptibility and progression, and variants in COL4A1 cause one of the most common forms of genetic cSVD. COL4A1 encodes the α1 chain of collagen type IV which is the major structural component of the basement membrane, a specialised extracellular matrix (ECM) structure, in the vasculature. In addition to this vascular basement membrane (vBM), in the central nervous system (CNS), the neurovascular unit (NVU) also has a unique parenchymal basement membrane (pBM) that is largely produced by astrocytes and forms a critical interface anchoring astrocyte end-feet to vascular cells. Together, these BMs play essential roles in regulating blood-brain barrier (BBB) function. However, the role of the pBM in cSVD has been relatively less investigated compared to the vBM. The lack of relevant human disease models that faithfully recapitulate the pBM, makes it difficult to dissect pBM-related cell-cell and cell-matrix interactions specific to cSVD, hindering the identification of effective therapeutic targets. In this study, we hypothesised that astrocyte-mediated ECM remodelling contributes to BBB dysfunction in COL4A1 -associated cSVD. To investigate this, human induced pluripotent stem cells (iPSCs) derived from a patient carrying the COL4A1 ^{G 755 R} variant and its isogenic control line were differentiated into astrocytes and brain microvascular endothelial cells (BMECs). Comparing to isogenic controls, the COL4A1 ^{G 755 R} astrocytes significantly reduced the expression of ECM-related genes and increased glutamate uptake. ECM preparations from COL4A1 ^{G 755 R} astrocytes significantly damaged the tight junction (TJ) structure formed by control iPSC-BMECs and failed to rescue the TJ integrity in COL4A1 ^{G 755 R} BMECs. The secretome from COL4A1 ^{G 755 R} astrocytes exacerbated the ECM defects in COL4A1 ^{G 755 R} BMECs. Most importantly, COL4A1 ^{G 755 R} astrocytes exhibited reduced expression of HTRA1, a serine protease that regulates both ECM turnover and homeostasis, and increased TGF-β signalling. Functional rescue by recombinant human HTRA1 protein rescues TJ defects in COL4A1 ^{G 755 R} BMECs, and normalized TGF-β signalling and glutamate uptake in COL4A1 ^{G 755 R} astrocytes. Together, these findings define a previously unrecognised astrocyte-driven pBM mechanism in COL4A1 -associated cSVD and highlight HTRA1 in ECM remodelling as a therapeutic target for cSVD.