Map3k3  I441M Knock-In Mouse Model of Cerebral Cavernous Malformations.

BACKGROUND

Cerebral cavernous malformations (CCMs) refer to vascular dysplasia primarily found in the brain, affecting ≈0.5% of the population. A somatic Map3k3I441M mutation has been found in ≈40% of patients with sporadic CCMs, which were typically accompanied by somatic gain-of-function mutations in PIK3CA. Although mouse models of adeno-associated virus-BR1-mediated mutant overexpression have been reported, these models have limitations in representing clinical specimens of CCMs, which typically harbor single allele mutation in Map3k3. A Map3k3I441M knock-in murine model of CCMs has not yet been established.

METHODS

The Map3k3I441M knock-in mice were crossed with Cdh5-creERT2 mice to induce mutant gene expression specifically in endothelial cells. Subsequently, Map3k3I441M mice were bred with Ptenfl/fl mice to generate Map3k3I441M; Ptenfl/fl mice. In both murine models, CCM lesions were examined using magnetic resonance imaging, while single-cell RNA sequencing and immunostaining were utilized to investigate the pathomechanism of the mutation. Finally, we administered an mTOR (mechanistic target of rapamycin) inhibitor to explore its therapeutic effect on lesions of both murine models.

RESULTS

Both endothelial Map3k3I441M mutant juvenile mice and Map3k3I441M; Ptenfl/fl mice developed abnormal lesions with human CCM characteristics. In Map3k3I441M mice, the mutant promoted endothelial apoptosis, while activation of the PI3K (phosphatidylinositol 3-kinase) pathway was able to activate the downstream AKT (protein kinase B)/mTOR/p-S6 (phosphorylated S6 ribosomal protein) pathway and upregulate VEGFA (vascular endothelial growth factor A) expression, counteracting apoptosis, and facilitating lesion progression. The activation of PI3K signaling is required for Map3k3I441M to generate CCM-like lesions in adult mice. Finally, we demonstrated that rapamycin effectively inhibited the formation of lesions in the Map3k3I441M mice and Map3k3I441M; Ptenfl/fl mice.

CONCLUSIONS

Map3k3I441M heterozygous is sufficient to induce lesions in juvenile mice, while the additional activation of PI3K signaling is required for the effective formation of CCMs at the adult stage. The Map3k3I441M murine model provides a preclinical model for further mechanistic and therapeutic studies of CCMs.