Evidences of PIEZO1 involvement in cerebral cavernous malformation pathogenesis

Dear Editor, Familial cerebral cavernous malformations (CCM) are inherited vascular lesions affecting brain capillaries. Involved vessels are tangled and enlarged due to cell junction impairment. This results in an increased permeability of blood-brain barrier (BBB), further worsened by deficiency of pericytes. Germ-line mutations at the three loci KRIT1/ CCM1 (HGNC ID: 1573; 7q11.2-21), MGC4607/CCM2 (HGNC ID: 21708; 7p13) and PDCD10/CCM3 (HGNC ID: 8761; 3q26.1) have been linked to this autosomal dominant condition. However, several studies report absence of germline mutations in about 6–10% of patients with familial history of CCM, making existence of a fourth CCM locus an increasingly accredited hypothesis (Scimone et al., 2017). However, to date, there are not data in literature reporting novel potential candidate genes. Here, we introduce preliminary results showing the mechano- sensitive cation channel encoding gene, PIEZO1 (HGNC ID: 28993; 16q24.3) as a potential locus involved in CCM onset. Whole exome sequencing (WES) and segregation analysis of a family with positive hereditary history of CCM and carrying no mutations in the CCM genes highlighted the novel c.3229C>A missense mutation in the PIEZO1 locus (Fig. 1a). It affects the exon 21 and results in the p.Phe991Leu amino acid substitution and it segregates within the family in all affected members. This novel mutation was predicted to be probably damaging, with high score, by the two prediction tools MutationTaster (htt ps://www.mutationtaster.org/) and PolyPhen2 (http://genetics.bwh. harvard.edu/pph2/). Multiple alignment, indeed, showed as the 991F amino acid is highly conserved in PIEZO1 protein, in several mammalian and bird species (Fig. 1b). PIEZO1 encodes for a mechanosensitive Ca2+ ion channel that links mechanical forces, as blood flow or shear stress, to biological signals. In endothelial cells, Ca2+ ion homeostasis contributes to maintaining architecture of cytoskeleton, adherens and tight junc- tions, regulating BBB. Both increase and decrease in calcium concentration within endo- thelial cells cause cell junction disorganization and cytoskeleton remodeling, resulting in BBB loss of properties (Stoica et al., 2021). Although dysfunction of several ion channels has been associated to perturbation of Ca2+ signaling in some nervous pathological conditions such as epilepsy and neurodegenerative diseases, role of PIEZO1 is still poorly investigated (Velasco-Estevez et al., 2019). It is known that it is expressed on the surface of several endothelial cells, as aorta ones and Human Umbilical Vein Endothelial Cells (HUVECs), where contribute to maintenance of cell polarity, driven by wall shear stress (Kang et al., 2019). Moreover, it was recently shown that PIEZO1 drives sprouting angiogenesis during brain development in zebrafish and homozygous knock-down larvae fail in development of vasculature patterns (Liu et al., 2020). Finally, a very recent study performed by Harraz et al. highlighted that Piezo1 drives Ca2+ signals also in brain capillaries modulating cerebral blood flow (Harraz et al., 2022). Taken together, all these data suggest a possible involvement of PIEZO1 in cerebral cavernous malformation pathogenesis, being CCM lesions characterized by enlarged capillaries with defective junctions and increased perme- ability and they are prone to hemorrhagic events. The three CCM genes, indeed, encode for proteins that co-localize at adherens junctions where they the CCM signaling complex (CSC). The CSC contributes to main- tenance of cell-cell junction integrity and it is also involved in β-integrin transduction signaling, encouraging endothelial cell-extracellular ma- trix adhesion (Abou-Fadel et al., 2019). For this reason, by real time-PCR we measured the expression level of the three CCM genes in Human Brain Microvascular Endothelial Cells (HBMECs) after treatment with 1.5 μM Yoda1. Yoda1 is a small synthetic compound acting as a PIEZO1 agonist, mimicking the constitutive cation channel activation. Interest- ingly, both KRIT1 and CCM2 gene expression decreased in treated HBMECs, following 3 h exposure. In contrast, PDCD10 expression did not undergo to significative change (Fig. 1c). This event can result in gain of BBB permeability, suggesting a possible role for PIEZO1 in CCM development. Data here reported are preliminary and further studies are required to confirm this hypothesis. In particular, it remain to be elucidated the expression trend of the three CCM genes following both shorter a longer time scales after Yoda1 treatment. Moreover, it is not yet proven if this novel mutation results in gain or loss of function for the mechanosensitive channel Piezo1. Therefore, we also aim to charac- terize the mutation in order to better understand the mechanism by which it can results in CCM phenotype. Therefore, we believe that this could be a valid field for further investigation in order to clarify new mechanism in CCM pathogenesis.