Defective autophagy is a key feature of cerebral cavernous malformations.

Saverio Marchi,Mariangela Corricelli,Letizia Ferroni,Saverio Francesco Retta,Alessandra Pittaro,Carlotta Giorgi,Elisabetta Dejana,Luca Bravi,Simone Patergnani,Eliana Trapani,Alessandro Rimessi,Sonia Missiroli,Barbara Zavan,Paolo Pinton,Simona Delle Monache,Luca Goitre,Paola Cassoni,Lorenza Trabalzini

doi:10.15252/emmm.201505316

Abstract

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

Highlights

We show that human Cerebral Cavernous Malformation (CCM) lesions display increased levels of p62/SQSTM1, an autophagic marker that accumulates when autophagy is inhibited, and demonstrate that both KRIT1 and CCM3 loss-of-function impair autophagy through the upregulation of the mechanistic target of rapamycin pathway, leading to a defective quality control system and the accumulation of aberrant and aggregated proteins
To study the contribution of autophagy to CCM pathogenesis, we investigated whether KRIT1 down-regulation would lead to the impairment of autophagy in endothelial cell lines
The autophagy protein microtubule-associated protein 1 light chain 3 (LC3) is present in the cytosol in the LC3-I form, until it is modified to a cleaved and lipidated membrane-bound form (LC3-II), which is localized to autophagosomes

Summary

Introduction

Cerebral cavernous malformations (CCMs; OMIM 116860), which are known as cavernous angiomas or cavernomas, are major vascular malformations consisting of closely clustered, abnormally dilated, and leaky capillary channels (caverns) lined by a thin endothelium and devoid of normal vessel structural components (Clatterbuck et al, 2001; Gault et al, 2004; Batra et al, 2009; Cavalcanti et al, 2012). No direct therapeutic approaches for CCM disease exist other than the surgical removal of accessible lesions in patients with recurrent hemorrhage or intractable seizures. The suppression of autophagy causes the accumulation of proteins and potentially hazardous intracellular structures, thereby inducing high levels of metabolic stress and limiting organelle functionality. We show that human CCM lesions display increased levels of p62/SQSTM1, an autophagic marker that accumulates when autophagy is inhibited, and demonstrate that both KRIT1 and CCM3 loss-of-function impair autophagy through the upregulation of the mechanistic target of rapamycin (mTOR) pathway, leading to a defective quality control system and the accumulation of aberrant and aggregated proteins. Our data raise the possibility that therapeutic activation of autophagy might prevent or reverse adverse clinical outcomes, improving the long-term prognosis of CCM patients

Results and Discussion

80 ATG7 siRNA

Materials and Methods

Results

Conflict of interest