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Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1−/− iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1−/− precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.

KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell–cell and cell–matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein., Summary: A novel role for PKC signaling in triggering Ser/Thr phosphorylation and regulating nucleocytoplasmic shuttling of KRIT1, a major protein with pleiotropic functions associated with human diseases.

This article contains additional data related to the original research article entitled “KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: implication for Cerebral Cavernous Malformation disease” (Antognelli et al., 2017) [1].Data were obtained by si-RNA-mediated gene silencing, qRT-PCR, immunoblotting, and immunohistochemistry studies, and enzymatic activity and apoptosis assays. Overall, they support, complement and extend original findings demonstrating that KRIT1 loss-of-function induces a redox-sensitive and JNK-dependent sustained upregulation of the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), and a drop in intracellular levels of AP-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that sensitizes cells to oxidative DNA damage and apoptosis.In particular, immunoblotting analyses of Nrf2, Glo1, AP-modified Hsp70 and Hsp27 proteins, HO-1, phospho-c-Jun, phospho-ERK5, and KLF4 expression levels were performed both in KRIT1-knockout MEF cells and in KRIT1-silenced human brain microvascular endothelial cells (hBMEC) treated with the antioxidant Tiron, and compared with control cells. Moreover, immunohistochemistry analysis of Nrf2, Glo1, phospho-JNK, and KLF4 was performed on histological samples of human CCM lesions. Finally, the role of Glo1 in the downregulation of AP-modified Hsp70 and Hsp27 proteins, and the increase in apoptosis susceptibility associated with KRIT1 loss-of-function was addressed by si-RNA-mediated Glo1 gene silencing in KRIT1-knockout MEF cells. Keywords: Cerebrovascular disease, Cerebral cavernous malformations, CCM1/KRIT1, Oxidative stress, Antioxidant defense, Adaptive redox homeostasis, Redox signaling, Nuclear factor erythroid 2-related factor 2 (Nrf2), c-Jun, Glyoxalase 1 (Glo1), Heme oxygenase-1 (HO-1), Argpyrimidine-modified heat-shock proteins, Oxidative DNA damage and apoptosis

Cerebral cavernous malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or can be inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM disease exhibits a range of different phenotypes, including wide interindividual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Mutations of the KRIT1 gene account for over 50% of familial cases. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered homeostasis of intracellular reactive oxygen species (ROS) and abnormal activation of redox-sensitive transcription factors, which collectively result in pro-oxidative, pro-inflammatory, and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease. Consistently, these original discoveries have been confirmed and extended by subsequent findings showing mechanistic relationships between pleiotropic redox-dependent effects of KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis. In this chapter, we describe few basic methods used for qualitative and quantitative analysis of intracellular ROS in cellular models of CCM disease.

Cerebral cavernous malformations (CCMs) is a disorder of endothelial cells predominantly localized in the brain. Although a complete inactivation of each CCM protein has been found in the affected endothelium of diseased patients and a necessary and additional role of microenvironment has been demonstrated to determine in vivo the occurrence of vascular lesions, a microvascular endothelial model based on knockdown of a CCM gene represents today a convenient method to study some of critical signaling events regulating pathogenesis of CCM. For these reasons, in our laboratory we developed a microvascular cerebral endothelial model of Krit1 deficiency performing silencing experiments of CCM1 gene (Krit1) with siRNA procedure.

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3–0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies., Graphical abstract Schematic models representing adaptive redox responses associated with KRIT1 loss-of-function. KRIT1 loss-of-function causes a persistent activation of the redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS* and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp70 and AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. Inter-individual differences in Nrf2-mediated adaptive defense mechanisms might influence susceptibility to CCM disease onset and progression. *The generic ROS term refers to O2•−and H2O2as well as to putative secondary oxidative products that might be implicated without certainty.fx1, Highlights • KRIT1 loss causes a chronic adaptive redox response based on the JNK-Nrf2-Glo1 axis. • Phospho-JNK, Nrf2 and Glo1 are upregulated in endothelial cells lining human CCMs. • Defective autophagy contributes to the sustained upregulation of the Nrf2-Glo1 axis. • Nrf2-Glo1 upregulation causes a drop of AP-modified Hsp70 and Hsp27 proteins. • Sustained Nrf2-Glo1 activation sensitizes cells to oxidative stress and apoptosis.

Graphical abstract Towards a unifying mechanism for CCM disease pathogenesis and treatment. CCM proteins play pleiotropic roles in distinct redox-sensitive pathways by modulating the fine-tuned crosstalk between redox signaling and autophagy. Effective autophagy removes ROS-generating cellular trash, including damaged mitochondria, to rejuvenate cell environment, thus serving a cytoprotective function for the maintenance of endothelial cell monolayer integrity and functionality and blood–brain barrier (BBB) stability even under adverse stress conditions. Loss-of-function of a CCM protein (e.g., KRIT1) causes defective autophagy and altered redox signaling, affecting BBB stability and sensitizing endothelial cells to local oxidative stress and inflammatory events, which may act as key pathogenic determinants of focal formation and progression of CCM lesions. The common capacity to modulate the interplay between autophagy and redox signaling reconciles the distinct pharmacological approaches proposed so far for CCM disease prevention and treatment., Highlights • CCM proteins play pleiotropic roles in various redox-sensitive signaling pathways. • CCM proteins modulate the crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses. • Oxidative stress and inflammation are emerging as key focal determinants of CCM lesion formation, progression and severity. • The pleiotropic functions of CCM proteins may prevent vascular dysfunctions triggered by local oxidative stress and inflammatory events. • The distinct therapeutic compounds proposed so far for CCM disease share the ability to modulate redox signaling and autophagy., Cerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity. This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies.