CCM3/PDCD10 heterodimerizes with germinal center kinase III (GCKIII) proteins using a mechanism analogous to CCM3 homodimerization

CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization.     

Ccm1 is required for arterial morphogenesis: implications for the etiology of human cavernous malformations

Hemorrhagic stroke is a significant cause of morbidity and mortality in children, and is frequently associated with intracranial vascular malformations. One prevalent form of these vascular malformations, cerebral cavernous malformation, is characterized by thin-walled vascular cavities that hemorrhage and has been linked to loss-of-function mutations in CCM1. The neural and epithelial expression of CCM1 in adulthood suggests that cavernous malformations may be the result of primary neural defects. In this study, we generated mice lacking Ccm1 and demonstrate that Ccm1 is ubiquitously expressed early in embryogenesis and is essential for vascular development. Homozygous mutant embryos die in mid-gestation and the first detectable defects are exclusively vascular in nature. The precursor vessels of the brain become dilated starting at E8.5, reminiscent of the intracranial vascular defects observed in the human disease. In addition, there is marked enlargement and increased endothelial proliferation of the caudal dorsal aorta, as well as variable narrowing of the branchial arch arteries and proximal dorsal aorta. These vascular defects are not secondary to primary neural defects, as neural morphology and marker expression are normal even subsequent to the onset of vascular pathology. The defects in the vascular structure of embryos lacking Ccm1 are associated with early downregulation of artery-specific markers, including the Efnb2- and Notch-related genes. Finally, consistent with the murine data, we found that there is an analogous reduction in Notch gene expression in arterioles from humans with mutations in CCM1. Our studies suggest that cavernous malformations result from primary vascular rather than neural defects.     

Neuronal expression of the Ccm2 gene in a new mouse model of cerebral cavernous malformations

Cerebral cavernous malformations are vascular defects of the central nervous system consisting of clusters of dilated vessels that are subject to frequent hemorrhaging. The genes mutated in three forms of autosomal dominant cerebral cavernous malformations have been cloned, but it remains unclear which cell type is ultimately responsible for the lesion. In this article we describe mice with a gene trap insertion in the Ccm2 gene. Consistent with the human phenotype, heterozygous animals develop cerebral vascular malformations, although penetrance is low. β-galactosidase activity in heterozygous brain and in situ hybridization in wild-type brain revealed Ccm2 expression in neurons and choroid plexus but not in vascular endothelium of small vessels in the brain. The expression pattern of Ccm2 is similar to that of the Ccm1 gene and its interacting protein ICAP1 (Itgb1bp1). These data suggest that cerebral cavernous malformations arise as a result of defects in the neural parenchyma surrounding the vascular endothelial cells in the brain. Nicholas W. Plummer, Teresa L. Squire and Sudha Srinivasan contributed equally to this work.     

Kinases in cerebral cavernous malformations: Pathogenesis and therapeutic targets

Cerebral cavernous malformations (CCMs) are low-flow, hemorrhagic vascular lesions of the central nervous system of genetic origin, which can cause stroke-like symptoms and seizures. From the identification of CCM1, CCM2 and CCM3 as genes related to disease progression, molecular and cellular mechanisms for CCM pathogenesis have been established and the search for potential drugs to target CCM has begun. Broadly speaking, kinases are the major group signaling in CCM pathogenesis. These include the MEKK3/MEK5/ERK5 cascade, Rho/Rock signaling, CCM3/GCKIII signaling, PI3K/mTOR signaling, and others. Since the discovery of Rho/Rock in CCM pathogenesis, inhibitors for Rho signaling and subsequently other components in CCM signaling were discovered and applied in preclinical and clinical trials to ameliorate CCM progression. This review discusses the general aspects of CCM disease, kinase-mediated signaling in CCM pathogenesis and the current state of potential treatment options for CCM. It is suggested that kinase target drug development in the context of CCM might facilitate and meet the unmet requirement – a non-surgical option for CCM disease.     

Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization

Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.     

CCM1–ICAP-1 complex controls β1 integrin–dependent endothelial contractility and fibronectin remodeling

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1–deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.     

Mutations in a gene encoding a novel protein containing a phosphotyrosine-binding domain cause type 2 cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in hemorrhagic stroke, seizures, recurrent headaches, and focal neurologic deficits. Mutations in the gene KRIT1 are responsible for type 1 CCM (CCM1). We report that a novel gene, MGC4607, exhibits eight different mutations in nine families with type 2 CCM (CCM2). MGC4607, similar to the KRIT1 binding partner ICAP1alpha, encodes a protein with a phosphotyrosine-binding domain. This protein may be part of the complex pathway of integrin signaling that, when perturbed, causes abnormal vascular morphogenesis in the brain, leading to CCM formation.     

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.     

Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development

Cerebral Cavernous Malformation (CCM) is a disease characterized by capillary-venous lesions mostly located in the central nervous system. It occurs both as a sporadic and hereditary autosomal dominant condition. Three CCM genes have been identified and shown to encode the KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3) proteins whose functions are so far unknown. In an attempt to get some insight into the role of the 3 CCM genes, we used in situ hybridization to conduct a comparative analysis of their expression pattern at several time points during murine embryonic, postnatal and adult stages particularly within the central nervous system. A strong expression of the 3 Ccm genes was detected in the various neuronal cell layers of the brain, cerebellum and spinal cord, from embryonic to adult life. By E14.5 a moderate labelling was observed in the heart, arterial and venous large vessels with all 3 Ccm probes. Ccm2 and Ccm3 mRNAs, but not Ccm1, were clearly detected within meningeal and parenchymal cortical vessels at P8. This expression was no more detected by P19 and in adult murine brain, strongly suggesting a role for these 2 proteins in the intensive angiogenesis process occuring within the central nervous system during this period.     

Cloning and embryonic expression of zebrafish neuropilin genes

Neuropilin (Nrp), a cell surface receptor for class 3 semaphorins and for certain heparin forms of vascular endothelial growth factors, functions in many biological processes including axon guidance, neural cell migration and angiogenesis in the development of the nervous system and the cardiovascular system. To understand the role of neuropilins in zebrafish embryogenesis, we have cloned three zebrafish neuropilin homologues, nrp1b, nrp2a and nrp2b. Based on synteny, zebrafish nrp1b and the previously cloned nrp1a are orthologous to human nrp1, and zebrafish nrp2a and 2b orthologous to human nrp2. We have characterized the expression patterns of these four zebrafish neuropilin genes in wild type embryos from the beginning of somitogenesis to 48 h post-fertilization. Zebrafish nrp1a is expressed in the neural tube including telencephalon, epithalamus, cells along the axonal trajectory of the posterior commissure and the medial longitudinal fascicle, hindbrain neurons, vagus motor neurons and spinal motoneurons. Zebrafish nrp1b is expressed in the nose, the cranial neural crest cell (NCC) derived tissue underlying the hypothalamus, endothelial precursors and the trunk and tail vasculature. Zebrafish nrp2a is expressed in telencephalon, anterior pituitary, oculomotor and trochlear motor neurons, cells along the supra-optic and posterior commissures, hindbrain rhombomere 1, hindbrain neurons, cranial NCCs and sclerotome. Zebrafish nrp2b is expressed in telencephalon, thalamus, hypothalamus, epiphysis, cells along the anterior and posterior commissures, post-optic and supra-optic commissures and the olfactory axonal trajectory, hindbrain neurons, cranial NCCs, somites and spinal cord neurons.     

Structural basis for small G protein effector interaction of Ras-related protein 1 (Rap1) and adaptor protein Krev interaction trapped 1 (KRIT1)

Cerebral cavernous malformations (CCMs) affect 0.1–0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Å co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain “modules.”     

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.     

Signalling through cerebral cavernous malformation protein networks

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to haemorrhages, stroke, epilepsy and focal neurological deficits. CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2 or CCM3. These widely expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and (either alone or in complex) modulate signalling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. Recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease. Here, we review current knowledge of CCM protein signalling with a focus on three pathways which have generated the most interest—the RhoA–ROCK, MEKK3–MEK5–ERK5–KLF2/4 and cell junctional signalling pathways—but also consider ICAP1-β1 integrin and cdc42 signalling. We discuss emerging links between these pathways and the processes that drive disease pathology and highlight important open questions—key among them is the role of subcellular localization in the control of CCM protein activity.     

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signalling pathway

Spontaneous intracranial hemorrhage is a debilitating form of stroke, often leading to death or permanent cognitive impairment. Many of the causative genes and the underlying mechanisms implicated in developmental cerebral-vascular malformations are unknown. Recent in vitro and in vivo studies in mice have shown inhibition of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway to be effective in stabilizing cranial vessels. Using a combination of pharmacological and genetic approaches to specifically inhibit the HMGCR pathway in zebrafish (Danio rerio), we demonstrate a requirement for this metabolic pathway in developmental vascular stability. Here we report that inhibition of HMGCR function perturbs cerebral-vascular stability, resulting in progressive dilation of blood vessels, followed by vessel rupture, mimicking cerebral cavernous malformation (CCM)-like lesions in humans and murine models. The hemorrhages in the brain are rescued by prior exogenous supplementation with geranylgeranyl pyrophosphate (GGPP), a 20-carbon metabolite of the HMGCR pathway, required for the membrane localization and activation of Rho GTPases. Consistent with this observation, morpholino-induced depletion of the β-subunit of geranylgeranyltransferase I (GGTase I), an enzyme that facilitates the post-translational transfer of the GGPP moiety to the C-terminus of Rho family of GTPases, mimics the cerebral hemorrhaging induced by the pharmacological and genetic ablation of HMGCR. In embryos with cerebral hemorrhage, the endothelial-specific expression of cdc42, a Rho GTPase involved in the regulation of vascular permeability, was significantly reduced. Taken together, our data reveal a metabolic contribution to the stabilization of nascent cranial vessels, requiring protein geranylgeranylation acting downstream of the HMGCR pathway.     

Proteomic identification of the cerebral cavernous malformation signaling complex

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.     

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.     

Coagulation factor III (tissue factor) is required for vascularization in zebrafish embryos

Tissue factor (coagulation factor III) is a cell surface receptor for coagulation factor VII/VIIa; it was initially recognized as an initiator of the extrinsic coagulation pathway. Recently, the zebrafish tissue factor gene (TF) has been cloned. Paralogs encode coagulation factors IIIa and IIIb; both show remarkable sequence identity to the human and mouse coagulation factor III gene. It has been reported that TF could have additional properties that are essential for normal embryonic development, since knockout of the murine coagulation factor III gene resulted in 90% embryonic lethality. We examined the role of coagulation factor IIIb (f3b) during zebrafish embryonic development. Expression analysis revealed that endogenous f3b was chronologically expressed in the pectoral fins and in the vicinity of the pharynx. Knockout of f3b by injection of an f3b morpholino at the one-to-two cell stage caused distinctive morphological defects in embryos, including edema in the fourth brain ventricle at early embryonic stages and occasional bleeding at later stages. Furthermore, f3b morphants displayed abnormal vascular patterning. We conclude that f3b is required for brain vascular development and for development of part of the somatic vasculature during embryogenesis in the zebrafish.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.