Two different points of view on signal transduction: defective autophagy as a key feature of Cerebral Cavernous Malformations and c-Src as modulator of intracellular Ca2+ homeostasis

Nel primo progetto abbiamo indagato il coinvolgimento dell’autofagia nella patogenesi delle Malformazioni Cavernose Cerebrali (CCM). CCM è una grave malattia cerebrovascolare che colpisce circa lo 0.3-0.5% della popolazione ed è caratterizzata da capillari dilatati e fragili che predispongono a convulsioni, deficit neurologici e fatali emorragie intracerebrali. CCM è una malattia genetica che può insorgere in modo sporadico o essere ereditata con modalità di trasmissione autosomica dominante. La causa è stata identificata in mutazioni che determinano la perdita di tre geni, KRIT1 (CCM1), CCM2 (MGC4607) e PDCD10 (CCM3), che si verificano nelle forme sia sporadiche sia familiari. L'autofagia è un processo di degradazione che preserva l'omeostasi intracellulare e svolge funzioni essenziali di controllo qualità. Infatti, diversi studi hanno identificato un’associazione tra disregolazione dell’autofagia e diverse patologie umane. Nel nostro lavoro mostriamo che la perdita del gene KRIT1 sopprime l'autofagia, portando ad un aberrante accumulo dell'adattatore autofagico p62/SQSTM1, difetti nei sistemi di controllo qualità e aumento dello stress intracellulare. La perdita di funzione della proteina KRIT1 attiva il pathway mTOR-ULK1, il principale regolatore dell'autofagia, e il trattamento con inibitori di mTOR reverte alcuni dei fenotipi molecolari e cellulari associati alle CCM. La riduzione nei livelli di autofagia è evidente anche in cellule endoteliali umane in cui è stato silenziato CCM2, in cellule e in tessuti di topo CCM3 knockout endotelio-specifico, e in lesioni CCM umane. Inoltre, la deregolazione dell'autofagia è altamente correlata alla transizione endoteliale-mesenchimale, evento cruciale che contribuisce alla progressione delle CCM. Complessivamente, i nostri dati indicano un ruolo chiave per la deregolazione dell’autofagia nella patogenesi delle CCM, fornendo così nuove possibilità per lo sviluppo di strategie farmacologiche per prevenire o contrastare gli esiti clinici avversi conseguenti alle lesioni. [da: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17]. Il secondo progetto è focalizzato sullo studio del ruolo del proto-oncogene c-Src nella modulazione del segnale Ca2+ intracellulare. c-Src è una tirosin chinasi di tipo non recettoriale e svolge un ruolo chiave in diverse vie di segnalazione coinvolte in eventi cellulari fondamentali, quali crescita cellulare, sopravvivenza, adesione, migrazione e invasione. È nota la correlazione diretta tra deregolazione o sovraespressione di c-Src e diversi tumori umani. Il calcio (Ca2+) è un secondo messaggero intracellulare altamente versatile che agisce in funzioni cellulari cruciali. Durante la carcinogenesi e la progressione del tumore, il segnale Ca2+ viene rimodellato in modo tale da supportare la maggior parte delle caratteristiche tipiche del cancro. In questo progetto abbiamo identificato una funzione chiave per c-Src nella modulazione dell'omeostasi del Ca2+ intracellulare. c-Src riduce il rilascio di Ca2+ dal reticolo endoplasmatico (ER) in seguito a stimolazione con agonista e altera i livelli intracellulari di Ca2+. Tale regolazione del segnale Ca2+ mediata da c-Src è strettamente dipendente dalla sua attività catalitica e dalla sua localizzazione. I nostri risultati hanno stabilito che c-Src controlla i livelli di Ca2+ intracellulare attraverso la fosforilazione di uno specifico target. Questa fosforilazione diretta media l'effetto regolatorio di c-Src sul rilascio di Ca2+ dal ER, influenzando quindi il segnale Ca2+ negli altri compartimenti. Nel complesso il nostro lavoro ha identificato un nuovo ruolo per c-Src nel controllo delle dinamiche del Ca2+ e contribuisce a chiarire in modo più dettagliato come c-Src agisca sia in un contesto fisiologico sia patologico.In the first project we investigated the involvement of the autophagy in the pathogenesis of the Cerebral Cavernous Malformation (CCM). Cerebral cavernous malformation 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 molecular 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. [From: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17]. The second project was focused on the investigation of a potential role for the proto-oncogene c-Src in the modulation of intracellular Ca2+ signalling. c-Src is a non-receptor tyrosine kinase that plays a pivotal role in several signalling pathways involved in fundamental cellular events, including cell growth, survival, cell adhesion, migration and invasion. It is well established the link existing between c-Src deregulation or overexpression and several human cancers. Calcium (Ca2+) is a highly versatile intracellular second messenger that acts in crucial cellular functions. During carcinogenesis and tumour progression, Ca2+ signalling is significantly remodelled in a way that sustains most of typical cancer hallmarks, as uncontrolled proliferation and evasion of programmed cell death. We identified a key function for c-Src in the modulation of intracellular Ca2+ homeostasis. c-Src downregulates the Ca2+ release from the endoplasmic reticulum (ER) upon agonist stimulation and significantly alters the intracellular Ca2+ levels. c-Src regulation of Ca2+ signalling is strictly dependent on its catalytic activity and subcellular localization. Our results established that c-Src controls Ca2+ handling through phosphorylation of a specific molecular target. This direct phosphorylation mediates the c-Src effect on Ca2+ release from the intracellular store, thus affecting Ca2+ homeostasis at the other intracellular compartments. Overall, our work identified a new role for c-Src in the control of the intracellular Ca2+ dynamics and contribute to deeply understand how c-Src acts in the context of its physiological and pathological functions

Beyond multiple mechanisms and a unique drug: Defective autophagy as pivotal player in cerebral cavernous malformation pathogenesis and implications for targeted therapies

Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease of proven genetic origin affecting 0.3-0.5% of the general population. It is characterized by abnormally enlarged and leaky capillaries, which predispose to seizures, focal neurological deficits and intracerebral hemorrhage. Causative loss-of-function mutations have been identified in 3 genes, KRIT1 (CCM1), CCM2 and PDCD10 (CCM3). While providing new options for the development of pharmacological therapies, recent advances in knowledge of the functions of these genes have clearly indicated that they exert pleiotropic effects on several biological pathways. Recently, we found that defective autophagy is a common feature of loss-of-function mutations of the 3 known CCM genes, and underlies major phenotypic signatures of CCM disease, including endothelial-to-mesenchymal transition and enhanced ROS production, suggesting a unifying pathogenetic mechanism and reconciling the distinct therapeutic approaches proposed so far. In this invited review, we discuss autophagy as a possible unifying mechanism in CCM disease pathogenesis, and new perspectives and avenues of research for disease prevention and treatment, including novel potential drug repurposing and combination strategies, and identification of genetic risk factors as basis for development of personalized medicine approaches

Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth

Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development

Role of Mitochondria-Associated ER Membranes in Calcium Regulation in Cancer-Specific Settings

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are highly specialized subcellular compartments that are shaped by ER subdomains juxtaposed to mitochondria but are biochemically distinct from pure ER and pure mitochondria. MAMs are enriched in enzymes involved in lipid synthesis and transport, channels for calcium transfer, and proteins with oncogenic/oncosuppressive functions that modulate cell signaling pathways involved in physiological and pathophysiological processes. The term "cancer" denotes a group of disorders that result from uncontrolled cell growth driven by a mixture of genetic and environmental components. Alterations in MAMs are thought to account for the onset as well as the progression and metastasis of cancer and have been a focus of investigation in recent years. In this review, we present the current state of the art regarding MAM-resident proteins and their relevance, alterations, and deregulating functions in different types of cancer from a cell biology and clinical perspective

Role of Mitochondria-Associated ER Membranes in Calcium Regulation in Cancer-Specific Settings

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are highly specialized subcellular compartments that are shaped by ER subdomains juxtaposed to mitochondria but are biochemically distinct from pure ER and pure mitochondria. MAMs are enriched in enzymes involved in lipid synthesis and transport, channels for calcium transfer, and proteins with oncogenic/oncosuppressive functions that modulate cell signaling pathways involved in physiological and pathophysiological processes. The term "cancer" denotes a group of disorders that result from uncontrolled cell growth driven by a mixture of genetic and environmental components. Alterations in MAMs are thought to account for the onset as well as the progression and metastasis of cancer and have been a focus of investigation in recent years. In this review, we present the current state of the art regarding MAM-resident proteins and their relevance, alterations, and deregulating functions in different types of cancer from a cell biology and clinical perspective.status: publishe

Defective autophagy is a key feature of cerebral cavernous malformations

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

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca(2+)-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development