Ragopathies and the rising influence of RagGTPases on human diseases.

RagGTPases (Rags) play an essential role in the regulation of cell metabolism by controlling the activities of both mechanistic target of rapamycin complex 1 (mTORC1) and Transcription factor EB (TFEB). Several diseases, herein named ragopathies, are associated to Rags dysfunction. These diseases may be caused by mutations either in genes encoding the Rags, or in their upstream regulators. The resulting phenotypes may encompass a variety of clinical features such as cataract, kidney tubulopathy, dilated cardiomyopathy and several types of cancer. In this review, we focus on the key clinical, molecular and physio-pathological features of ragopathies, aiming to shed light on their underlying mechanisms.

The SGLT2 inhibitor dapagliflozin improves kidney function in glycogen storage disease XI.

Glycogen storage disease XI, also known as Fanconi-Bickel syndrome (FBS), is a rare autosomal recessive disorder caused by mutations in the SLC2A2 gene that encodes the glucose-facilitated transporter type 2 (GLUT2). Patients develop a life-threatening renal proximal tubule dysfunction for which no treatment is available apart from electrolyte replacement. To investigate the renal pathogenesis of FBS, SLC2A2 expression was ablated in mouse kidney and HK-2 proximal tubule cells. GLUT2Pax8Cre+ mice developed time-dependent glycogen accumulation in proximal tubule cells and recapitulated the renal Fanconi phenotype seen in patients. In vitro suppression of GLUT2 impaired lysosomal autophagy as shown by transcriptomic and biochemical analysis. However, this effect was reversed by exposure to a low glucose concentration, suggesting that GLUT2 facilitates the homeostasis of key cellular pathways in proximal tubule cells by preventing glucose toxicity. To investigate whether targeting proximal tubule glucose influx can limit glycogen accumulation and correct symptoms in vivo, we treated mice with the selective SGLT2 inhibitor dapagliflozin. Dapagliflozin reduced glycogen accumulation and improved metabolic acidosis and phosphaturia in the animals by normalizing the expression of Napi2a and NHE3 transporters. In addition, in a patient with FBS, dapagliflozin was safe, improved serum potassium and phosphate concentrations, and reduced glycogen content in urinary shed cells. Overall, this study provides proof of concept for dapagliflozin as a potentially suitable therapy for FBS.

m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.

The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m)-AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper.

Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs.

miR-17 and -20a Target the Neuron-Derived Orphan Receptor-1 (NOR-1) in Vascular Endothelial Cells.

Neuron-derived orphan receptor-1 (NOR-1) plays a major role in vascular biology by controlling fibroproliferative and inflammatory responses. Because microRNAs (miRNAs) have recently emerged as key players in the regulation of gene expression in the vasculature, here we have investigated the regulation of NOR-1 by miRNAs in endothelial cells. Computational algorithms suggest that NOR-1 could be targeted by members of the miR-17 family. Accordingly, ectopic over-expression of miR-17 or miR-20a in endothelial cells using synthetic premiRNAs attenuated the up-regulation of NOR-1 expression induced by VEGF (as evidenced by real time PCR, Western blot and immunocitochemistry). Conversely, the antagonism of these miRNAs by specific antagomirs prevented the down-regulation of NOR-1 promoted by miR-17 or miR-20a in VEGF-stimulated cells. Disruption of the miRNA-NOR-1 mRNA interaction using a custom designed target protector evidenced the selectivity of these responses. Further, luciferase reporter assays and seed-sequence mutagenesis confirmed that miR-17 and -20a bind to NOR-1 3'-UTR. Finally, miR-17 and -20a ameliorated the up-regulation of VCAM-1 mediated by NOR-1 in VEGF-stimulated cells. Therefore, miR-17 and -20a target NOR-1 thereby regulating NOR-1-dependent gene expression.

A highly secreted sulphamidase engineered to cross the blood-brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA.

Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs.

Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17.

RATIONALE: The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. OBJECTIVE: Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. METHODS AND RESULTS: Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. CONCLUSIONS: Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.

The microRNA 15a/16-1 cluster down-regulates protein repair isoaspartyl methyltransferase in hepatoma cells: implications for apoptosis regulation.

Asparaginyl deamidation, a spontaneous protein post-biosynthetic modification, determines isoaspartyl formation and structure-function impairment. The isoaspartyl protein carboxyl-O-methyltransferase (PCMT1; EC 2.1.1.77) catalyzes the repair of the isopeptide bonds at isoaspartyl sites, preventing deamidation-related functional impairment. Protein deamidation affects key apoptosis mediators, such as BclxL, thus increasing susceptibility to apoptosis, whereas PCMT1 activity may effectively counteract such alterations. The aim of this work was to establish the role of RNAi as a potential mechanism for regulating PCMT1 expression and its possible implications in apoptosis. We investigated the regulatory properties of the microRNA 15a/16-1 cluster on PCMT1 expression on HepG2 cells. MicroRNA 15a or microRNA 16-1 transfection, as well as their relevant antagonists, showed that PCMT1 is effectively regulated by this microRNA cluster. The direct interaction of these two microRNAs with the seed sequence at the 3' UTR of PCMT1 transcripts was demonstrated by the luciferase assay system. The role of PCMT1 down-regulation in conditioning the susceptibility to apoptosis was investigated using various specific siRNA or shRNA approaches, to prevent non-PCMT1-specific pleiotropic effects to take place. We found that PCMT1 silencing is associated with an increase of the BclxL isoform reported to be inactivated by deamidation, thus making cells more susceptible to apoptosis induced by cisplatinum. We conclude that PCMT1 is effectively regulated by the microRNA 15a/16-1 cluster and is involved in apoptosis by preserving the structural stability and biological function of BclxL from deamidation. Control of PCMT1 expression by microRNA 15a/16-1 may thus represent a late checkpoint in apoptosis regulation.