The PINK1-Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells

Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in PINK1 -Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential mitophagy defects. We aimed to characterise the expression of the PINK1 -Parkin mitophagy machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein expression levels were characterised with reverse transcriptase quantitative PCR and western blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable and PINK1 mRNA levels were remarkably low in control PBMCs. Our findings suggest that the PINK1 -Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable biosample for analysis of mitophagy function in Parkinson disease patients

The glucocerobrosidase E326K variant predisposes to Parkinson's disease, but does not cause Gaucher's disease

Heterozygous loss-of-function mutations in the acid beta-glucocerebrosidase (GBA1) gene, responsible for the recessive lysosomal storage disorder, Gaucher's disease (GD), are the strongest known risk factor for Parkinson's disease (PD). Our aim was to assess the contribution of GBA1 mutations in a series of early-onset PD

LRRK2 deficiency induced mitochondrial Ca2+ efflux inhibition can be rescued by Na+/Ca2+/Li+ exchanger upregulation

Variants of leucine-rich repeat kinase 2 (lrrk2) are associated with an increased risk in developing Parkinson’s disease (PD). Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy

Structural Biology of Human H3K9 Methyltransferases

SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Enhanced version: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available i

Quantifying the Evolution of Vascular Barrier Disruption in Advanced Atherosclerosis with Semipermeant Nanoparticle Contrast Agents

Acute atherothrombotic occlusion in heart attack and stroke implies disruption of the vascular endothelial barrier that exposes a highly procoagulant intimal milieu. However, the evolution, severity, and pathophysiological consequences of vascular barrier damage in atherosclerotic plaque remain unknown, in part because quantifiable methods and experimental models are lacking for its in vivo assessment.To develop quantitative nondestructive methodologies and models for detecting vascular barrier disruption in advanced plaques.Sustained hypercholesterolemia in New Zealand White (NZW) rabbits for >7-14 months engendered endothelial barrier disruption that was evident from massive and rapid passive penetration and intimal trapping of perfluorocarbon-core nanoparticles (PFC-NP: ∼250 nm diameter) after in vivo circulation for as little as 1 hour. Only older plaques (>7 mo), but not younger plaques (<3 mo) demonstrated the marked enhancement of endothelial permeability to these particles. Electron microscopy revealed a complex of subintimal spongiform channels associated with endothelial apoptosis, superficial erosions, and surface-penetrating cholesterol crystals. Fluorine ((19)F) magnetic resonance imaging and spectroscopy (MRI/MRS) enabled absolute quantification (in nanoMolar) of the passive permeation of PFC-NP into the disrupted vascular lesions by sensing the unique spectral signatures from the fluorine core of plaque-bound PFC-NP.The application of semipermeant nanoparticles reveals the presence of profound barrier disruption in later stage plaques and focuses attention on the disrupted endothelium as a potential contributor to plaque vulnerability. The response to sustained high cholesterol levels yields a progressive deterioration of the vascular barrier that can be quantified with fluorine MRI/MRS of passively permeable nanostructures. The possibility of plaque classification based on the metric of endothelial permeability to nanoparticles is suggested

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Morphological flexibility of Cocconeis placentula nanostructure along a natural salinity gradient.

International audienceDiatoms possess a silica frustule decorated with unique patterns of nanosize features. Here, we show for the first time from in situ samples that the size of the nanopores present at the surface of the diatom Cocconeis placentula Ehrenb. varies with fluctuating salinity levels. The observed reduction in nanopore size with decreasing salinity agrees with previous laboratory experiments. We also uniquely combined our observations with theoretical considerations to demonstrate that the decrease in the diffusive layer thickness is compensated for by the changes in pore size, which maintain a steady diffusive flux toward the diatom's cell at different salinities. This process allows diatoms to absorb similar amount of nutrients whatever the salinity and as such to increase their ecological competitiveness in fluctuating environments. These results further suggest that the overall ecological success of diatoms, and their ability to react to environmental changes, may be controlled by the flexibility of the morphological characteristics of their frustules