Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals

The recent identification of the mitochondrial Ca(2+) uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca(2+) uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca(2+) levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca(2+), resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca(2+) overload when cytoplasmic Ca(2+) levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca(2+) and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca(2+) uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders

Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins

Gut mucosal DAMPs in IBD: From mechanisms to therapeutic implications

Endogenous damage-associated molecular patterns (DAMPs) are released during tissue damage and have increasingly recognized roles in the etiology of many human diseases. The inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), are immune-mediated conditions where high levels of DAMPs are observed. DAMPs such as calprotectin (S100A8/9) have an established clinical role as a biomarker in IBD. In this review, we use IBD as an archetypal common chronic inflammatory disease to focus on the conceptual and evidential importance of DAMPs in pathogenesis and why DAMPs represent an entirely new class of targets for clinical translation. </p

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

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Mitochondrial damage-associated molecular patterns (DAMPs) in inflammatory bowel disease

Background The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn’s disease (CD) are chronic relapsing inflammatory disorders which have a rising incidence and cause significant morbidity. There are currently several treatment options with many more in the drug pipeline, but there are a lack of accurate biomarkers for decisions on treatment choice, assessment of disease activity and prognostication. There is a growing interest and desire for personalised or ‘precision’ medicine in IBD where novel biomarkers may help individualise IBD care in terms of diagnosis, choice of therapy, monitoring of response and detection of relapse. One class of functionally active biomarkers which have yet to be thoroughly investigated in IBD is damage-associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA). It has been recently shown that gut mitochondrial dysfunction can result in loss of epithelial barrier function and the development of colitis. Mitochondrial DAMPs have recently been described as elevated in several inflammatory diseases. Hypothesis The primary hypothesis of this thesis is that circulating levels of mtDNA is elevated in IBD. Secondary hypotheses are: (a) levels of other mitochondrial DAMPs are elevated in IBD, (b) circulating mtDNA can be used as a novel biomarker in IBD and (c) mtDNA is released locally at sites of inflammation in IBD. Methods Plasma and serum were collected prospectively from recruited IBD patients and non-IBD controls. Faeces and colonic tissue were collected from a subset of these patients. mtDNA in serum, plasma and faeces was measured using qPCR (amplifying COXIII/ND2 genes). Mass spectrometry was used to detect mitochondrial formylated peptides in the plasma of a subset of patients. IBD tissue was assessed for (a) mitochondrial damage using transmission electron microscopy (TEM) and (b) TLR9 expression, the target for mtDNA. Results 97 patients with IBD (67 UC and 30 CD), and 40 non-IBD controls were recruited. Plasma mtDNA levels were increased in UC and CD (both p<0.0001) compared to non-IBD controls; with significant correlations with blood (CRP, albumin, white cell count), clinical and endoscopic markers of severity; and disease activity. In active UC, we detected significantly higher circulating mitochondrial formylated peptides and faecal mtDNA levels (vs. non-IBD controls [p<0.01 and <0.0001 respectively]) with demonstrable TEM evidence of intestinal mucosal mitochondrial damage. In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC/CD compared to controls (both p<0.05). Conclusions Taken together, the findings suggest mtDNA is released during active inflammation in inflammatory bowel disease and is a potential novel mechanistic biomarker

Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations.

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation

Mitochondria in motor nerve terminals: function in health and in mutant superoxide dismutase 1 mouse models of familial ALS

Mitochondria contribute to neuronal function not only via their ability to generate ATP, but also via their ability to buffer large Ca(2+) loads. This review summarizes evidence that mitochondrial Ca(2+) sequestration is especially important for sustaining the function of vertebrate motor nerve terminals during repetitive stimulation. Motor terminal mitochondria can sequester large amounts of Ca(2+) because they have mechanisms for limiting both the mitochondrial depolarization and the increase in matrix free [Ca(2+)] associated with Ca(2+) influx. In mice expressing mutations of human superoxide dismutase −1 (SOD1) that cause some cases of familial amyotrophic lateral sclerosis (fALS), motor terminals degenerate well before the death of motor neuron cell bodies. This review presents evidence for early and progressive mitochondrial dysfunction in motor terminals of mutant SOD1 mice (G93A, G85R). This dysfunction would impair mitochondrial ability to sequester stimulation-associated Ca(2+) loads, and thus likely contributes to the early degeneration of motor terminals