[Show abstract][Hide abstract] ABSTRACT: The mitochondrial permeability transition pore (PT pore) is a recognised drug target for neurodegenerative conditions such
as multiple sclerosis (MS) and for ischaemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin
D (CypD, ppif) is a positive regulator of the pore and genetic downregulation or knockout improves outcomes in disease models.
Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and
exhibit significant off target effects, immune-suppression and toxicity. We therefore designed and synthesised a new mitochondrially-targeted
CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine (CsA). X-ray analysis was used to validate the
design concept and biological evaluation revealed selective cellular inhibition of CypD and the PT pore with reduced cellular
toxicity compared to CsA. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple
sclerosis (MS), JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression.
These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium
as a mitochondrial targeting group for in vivo use.
Full-text · Article · Dec 2015 · Journal of Biological Chemistry
[Show description][Hide description] DESCRIPTION: Resolution : 1.25Å(reported) This is a wwPDB validation summary report for a publicly released PDB entry. We welcome your comments at email@example.com A user guide is available at http://wwpdb.org/ValidationPDFNotes.html
[Show abstract][Hide abstract] ABSTRACT: Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. While restoring ionic gradients is energetically costly, a rise in intracellular Ca(2+) acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca(2+) stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca(2+) -regulated mitochondrial carriers, while an increase in matrix Ca(2+) stimulates the citric acid cycle and the ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca(2+) and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K(+) -depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 KO neurons reflects impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca(2+) influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca(2+) ]. Although the physiological importance of the MCU complex (MCUC) in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca(2+) signaling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCUC, are associated with neuromuscular disease. In patient derived MICU1 deficient fibroblasts, resting matrix Ca(2+) is increased and mitochondria fragmented. Thus, the fine tuning of Ca(2+) signals plays a key role in shaping mitochondrial bioenergetics. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · Mar 2015 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as /`accidental cell death/' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. /`Regulated cell death/' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects i
Full-text · Article · Sep 2014 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: Intracellular Ca(2+) signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca(2+) homeostasis has been shown to determine cell fate in resting conditions as well as in response to stress. The endoplasmic reticulum and mitochondria represent key hubs of cellular metabolism and Ca(2+) signaling. However, it has been challenging to specifically detect highly localized Ca(2+) fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca(2+) indicators that can be targeted to specific subcellular compartments have been developed over the past two decades. While the use of these probes for measuring agonist-induced Ca(2+) signals in various organelles has been extensively described, the assessment of basal Ca(2+) concentrations within specific organelles is often disregarded, in spite of the fact that this parameter is vital for several metabolic functions, including the enzymatic activity of mitochondrial dehydrogenases of the Krebs cycle and protein folding in the endoplasmic reticulum. Here, we provide an overview on genetically engineered, organelle-targeted fluorescent Ca(2+) probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca(2+) concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca(2+) levels affect the metabolic profile of cancer cells.
No preview · Article · Jun 2014 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.
[Show abstract][Hide abstract] ABSTRACT: NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.
Full-text · Article · May 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.
Full-text · Article · May 2014 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Novel therapeutic targets are required to protect the heart against cell death from acute ischemia-reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia-reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1(L166P) and DJ-1(Cys106A) mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.
Full-text · Article · Feb 2014 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca(2+) fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.Cell Death and Differentiation advance online publication, 24 January 2014; doi:10.1038/cdd.2013.193.
Full-text · Article · Jan 2014 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.
Full-text · Article · Aug 2013 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.
[Show abstract][Hide abstract] ABSTRACT: We are currently witnessing a stream of major discoveries describing the elementary constituents of complex mitochondrial machineries that define the shape, dynamics and intracellular distribution of the organelle. On the basis of these findings the next wave of research holds the promise of unveiling the so far hidden relationships between the fascinating ultrastructural mitochondrial anatomy and its key roles in many areas of cell biology.
No preview · Article · May 2013 · Current opinion in cell biology