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PINK1, mutated in familial forms of Parkinson's disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions.
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Leucine-rich repeat kinase 2 (LRRK2) is one of the most promising targets for Parkinson's disease. LRRK2-targeting strategies have primarily focused on type 1 kinase inhibitors, which, however, have limitations as the inhibited protein can interfere with natural mechanisms, which could lead to undesirable side effects. Herein, we report the development of LRRK2 proteolysis targeting chimeras (PROTACs), culminating in the discovery of degrader XL01126, as an alternative LRRK2-targeting strategy. Initial designs and screens of PROTACs based on ligands for E3 ligases von Hippel-Lindau (VHL), Cereblon (CRBN), and cellular inhibitor of apoptosis (cIAP) identified the best degraders containing thioether-conjugated VHL ligand VH101. A second round of medicinal chemistry exploration led to qualifying XL01126 as a fast and potent degrader of LRRK2 in multiple cell lines, with DC50 values within 15-72 nM, Dmax values ranging from 82 to 90%, and degradation half-lives spanning from 0.6 to 2.4 h. XL01126 exhibits high cell permeability and forms a positively cooperative ternary complex with VHL and LRRK2 (α = 5.7), which compensates for a substantial loss of binary binding affinities to VHL and LRRK2, underscoring its strong degradation performance in cells. Remarkably, XL01126 is orally bioavailable (F = 15%) and can penetrate the blood-brain barrier after either oral or parenteral dosing in mice. Taken together, these experiments qualify XL01126 as a suitable degrader probe to study the noncatalytic and scaffolding functions of LRRK2 in vitro and in vivo and offer an attractive starting point for future drug development.
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Barrera Hematoencefálica , Ubiquitina-Proteína Ligasas , Animales , Ratones , Barrera Hematoencefálica/metabolismo , Leucina , Ligandos , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Sulfuros , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activationdependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.
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Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study, we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild-type LRRK2 compared with pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast with Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aid with the development of more selective Type II LRRK2 inhibitors.
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Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mitofagia/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Benzamidas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Imidazoles/farmacología , Indazoles/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Ratones Endogámicos C57BL , Mitofagia/genética , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Enfermedad de Parkinson , Fosforilación/efectos de los fármacos , Fosforilación/genética , Pirazoles/farmacología , Piridazinas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Transducción de Señal/genética , TransfecciónRESUMEN
Parkinson's disease (PD) is a major and progressive neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and/or impaired lysosomal degradation - key features of the autophagy of mitochondria, known as mitophagy. Here, we investigated the role of LRRK2, a protein kinase frequently mutated in PD, in this process in vivo. Using mitophagy and autophagy reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. In support of this, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued the mitophagy defects observed in LRRK2 G2019S mice. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy, a process with strong links to idiopathic Parkinson's disease, and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy.
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Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Mitofagia/genética , Enfermedad de Parkinson/fisiopatología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitofagia/efectos de los fármacos , Mutación , Enfermedad de Parkinson/genéticaRESUMEN
Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.
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Interleucina-15/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Granzimas/genética , Granzimas/metabolismo , Humanos , Interleucina-15/genética , Linfocitos Intraepiteliales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Previous studies suggest that statins may disturb skeletal muscle lipid metabolism potentially causing lipotoxicity with insulin resistance. We investigated this possibility in wild-type mice (WT) and mice with skeletal muscle PGC-1α overexpression (PGC-1α OE mice). In WT mice, simvastatin had only minor effects on skeletal muscle lipid metabolism but reduced glucose uptake, indicating impaired insulin sensitivity. Muscle PGC-1α overexpression caused lipid droplet accumulation in skeletal muscle with increased expression of the fatty acid transporter CD36, fatty acid binding protein 4, perilipin 5 and CPT1b but without significant impairment of muscle glucose uptake. Simvastatin further increased the lipid droplet accumulation in PGC-1α OE mice and stimulated muscle glucose uptake. In conclusion, the impaired muscle glucose uptake in WT mice treated with simvastatin cannot be explained by lipotoxicity. PGC-1α OE mice are protected from lipotoxicity of fatty acids and triglycerides by increased the expression of FABP4, formation of lipid droplets and increased expression of CPT1b.
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Metabolismo de los Lípidos/efectos de los fármacos , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Simvastatina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Antígenos CD36/genética , Antígenos CD36/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Colesterol/sangre , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/sangre , Glucosa/metabolismo , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Lipoproteína Lipasa/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Tamaño de los Órganos/efectos de los fármacos , Perilipina-5/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/sangreRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects around 2% of individuals over 60 years old. It is characterised by the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, which is thought to account for the major clinical symptoms such as tremor, slowness of movement and muscle stiffness. Its aetiology is poorly understood as the physiological and molecular mechanisms leading to this neuronal loss are currently unclear. However, mitochondrial and lysosomal dysfunction seem to play a central role in this disease. In recent years, defective mitochondrial elimination through autophagy, termed mitophagy, has emerged as a potential contributing factor to disease pathology. PINK1 and Parkin, two proteins mutated in familial PD, were found to eliminate mitochondria under distinct mitochondrial depolarisation-induced stress. However, PINK1 and Parkin are not essential for all types of mitophagy and such pathways occur in most cell types and tissues in vivo, even in the absence of overt mitochondrial stress - so-called basal mitophagy. The most common mutation in PD, that of glycine at position 2019 to serine in the protein kinase LRRK2, results in increased activity and this was recently shown to disrupt basal mitophagy in vivo. Thus, different modalities of mitophagy are affected by distinct proteins implicated in PD, suggesting impaired mitophagy may be a common denominator for the disease. In this short review, we discuss the current knowledge about the link between PD pathogenic mutations and mitophagy, with a particular focus on LRRK2.
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Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mitofagia/genética , Mutación , Enfermedad de Parkinson/genética , Animales , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Several studies showed an increased risk for diabetes with statin treatment. PGC-1α is an important regulator of muscle energy metabolism and mitochondrial biogenesis. Since statins impair skeletal muscle PGC-1α expression and reduced PGC-1α expression has been observed in diabetic patients, we investigated the possibility that skeletal muscle PGC1α expression influences the effect of simvastatin on muscle glucose metabolism. Mice with muscle PGC-1α knockout (KO) or PGC-1α overexpression (OE), and wild-type (WT) mice were investigated. Mice were treated orally for 3 weeks with simvastatin (5 mg/kg/day) and investigated by intraperitoneal glucose tolerance (iGTT), in vivo skeletal muscle glucose uptake, muscle glycogen content, and Glut4 and hexokinase mRNA and protein expression. Simvastatin impaired glucose metabolism in WT mice, as manifested by increased glucose blood concentrations during the iGTT, decreased skeletal muscle glucose uptake and glycogen stores. KO mice showed impaired glucose homeostasis with increased blood glucose concentrations during the iGTT already without simvastatin treatment and simvastatin induced a decrease in skeletal muscle glucose uptake. In OE mice, simvastatin treatment increased blood glucose and insulin concentrations during the iGTT, and increased skeletal muscle glucose uptake, glycogen stores, and Glut4 and hexokinase protein expression. In conclusion, simvastatin impaired skeletal muscle insulin sensitivity in WT mice, while KO mice exhibited impaired skeletal muscle insulin sensitivity already in the absence of simvastatin. In OE mice, simvastatin augmented muscular glucose uptake but impaired whole-body insulin sensitivity. Thus, simvastatin affected glucose homeostasis depending on PGC-1α expression.
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Statins lower the serum low-density lipoprotein cholesterol and prevent cardiovascular events by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA reductase. Although the safety of statins is documented, many patients ingesting statins may suffer from skeletal muscle-associated symptoms (SAMS). Importantly, SAMS are a common reason for stopping the treatment with statins. Statin-associated muscular symptoms include fatigue, weakness and pain, possibly accompanied by elevated serum creatine kinase activity. The most severe muscular adverse reaction is the potentially fatal rhabdomyolysis. The frequency of SAMS is variable but in up to 30% of the patients ingesting statins, depending on the population treated and the statin used. The mechanisms leading to SAMS are currently not completely clarified. Over the last 15 years, several research articles focused on statin-induced mitochondrial dysfunction as a reason for SAMS. Statins can impair the function of the mitochondrial respiratory chain, thereby reducing ATP and increasing ROS production. This can induce mitochondrial membrane permeability transition, release of cytochrome c into the cytosol and induce apoptosis. In parallel, statins inhibit activation of Akt, mainly due to reduced function of mTORC2, which may be related to mitochondrial dysfunction. Mitochondrial dysfunction by statins is also responsible for activation of AMPK, which is associated with impaired activation of mTORC1. Reduced activation of mTORC1 leads to increased skeletal muscle protein degradation, impaired protein synthesis and stimulation of apoptosis. In this paper, we discuss some of the different hypotheses how statins affect skeletal muscle in more detail, focusing particularly on those related to mitochondrial dysfunction and the impairment of the Akt/mTOR pathway.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Músculo Esquelético/efectos de los fármacos , Enfermedades Musculares/inducido químicamente , Animales , Humanos , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismoRESUMEN
Mitophagy is a natural phenomenon and entails the lysosomal degradation of mitochondria by the autophagy pathway. In recent years, the development of fluorescent pH-sensitive mitochondrial reporters has greatly facilitated the monitoring of mitophagy by distinguishing between cytosolic mitochondria or those delivered to acidic lysosomes. We recently published the mito-QC reporter, which consists of a mitochondrial outer membrane-localised tandem mCherry-GFP tag. This allows the quantification of mitophagy via the increase in red-only mCherry signal that arises when the GFP signal is quenched upon mitochondrial delivery to lysosomes. Here we develop a macro for FIJI, the mito-QC Counter, and describe its use to allow reliable and consistent semi-automated quantification of mitophagy. In this methods article we describe step-by-step how to detect and quantify mitophagy and show that mitophagy levels can be reliably calculated in different cell lines and under distinct stimuli. Finally, we show that the mito-QC Counter can be used to quantify mitophagy in tissues of mito-QC transgenic mice. We demonstrate that mitophagy levels in skeletal muscle correlates with glycolytic activity. Our present data show that the mito-QC Counter macro for FIJI enables the robust quantification of mitophagy both in vitro and in vivo.
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Autofagia/fisiología , Lisosomas , Microscopía Fluorescente/métodos , Mitocondrias , Mitofagia/fisiología , Animales , Transporte Biológico/fisiología , Línea Celular , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Recambio MitocondrialRESUMEN
AIM: Statins decrease cardiovascular complications, but can induce myopathy. Here, we explored the implication of PGC-1α in statin-associated myotoxicity. METHODS: We treated PGC-1α knockout (KO), PGC-1α overexpression (OE) and wild-type (WT) mice orally with 5 mg simvastatin kg-1 day-1 for 3 weeks and assessed muscle function and metabolism. RESULTS: In WT and KO mice, but not in OE mice, simvastatin decreased grip strength, maximal running distance and vertical power assessed by ergometry. Post-exercise plasma lactate concentrations were higher in WT and KO compared to OE mice. In glycolytic gastrocnemius, simvastatin decreased mitochondrial respiration, increased mitochondrial ROS production and free radical leak in WT and KO, but not in OE mice. Simvastatin increased mRNA expression of Sod1 and Sod2 in glycolytic and oxidative gastrocnemius of WT, but decreased it in KO mice. OE mice had a higher mitochondrial DNA content in both gastrocnemius than WT or KO mice and simvastatin exhibited a trend to decrease the citrate synthase activity in white and red gastrocnemius in all treatment groups. Simvastatin showed a trend to decrease the mitochondrial volume fraction in both muscle types of all treatment groups. Mitochondria were smaller in WT and KO compared to OE mice and simvastatin further reduced the mitochondrial size in WT and KO mice, but not in OE mice. CONCLUSIONS: Simvastatin impairs skeletal muscle function, muscle oxidative metabolism and mitochondrial morphology preferentially in WT and KO mice, whereas OE mice appear to be protected, suggesting a role of PGC-1α in preventing simvastatin-associated myotoxicity.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Músculo Esquelético/lesiones , Miotoxicidad/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Simvastatina/efectos adversos , Animales , Citrato (si)-Sintasa/metabolismo , ADN Mitocondrial/metabolismo , Glutatión/metabolismo , Glucólisis/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Ácido Láctico/sangre , Ratones , Ratones Noqueados , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Condicionamiento Físico Animal/fisiología , Simvastatina/administración & dosificaciónRESUMEN
Statins inhibit cholesterol biosynthesis and lower serum LDL-cholesterol levels. Statins are generally well tolerated, but can be associated with potentially life-threatening myopathy of unknown mechanism. We have shown previously that statins impair PGC-1ß expression in human and rat skeletal muscle, suggesting that PGC-1ß may play a role in statin-induced myopathy. PGC-1ß is a transcriptional co-regulator controlling the expression of important genes in mitochondrial biogenesis, antioxidative capacity and energy metabolism. The principle aim of the current study was to investigate the interaction between atorvastatin and PGC-1ß in more detail. We therefore treated wild-type mice and mice with selective skeletal muscle knockout of PGC-1ß (PGC-1ß(i)skm-/- mice) with oral atorvastatin (5 mg/kg/day) for 2 weeks. At the end of treatment, we determined body parameters, muscle function, structure, and composition as well as the function of muscle mitochondria, mitochondrial biogenesis and activation of apoptotic pathways. In wild-type mice, atorvastatin selectively impaired mitochondrial function in glycolytic muscle and caused a conversion of oxidative type IIA to glycolytic type IIB myofibers. Conversely, in oxidative muscle of wild-type mice, atorvastatin enhanced mitochondrial function via activation of mitochondrial biogenesis pathways and decreased apoptosis. In PGC-1ß(i)skm-/- mice, atorvastatin induced a switch towards glycolytic fibers, caused mitochondrial dysfunction, increased mitochondrial ROS production, impaired mitochondrial proliferation and induced apoptosis in both glycolytic and oxidative skeletal muscle. Our work reveals that atorvastatin mainly affects glycolytic muscle in wild-type mice and demonstrates the importance of PGC-1ß for oxidative muscle integrity during long-term exposure to a myotoxic agent.
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Atorvastatina/toxicidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Músculo Esquelético/efectos de los fármacos , Miotoxicidad/etiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Atorvastatina/metabolismo , Femenino , Peróxido de Hidrógeno/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Cadenas Pesadas de Miosina/metabolismo , Miotoxicidad/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genéticaRESUMEN
Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.
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Mitocondrias/metabolismo , Mitofagia , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fosforilación/genética , Proteínas Quinasas/genética , Serina/genética , Serina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Dysregulated mitophagy has been linked to Parkinson's disease (PD) due to the role of PTEN-induced kinase 1 (PINK1) in mediating depolarization-induced mitophagy in vitro. Elegant mouse reporters have revealed the pervasive nature of basal mitophagy in vivo, yet the role of PINK1 and tissue metabolic context remains unknown. Using mito-QC, we investigated the contribution of PINK1 to mitophagy in metabolically active tissues. We observed a high degree of mitophagy in neural cells, including PD-relevant mesencephalic dopaminergic neurons and microglia. In all tissues apart from pancreatic islets, loss of Pink1 did not influence basal mitophagy, despite disrupting depolarization-induced Parkin activation. Our findings provide the first in vivo evidence that PINK1 is detectable at basal levels and that basal mammalian mitophagy occurs independently of PINK1. This suggests multiple, yet-to-be-discovered pathways orchestrating mammalian mitochondrial integrity in a context-dependent fashion, and this has profound implications for our molecular understanding of vertebrate mitophagy.
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Mitofagia , Especificidad de Órganos , Proteínas Quinasas/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Ratones Noqueados , Microglía/metabolismo , Estrés FisiológicoRESUMEN
Whether and how moderate exercise might allow for accelerated limb recovery in chronic critical limb ischemia (CLI) remains to be determined. Chronic CLI was surgically induced in mice, and the effect of moderate exercise (training five times per week over a 3-week period) was investigated. Tissue damages and functional scores were assessed on the 4th, 6th, 10th, 20th, and 30th day after surgery. Mice were sacrificed 48 h after the last exercise session in order to assess muscle structure, mitochondrial respiration, calcium retention capacity, oxidative stress and transcript levels of genes encoding proteins controlling mitochondrial functions (PGC1α, PGC1ß, NRF1) and anti-oxidant defenses markers (SOD1, SOD2, catalase). CLI resulted in tissue damages and impaired functional scores. Mitochondrial respiration and calcium retention capacity were decreased in the ischemic limb of the non-exercised group (Vmax = 7.11 ± 1.14 vs. 9.86 ± 0.86 mmol 02/min/g dw, p < 0.001; CRC = 7.01 ± 0.97 vs. 11.96 ± 0.92 microM/mg dw, p < 0.001, respectively). Moderate exercise reduced tissue damages, improved functional scores, and restored mitochondrial respiration and calcium retention capacity in the ischemic limb (Vmax = 9.75 ± 1.00 vs. 9.82 ± 0.68 mmol 02/min/g dw; CRC = 11.36 ± 1.33 vs. 12.01 ± 1.24 microM/mg dw, respectively). Exercise also enhanced the transcript levels of PGC1α, PGC1ß, NRF1, as well as SOD1, SOD2, and catalase. Moderate exercise restores mitochondrial respiration and calcium retention capacity, and it has beneficial functional effects in chronic CLI, likely by stimulating reactive oxygen species-induced biogenesis and anti-oxidant defenses. These data support further development of exercise therapy even in advanced peripheral arterial disease.
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Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-ß induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-ß induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.
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Dermatomiositis/metabolismo , Inflamación/metabolismo , Interferón beta/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Adulto , Anciano , Animales , Línea Celular , Citocinas/sangre , Dermatomiositis/tratamiento farmacológico , Dermatomiositis/patología , Femenino , Depuradores de Radicales Libres/farmacología , Adyuvante de Freund , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Debilidad Muscular/tratamiento farmacológico , Debilidad Muscular/metabolismo , Debilidad Muscular/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedad Autoinmune Experimental del Sistema Nervioso/tratamiento farmacológico , Enfermedad Autoinmune Experimental del Sistema Nervioso/metabolismo , Enfermedad Autoinmune Experimental del Sistema Nervioso/patología , TranscriptomaRESUMEN
BACKGROUND: Protection against acute skeletal muscle metabolic dysfunction and oxidative stress could be a therapeutic target in volume expansion for severely bleeding patients. OBJECTIVES: This experimental pilot study in swine aims at comparing 130/0.4 hydroxyethyl starch (HES) with 4% albumin along with crystalloid perfusion for first-line volume expansion in haemorrhagic shock with a particular emphasis on oxidative stress and muscular mitochondrial function. DESIGN: Randomised experimental study. SETTING: Digestive Cancer Research Institute Preclinical Laboratory, Strasbourg University Hospital, France, from February 2012 to June 2013. ANIMALS: Twenty large white pigs. INTERVENTION: Pressure-controlled haemorrhagic shock and volume resuscitation using either 4% human serum albumin or 130/0.4 HES along with crystalloid perfusion were performed in 20 large white pigs. MAIN OUTCOME MEASURES: Muscular biopsy of gastrocnemius muscle was performed for metabolomics screening, mitochondrial respiratory chain assessment and electron spin resonance reactive oxygen species production along with arterial and venous reactive oxygen species production at baseline, at the completion of shock, at 90âmin and at 180âmin after volume expansion. RESULTS: There was no difference between the two groups in measurements of skeletal muscle superoxide production. In a pooled analysis, there was a statistically significant decrease in gastrocnemius muscle creatine content from baseline to 90âmin (Pâ<â0.05) and 180âmin (Pâ<â0.05). Muscular lactate content and mitochondrial respiratory chain oxidative capacity remained constant at the respective time points. CONCLUSION: In this pilot experimental study in swine, during pressure-controlled haemorrhagic shock treated with either albumin or 130/0.4 HES in conjunction with crystalloid perfusion, skeletal muscle metabolic profile was unaltered. ETHICAL APPROVAL NUMBER: 38.2012.01.031.
Asunto(s)
Derivados de Hidroxietil Almidón/administración & dosificación , Soluciones Isotónicas/administración & dosificación , Músculo Esquelético/efectos de los fármacos , Resucitación/métodos , Albúmina Sérica Humana/administración & dosificación , Choque Hemorrágico/tratamiento farmacológico , Animales , Soluciones Cristaloides , Quimioterapia Combinada , Femenino , Humanos , Masculino , Metabolómica/métodos , Músculo Esquelético/metabolismo , Sustitutos del Plasma/administración & dosificación , Distribución Aleatoria , Choque Hemorrágico/metabolismo , PorcinosRESUMEN
PURPOSE: The effects of carnitine depletion upon exercise performance and skeletal muscle mitochondrial function remain largely unexplored. We therefore investigated the effect of N-trimethyl-hydrazine-3-propionate (THP), a carnitine analog inhibiting carnitine biosynthesis and renal carnitine reabsorption, on physical performance and skeletal muscle mitochondrial function in rats. METHODS: Male Sprague Dawley rats were treated daily with water (control rats; n = 12) or with 20 mg/100 g body weight THP (n = 12) via oral gavage for 3 weeks. Following treatment, half of the animals of each group performed an exercise test until exhaustion. RESULTS: Distance covered and exercise performance were lower in THP-treated compared to control rats. In the oxidative soleus muscle, carnitine depletion caused atrophy (-24%) and impaired function of complex II and IV of the mitochondrial electron transport chain. The free radical leak (ROS production relative to oxygen consumption) was increased and the cellular glutathione pool decreased. Moreover, mRNA expression of markers of mitochondrial biogenesis and mitochondrial DNA were decreased in THP-treated compared to control rats. In comparison, in the glycolytic gastrocnemius muscle, carnitine depletion was associated with impaired function of complex IV and increased free radical leak, whilst muscle weight and cellular glutathione pool were maintained. Markers of mitochondrial proliferation and mitochondrial DNA were unaffected. CONCLUSIONS: Carnitine deficiency is associated with impaired exercise capacity in rats treated with THP. THP-induced carnitine deficiency is associated with impaired function of the electron transport chain in oxidative and glycolytic muscle as well as with atrophy and decreased mitochondrial DNA in oxidative muscle.
RESUMEN
AIMS: Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as in vivo and in vitro models. RESULTS: Statins increased mitochondrial H2O2 production, the Bax/Bcl-2 ratio, and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for 2 weeks at 10 mg/kg/day in rats increased H2O2 accumulation and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle, but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Cotreatment with the antioxidant, quercetin, at 25 mg/kg/day abolished these effects in plantaris. An in vitro study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway. INNOVATION: Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles. CONCLUSION: There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy.