Cristofol Vives-Bauza

PINK1-dependent recruitment of Parkin to mitochondria in mitophagy

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinsons disease. Upon a loss of mitochondrial membrane potential (ΔΨm) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨm relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨm. We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinsons disease.

Mitophagy: the latest problem for Parkinson's disease

Parkinsons disease (PD) is a common neurodegenerative disorder of unknown cause. Some familial forms of PD are provoked by mutations in the genes encoding for the PTEN (phosphatase and tensin homolog)-induced putative kinase-1 (PINK1) and Parkin. Mounting evidence indicates that PINK1 and Parkin might function in concert to modulate mitochondrial degradation, termed mitophagy. However, the molecular mechanisms by which PINK1/Parkin affect mitophagy are just beginning to be elucidated. Herein, we review the main advances in our understanding of the PINK1/Parkin pathway. Because of the phenotypic similarities among the different forms of PD, a better understanding of PINK1/Parkin biology might have far-reaching pathogenic and therapeutic implications for both the inherited and the sporadic forms of PD.

PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinsons disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation.

Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues

Publisher Summary This chapter discusses approaches to measure adenosine triphosphate (ATP) synthesis from mammalian cells and tissues and presents the procedures to estimate the steady-state content of ATP and other high-energy phosphates by high-performance liquid chromatography (HPLC). The isolation of highly coupled mitochondria from cultured cells involves delicate and time-consuming procedures; the results are sometimes inconsistent, leading to a potential lack of reproducibility. Measurement of ATP synthesis on whole cells requires a permeabilization step to allow for the penetration of hydrophilic substrates through biological membranes. In addition, the use of permeabilized cells rather than isolated mitochondria substantially reduces the number of cells required for each assay. Fluorimetry is a commonly used method to detect ATP produced by isolated mitochondria. Another method employed on isolated mitochondria is the incorporation of Pi into ADP and its subsequent transfer to glucose-6-phosphate by hexokinase, followed by extraction of unincorporated Pi and measurement of radioactivity in a scintillation counter. Among the different methods used to assay high-energy phosphates in biological samples, HPLC has the advantage of high sensitivity and efficiency because it allows for the simultaneous analyses of all species of phosphorylated nucleotides in one analysis. The chapter also describes the assay of ATP synthesis in five cell types—HeLa, COS-7, N2A, HEK 293T, and 143 B-derived cytoplasmic hybrids harboring either wild-type mitochondrial DNA (mtDNA) or the T8993G mtDNA mutation in the ATPase 6 gene, which is responsible for a mitochondrial disorder characterized by neuropathy, ataxia, and retinitis pigmentosa.

Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.

Lack of paternal inheritance of muscle mitochondrial DNA in sporadic mitochondrial myopathies.

In 2002, paternal inheritance of muscle mitochondrial DNA (mtDNA) was reported in a patient with exercise intolerance and a mitochondrial DNA (mtDNA) mutation restricted to skeletal muscle. To evaluate whether paternal inheritance is a common phenomenon, we studied 10 sporadic patients with skeletal muscle‐restricted mtDNA mutations: five harbored mtDNA point mutations in protein‐coding genes and five had single mtDNA deletions. We performed haplotype analysis and direct sequencing of the hypervariable regions 1 and 2 of the D‐loop in muscle and blood from the patients and, when available, in blood from their parents. We did not observe paternal inheritance in any of our patients.

Bilateral striatal necrosis associated with a novel mutation in the mitochondrial ND6 gene

We report the molecular findings in two independent patients presenting with progressive generalized dystonia and bilateral striatal necrosis in whom we have identified a mutation (T14487C) in the mitochondrial ND6 gene. The mutation is heteroplasmic in all samples analyzed, and it fulfills all accepted criteria of pathogenicity. Transmitochondrial cell lines harboring 100% mutant mitochondrial DNA showed a marked decrease in the activity of complex I of the respiratory chain supporting the pathogenic role of T14487C.

PINK1/Parkin direct mitochondria to autophagy.

Mutations in PTEN-induced putative kinase 1 (PINK1) and PARK2/Parkin cause autosomal recessive forms of Parkinson disease. In mammalian cells, cytosolic Parkin is selectively recruited to depolarized mitochondria, followed by a stimulation of mitochondrial autophagy. We show that Parkin translocation to mitochondria is mediated by PINK1, even in cells with normal mitochondrial membrane potential (ΔΨm). Once at the mitochondria, Parkin is in close proximity to PINK1, but Parkin does not catalyze PINK1 ubiquitination nor does PINK1 phosphorylate Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into large mitochondrial perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin and PINK1 modulate mitochondrial trafficking to the perinuclear region, a subcellular area associated with autophagy. Mutations in either Parkin or PINK1 impair this process and, consequently, mitochondrial turnover may be altered, inducing accumulation of defective mitochondria and, ultimately, causing neurodegeneration in Parkinson disease.

Measurements of the Antioxidant Enzyme Activities of Superoxide Dismutase, Catalase, and Glutathione Peroxidase

Publisher Summary This chapter describes the activity assays of the major reactive oxygen species (ROS) defense system enzymes—superoxide dismutases, glutathione peroxidases (GPx), and catalase (CAT)—in cultured cells, tissue homogenates, and mammalian mitochondria. To cope with the damaging actions of ROS, organisms have evolved a sophisticated ROS defense system (RDS), consisting of low-molecular-weight antioxidants, such as glutathione, ascorbic acid, tocopherol, and uric acid, and specialized ROS-detoxifying enzymes, such as SODs, CAT, GPxs, and various thio-, peroxi-, and glutaredoxins. These enzymes represent the primary line of ROS defense. Defense enzymes can remove ROS directly or can repair the damage to other macromolecules caused by ROS. Other enzymes are involved in the renewal of the reducing power of defense enzymes. Failure of RDS to cope with the intracellular ROS production results in oxidative stress, which contributes to the damage and death of cells. Therefore, measuring the activity of RDS enzymes is a valuable diagnostic tool to determine the role of the oxidative stress in the pathology of a particular disease.

A novel exon 3 mutation (D76V) in the SOD1 gene associated with slowly progressive ALS

INTRODUCTION: Details of the mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with the familial form of amyotrophic lateral sclerosis are currently being gathered in order better to understand the genotype-phenotype relationship in this disorder. We report on a large family with 15 affected individuals spanning five generations. RESULTS: A novel mutation in the exon 3 of the SOD1 gene, an A-to-T transversion at nucleotide position 696 in the heterozygous state leading to a D76V amino acid change, was identified in four family members. Affected individuals showed a homogeneous phenotype, characterized by initial symptoms in the lower limbs, clinical onset in the fifth decade of life, long survival and high penetrance. DISCUSSION: Our results are discussed in relation to the previously reported exon 3 SOD1 mutations, paying particular attention to the phenotypic characteristics of ALS-SOD1 patients.