Search

The 5703G>A mutation in the tRNA gene of mitochondrial DNA seems to show a tissue-specific phenotype: early age of clinical presentation, progressive external ophthalmoplegia, fatigability and ‘extremely thin appearance’. We report a second patient with the same mutation and phenotype. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown cause. For decades, a deficit in mitochondrial respiration was thought to be a key factor in PD neurodegeneration. However, excluding a few exceptions where a clinical picture of parkinsonism is associated with a mitochondrial DNA mutation, preclinical and clinical studies have failed to identify any genetic mutations in the genes encoding for the electron transport chain complexes in PD patients. More recently, it has been discovered that mutations in the genes encoding for Parkin, PINK1 and DJ1 are associated with familial forms of PD and with mitochondrial alterations, including morphological abnormalities. These results have led many researchers to revisit the question of mitochondrial biology as a primary mechanism in PD pathogenesis, this time from an angle of perturbation in mitochondrial dynamics and not from the angle of a deficit in respiration. [ABSTRACT FROM AUTHOR]

Background: Ischemic stroke (IS) represents a significant health burden globally, necessitating a better understanding of its genetic underpinnings to improve prevention and treatment strategies. Despite advances in IS genetics, studies focusing on the Spanish population and sex-stratified analyses are lacking., Methods: A case-control genome-wide association study was conducted with 9081 individuals (3493 IS cases and 5588 healthy controls). IS subtypes using Trial of ORG 10172 in Acute Stroke Treatment criteria were explored in a sex-stratified approach. Replication efforts involved the MEGASTROKE, GIGASTROKE, and the UK Biobank international cohorts. Post-genome-wide association study analysis included: in silico proteomic analysis, gene-based analysis, quantitative trait loci annotation, transcriptome-wide association analysis, and bioinformatic analysis using chromatin accessibility data., Results: Identified as associated with IS and its subtypes were 4 significant and independent loci. Replication confirmed 5p15.2 as a new locus associated with small-vessel occlusion stroke, with rs59970332-T as the lead variant (beta [SE], 0.13 [0.02]; P =4.34×10 -8 ). Functional analyses revealed CTNND2 given proximity and its implication in pathways involved in vascular integrity and angiogenesis. Integration of Hi-C data identified additional potentially modulated genes, and in silico proteomic analysis suggested a distinctive blood proteome profile associated with the lead variant. Gene-set enrichment analyses highlighted pathways consistent with small-vessel disease pathogenesis. Gene-based associations with known stroke-related genes such as F2 and FGG were also observed, reinforcing the relevance of our findings., Conclusions: We found CTNND2 as a potential key molecule in small-vessel occlusion stroke risk, and predominantly in males. This study sheds light on the genetic architecture of IS in the Spanish population, providing novel insights into sex-specific associations and potential molecular mechanisms. Further research, including replication in larger cohorts, is essential for a comprehensive understanding of these findings and for their translation to clinical practice., Competing Interests: Dr Arenillas reports compensation from Medtronic, Daiichi Sankyo, Boehringer Ingelheim, and Pfizer for consultant services; grants from AstraZeneca, Instituto de Salud Carlos III, European Commission and Gerencia de Salud Castilla y León; compensation from Philips for data and safety monitoring services; travel support from Daiichi Sankyo Company. Dr Ribo reports stock holdings in Methinks, Nora, and Ancaonda Biomed; compensation from Cerenovus, Vesalio, Stryker Corporation, Philips, Rapid Pulse, AptaTargets, and Medtronic MiniMed Inc for consultant services; compensation from Sensome for data and safety monitoring services. Dr Heitsch reports employment by Washington University School of Medicine in St. Louis; reports funding from National Institutes of Health and American Heart Association. Dr Dhar reports grants from National Institute of Neurological Disorders and Stroke. The other authors report no conflicts.

Background and Objectives: Post-ischemic stroke (IS) outcomes vary widely among individuals, independently of clinical factors. This variability could be related to epigenetic mechanisms that regulate biological processes involved in recovery after ischemia. While several microRNAs (miRNAs) and their target genes are implicated in the pathophysiology of IS, their role in functional outcomes remains unclear. Our aim is to identify potential miRNAs associated with the 3-month outcome in patients with IS., Methods: A discovery study was performed in patients with acute IS assessed at Hospital del Mar of Barcelona from 2009 to 2018. Main inclusion criteria were initial NIH Stroke Scale (NIHSS) score >2, hospital admission within 24 hours of IS onset, and previous functional independence. Poor 3-month outcome was defined as a modified Rankin Scale score >2. We performed miRNA next-generation sequencing on plasma samples obtained within 24 hours and performed a differential expression analysis for 2,083 miRNAs using the DESeq2 package. A replication stage was performed using real time-PCR in another multicenter cohort, with equivalent inclusion criteria and multivariate regression models. We also performed enrichment pathways analyses in both phases., Results: The discovery cohort included 215 patients with IS (mean age 74.7 ± 10.2 years, 54.9% male). Age, sex, and stroke severity (measured with the 24-hour NIHSS) were associated with the 3-month outcome ( p < 0.05). After adjusting for these potential confounders, we found 74 miRNAs significantly associated ( Q < 0.05) with the 3-month poor outcome. Pathway analysis revealed significant associations with pathways related to angiogenesis, neuronal morphogenesis, transforming growth factor β (TGF-β), endothelial development, and cognition. The replication included 191 patients (mean age 78.0 ± 6.0 years, 49.7% male). We analyzed 26 miRNAs selected from the discovery stage, and 5 miRNAs replicated their association with poor outcomes ( p < 0.05, fold change >1.7): miR-376c-3p, miR-4463, miR-199a-3p, miR-584-5p, and miR-134-5p., Discussion: We identified 5 miRNAs overexpressed in patients with poor 3-month outcomes after IS, which could be involved in biological processes such as cognition, neuronal morphogenesis, and TGF-β response, suggesting a potential role in brain recovery. These findings were not evaluated in infratentorial and lacunar strokes, which limits generalizability in these particular subtypes. Further investigation is needed to explore potential applicability of these findings.

During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6 h of stroke onset and NIHSS at 24 h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24 h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification., Methods: Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort ( n = 362,661) and AF cohort ( n = 1,030,836). We considered significant variants and replicated those variants with MTAG p -value < 5 × 10 -8 influencing both traits (GWAS-pairwise) with a p -value < 0.05 in the original GWAS and in an independent cohort ( n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort., Results: We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1 , and ZEB2 . KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension., Conclusion: The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cárcel-Márquez, Muiño, Gallego-Fabrega, Cullell, Lledós, Llucià-Carol, Sobrino, Campos, Castillo, Freijo, Arenillas, Obach, Álvarez-Sabín, Molina, Ribó, Jiménez-Conde, Roquer, Muñoz-Narbona, Lopez-Cancio, Millán, Diaz-Navarro, Vives-Bauza, Serrano-Heras, Segura, Ibañez, Heitsch, Delgado, Dhar, Krupinski, Delgado-Mederos, Prats-Sánchez, Camps-Renom, Blay, Sumoy, de Cid, Montaner, Cruchaga, Lee, Martí-Fàbregas and Férnandez-Cadenas.)

Haemorrhagic transformation is a complication of recombinant tissue-plasminogen activator treatment. The most severe form, parenchymal haematoma, can result in neurological deterioration, disability, and death. Our objective was to identify single nucleotide variations associated with a risk of parenchymal haematoma following thrombolytic therapy in patients with acute ischaemic stroke. A fixed-effect genome-wide meta-analysis was performed combining two-stage genome-wide association studies (n = 1904). The discovery stage (three cohorts) comprised 1324 ischaemic stroke individuals, 5.4% of whom had a parenchymal haematoma. Genetic variants yielding a P-value < 0.05 1 × 10-5 were analysed in the validation stage (six cohorts), formed by 580 ischaemic stroke patients with 12.1% haemorrhagic events. All participants received recombinant tissue-plasminogen activator; cases were parenchymal haematoma type 1 or 2 as defined by the European Cooperative Acute Stroke Study (ECASS) criteria. Genome-wide significant findings (P < 5 × 10-8) were characterized by in silico functional annotation, gene expression, and DNA regulatory elements. We analysed 7 989 272 single nucleotide polymorphisms and identified a genome-wide association locus on chromosome 20 in the discovery cohort; functional annotation indicated that the ZBTB46 gene was driving the association for chromosome 20. The top single nucleotide polymorphism was rs76484331 in the ZBTB46 gene [P = 2.49 × 10-8; odds ratio (OR): 11.21; 95% confidence interval (CI): 4.82-26.55]. In the replication cohort (n = 580), the rs76484331 polymorphism was associated with parenchymal haematoma (P = 0.01), and the overall association after meta-analysis increased (P = 1.61 × 10-8; OR: 5.84; 95% CI: 3.16-10.76). ZBTB46 codes the zinc finger and BTB domain-containing protein 46 that acts as a transcription factor. In silico studies indicated that ZBTB46 is expressed in brain tissue by neurons and endothelial cells. Moreover, rs76484331 interacts with the promoter sites located at 20q13. In conclusion, we identified single nucleotide variants in the ZBTB46 gene associated with a higher risk of parenchymal haematoma following recombinant tissue-plasminogen activator treatment., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Stroke is one of the most common causes of death and disability. Reperfusion therapies are the only treatment available during the acute phase of stroke. Due to recent clinical trials, these therapies may increase their frequency of use by extending the time-window administration, which may lead to an increase in complications such as hemorrhagic transformation, with parenchymal hematoma (PH) being the more severe subtype, associated with higher mortality and disability rates. Our aim was to find genetic risk factors associated with PH, as that could provide molecular targets/pathways for their prevention/treatment and study its genetic correlations to find traits sharing genetic background. We performed a GWAS and meta-analysis, following standard quality controls and association analysis (fastGWAS), adjusting age, NIHSS, and principal components. FUMA was used to annotate, prioritize, visualize, and interpret the meta-analysis results. The total number of patients in the meta-analysis was 2034 (216 cases and 1818 controls). We found rs79770152 having a genome-wide significant association (beta 0.09, p -value 3.90 × 10 -8 ) located in the RP11-362K2.2:RP11-767I20.1 gene and a suggestive variant (rs13297983: beta 0.07, p -value 6.10 × 10 -8 ) located in PCSK5 associated with PH occurrence. The genetic correlation showed a shared genetic background of PH with Alzheimer's disease and white matter hyperintensities. In addition, genes containing the ten most significant associations have been related to aggregated amyloid-β, tau protein, white matter microstructure, inflammation, and matrix metalloproteinases.

Background and Purpose: Large-scale observational studies of acute ischemic stroke (AIS) promise to reveal mechanisms underlying cerebral ischemia. However, meaningful quantitative phenotypes attainable in large patient populations are needed. We characterize a dynamic metric of AIS instability, defined by change in National Institutes of Health Stroke Scale score (NIHSS) from baseline to 24 hours baseline to 24 hours (NIHSS baseline - NIHSS 24hours = ΔNIHSS 6-24h ), to examine its relevance to AIS mechanisms and long-term outcomes., Methods: Patients with NIHSS prospectively recorded within 6 hours after onset and then 24 hours later were enrolled in the GENISIS study (Genetics of Early Neurological Instability After Ischemic Stroke). Stepwise linear regression determined variables that independently influenced ΔNIHSS 6 -24h . In a subcohort of tPA (alteplase)-treated patients with large vessel occlusion, the influence of early sustained recanalization and hemorrhagic transformation on ΔNIHSS 6-24h was examined. Finally, the association of ΔNIHSS 6 -24h with 90-day favorable outcomes (modified Rankin Scale score 0-2) was assessed. Independent analysis was performed using data from the 2 NINDS-tPA stroke trials (National Institute of Neurological Disorders and Stroke rt-PA)., Results: For 2555 patients with AIS, median baseline NIHSS was 9 (interquartile range, 4-16), and median ΔNIHSS 6 -24h was 2 (interquartile range, 0-5). In a multivariable model, baseline NIHSS, tPA-treatment, age, glucose, site, and systolic blood pressure independently predicted ΔNIHSS 6 -24h (R 2 =0.15). In the large vessel occlusion subcohort, early sustained recanalization and hemorrhagic transformation increased the explained variance (R 2 =0.27), but much of the variance remained unexplained. ΔNIHSS 6 -24h had a significant and independent association with 90-day favorable outcome. For the subjects in the 2 NINDS-tPA trials, ΔNIHSS 3 -24h was similarly associated with 90-day outcomes., Conclusions: The dynamic phenotype, ΔNIHSS 6-24h , captures both explained and unexplained mechanisms involved in AIS and is significantly and independently associated with long-term outcomes. Thus, ΔNIHSS 6 -24h promises to be an easily obtainable and meaningful quantitative phenotype for large-scale genomic studies of AIS.

Background and Purpose- Immune cells play a key role in the first 24h poststroke (acute phase), being associated with stroke outcome. We aimed to find genetic risk factors associated with leukocyte counts during the acute phase of stroke. Methods- Ischemic stroke patients with leukocyte counts data during the first 24h were included. Genome-wide association study and gene expression studies were performed. Results- Our genome-wide association study, which included 2064 (Discovery) and 407 (Replication) patients, revealed a new locus (14q24.3) associated with leukocyte counts. After Joint analysis (n=2471) 5 more polymorphisms reached genome-wide significance ( P <5×10 -8 ). The 14q24.3 locus was associated with acute stroke outcome (rs112809786, P =0.036) and with ACOT1 and PTGR2 gene expression. Previous polymorphisms associated with leukocyte counts in general-population did not show any significance in our study. Conclusions- We have found the first locus associated with leukocyte counts in ischemic stroke, also associated with acute outcome. Genetic analysis of acute endophenotypes could be useful to find the genetic factors associated with stroke outcome. Our findings suggested a different modulation of immune cells in stroke compared with healthy conditions.

Objective: To validate the Genot-PA score, a clinical-genetic logistic regression score that stratifies the thrombolytic therapy safety, in a new cohort of patients with stroke., Methods: We enrolled 1,482 recombinant tissue plasminogen activator (rtPA)-treated patients with stroke in Spain and Finland from 2003 to 2016. Cohorts were analyzed on the basis of ethnicity and therapy: Spanish patients treated with IV rtPA within 4.5 hours of onset (cohort A and B) or rtPA in combination with mechanical thrombectomy within 6 hours of onset (cohort C) and Finnish participants treated with IV rtPA within 4.5 hours of onset (cohort D). The Genot-PA score was calculated, and hemorrhagic transformation (HT) and parenchymal hematoma (PH) risks were determined for each score stratum., Results: Genot-PA score was tested in 1,324 (cohort A, n = 726; B, n = 334; C, n = 54; and D, n = 210) patients who had enough information to complete the score. Of these, 213 (16.1%) participants developed HT and 85 (6.4%) developed PH. In cohorts A, B, and D, HT occurrence was predicted by the score ( p = 2.02 × 10 -6 , p = 0.023, p = 0.033); PH prediction was associated in cohorts A through C ( p = 0.012, p = 0.034, p = 5.32 × 10 -4 ). Increased frequency of PH events from the lowest to the highest risk group was found (cohort A 4%-15.7%, cohort B 1.5%-18.2%, cohort C 0%-100%). The best odds ratio for PH prediction in the highest-risk group was obtained in cohort A (odds ratio 5.16, 95% confidence interval 1.46-18.08, p = 0.009)., Conclusion: The Genot-PA score predicts HT in patients with stroke treated with IV rtPA. Moreover, in an exploratory study, the score was associated with PH risk in mechanical thrombectomy-treated patients., (© 2019 American Academy of Neurology.)

Following the involvement of CHCHD10 in FrontoTemporal-Dementia-Amyotrophic Lateral Sclerosis (FTD-ALS) clinical spectrum, a founder mutation (p.Gly66Val) in the same gene was identified in Finnish families with late-onset spinal motor neuronopathy (SMAJ). SMAJ is a slowly progressive form of spinal muscular atrophy with a life expectancy within normal range. In order to understand why the p.Ser59Leu mutation, responsible for severe FTD-ALS, and the p.Gly66Val mutation could lead to different levels of severity, we compared their effects in patient cells. Unlike affected individuals bearing the p.Ser59Leu mutation, patients presenting with SMAJ phenotype have neither mitochondrial myopathy nor mtDNA instability. The expression of CHCHD10 S59L mutant allele leads to disassembly of mitochondrial contact site and cristae organizing system (MICOS) with mitochondrial dysfunction and loss of cristae in patient fibroblasts. We also show that G66V fibroblasts do not display the loss of MICOS complex integrity and mitochondrial damage found in S59L cells. However, S59L and G66V fibroblasts show comparable accumulation of phosphorylated mitochondrial TDP-43 suggesting that the severity of phenotype and mitochondrial damage do not depend on mitochondrial TDP-43 localization. The expression of the CHCHD10 G66V allele is responsible for mitochondrial network fragmentation and decreased sensitivity towards apoptotic stimuli, but with a less severe effect than that found in cells expressing the CHCHD10 S59L allele. Taken together, our data show that cellular phenotypes associated with p.Ser59Leu and p.Gly66Val mutations in CHCHD10 are different; loss of MICOS complex integrity and mitochondrial dysfunction, but not TDP-43 mitochondrial localization, being likely essential to develop a severe motor neuron disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The role of neuronal surface autoantibodies (NSAs) in non-encephalitic psychosis is of recent and controversial interest. Most of the studies relating NSAs with psychosis are retrospective and only focused on the N-methyl-d-aspartate glutamate receptor (NMDAR). Our goal was to evaluate the prevalence of IgG antibodies against the NMDAR NR1 subunit (NMDAR-Abs) along with five additional NSAs in 61 first psychotic episode patients and 47 matched controls. We found two patients positive for NMDAR-Abs (3.3%). One of them was eventually considered to have been misdiagnosed and reclassified as encephalitis. The other met the criteria for bipolar I disorder, presented no neurological symptoms and had a comorbid HIV infection of vertical transmission. This is the first reported case of an HIV-infected patient with psychosis associated with NSAs. This study shows that patients presenting with clinically incomplete forms of anti-NMDAR encephalitis, with predominant or isolated psychiatric symptoms, can remain undetected if no ancillary tests are performed. To improve patient diagnosis and treatment of individuals with a first psychotic episode, more detailed neurological examinations might be needed. Further studies are required to better clarify the role of NSAs in the neuropsychiatric effects of HIV infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Objective: Cases of narcolepsy in association with psychotic features have been reported but never fully characterized. These patients present diagnostic and treatment challenges and may shed new light on immune associations in schizophrenia., Method: Our case series was gathered at two narcolepsy specialty centers over a 9-year period. A questionnaire was created to improve diagnosis of schizophrenia or another psychotic disorder in patients with narcolepsy. Pathophysiological investigations included full HLA Class I and II typing, testing for known systemic and intracellular/synaptic neuronal antibodies, recently described neuronal surface antibodies, and immunocytochemistry on brain sections to detect new antigens., Results: Ten cases were identified, one with schizoaffective disorder, one with delusional disorder, two with schizophreniform disorder, and 6 with schizophrenia. In all cases, narcolepsy manifested first in childhood or adolescence, followed by psychotic symptoms after a variable interval. These patients had auditory hallucinations, which was the most differentiating clinical feature in comparison to narcolepsy patients without psychosis. Narcolepsy therapy may have played a role in triggering psychotic symptoms but these did not reverse with changes in narcolepsy medications. Response to antipsychotic treatment was variable. Pathophysiological studies did not reveal any known autoantibodies or unusual brain immunostaining pattern. No strong HLA association outside of HLA DQB1*06:02 was found, although increased DRB3*03 and DPA1*02:01 was notable., Conclusion: Narcolepsy can occur in association with schizophrenia, with significant diagnostic and therapeutic challenges. Dual cases maybe under diagnosed, as onset is unusually early, often in childhood. Narcolepsy and psychosis may share an autoimmune pathology; thus, further investigations in larger samples are warranted., (© 2014 American Academy of Sleep Medicine.)

Parkinson's 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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

For decades, it has been presumed that mitochondrial dysfunction, in the form of impaired complex I activity, may contribute to the cause of Parkinson disease (PD). ( 1) The discovery that several gene mutations cause familial forms of PD ( 1) has led to a renewed enthusiasm for the mitochondrial hypothesis of PD, but this time from a quite distinct and, perhaps, more realistic angle. Among these genes, those that code for PTEN-induced kinase-1 (PINK1) ( 2) and for the E3-ubiquitin ligase Parkin ( 3) did attract major interest from mitochondriologists, in part, because both proteins interact with each other and apparently function, genetically, within the same molecular pathway to modulate mitochondrial dynamics in Drosophila. ( 4-6).

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (DeltaPsi(m)) 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 DeltaPsi(m) relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal DeltaPsi(m). 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 Parkinson's disease.

In mammals, subunit c of the F(1)F(0)-ATP synthase has three isoforms (P1, P2, and P3). These isoforms differ by their cleavable mitochondrial targeting peptides, whereas the mature peptides are identical. To investigate this apparent genetic redundancy, we knocked down each of the three subunit c isoform by RNA interference in HeLa cells. Silencing any of the subunit c isoforms individually resulted in an ATP synthesis defect, indicating that these isoforms are not functionally redundant. We found that subunit c knockdown impaired the structure and function of the mitochondrial respiratory chain. In particular, P2 silencing caused defective cytochrome oxidase assembly and function. Because the expression of exogenous P1 or P2 was able to rescue the respective silencing phenotypes, but the two isoforms were unable to cross-complement, we hypothesized that their functional specificity resided in their targeting peptides. In fact, the expression of P1 and P2 targeting peptides fused to GFP variants rescued the ATP synthesis and respiratory chain defects in the silenced cells. Our results demonstrate that the subunit c isoforms are nonredundant, because they differ functionally by their targeting peptides, which, in addition to mediating mitochondrial protein import, play a yet undiscovered role in respiratory chain maintenance.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder associated with a loss of dopaminergic neurons. The role of mitochondria in the aetiology of PD has been questioned for decades, mostly from the perspective of bioenergetic failure. For decades, a deficit in mitochondrial respiration was thought to be a key factor in PD neurodegeneration. However, excluding a few exceptions where a clinical picture of parkinsonism is associated with a mitochondrial DNA mutation, preclinical and clinical studies have failed to identify any genetic mutations in the genes encoding for the electron transport chain complexes in PD patients. More recently, it has been discovered that mutations in the genes encoding for Parkin, PINK1 (PTEN-induced putative kinase-1) and DJ-1 are associated with familial forms of PD and with mitochondrial alterations, including morphological abnormalities. These results have led many researchers to revisit the question of mitochondrial biology as a primary mechanism in PD pathogenesis, this time from an angle of perturbation in mitochondrial dynamics and not from the angle of a deficit in respiration., (2010 Elsevier B.V. All rights reserved.)

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's 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 alpha-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and alpha-synclein aggregation.

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATP-dependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like age-related macular degeneration.

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.

The malondialdehyde-thiobarbituric acid assay is widely used to study lipid peroxidation. Among the various methods used to perform the assay, the most widely accepted is the quantification of malondialdehyde using the thiobarbituric acid reaction, followed by reversed-phase chromatography. However, unacceptable results may be obtained as malondialdehyde can be produced in vitro. To study the conditions that inhibit in vitro lipid peroxidation, malondialdehyde levels were measured in cultured cells using different concentrations of butylated hydroxytoluene, EDTA or a combination of both. Butylated hydroxytoluene alone inhibits in vitro lipid peroxidation effectively. EDTA reduces artificially produced malondialdehyde, but not totally. Finally, the combination of EDTA and butylated hydroxytoluene does not improve the results obtained using butylated hydroxytoluene alone. The conclusion is that in the malondialdehyde-thiobarbituric acid assay it is necessary to add an inhibitor of the in vitro lipid peroxidation and assay the necessary concentration depending on the specimen used.

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.

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.

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.

The pathogenesis of Parkinson's disease (PD) is largely unknown. Indirect evidence suggests that mutations in mitochondrial DNA (mtDNA) might play a role, but previous studies have not consistently associated any specific mutations with PD. However, these studies have generally been confined to limited areas of the mitochondrial genome. We therefore sequenced the entire mitochondrial genome from substantia nigra of 8 PD and 9 control subjects. Several sequence variants were distributed differently between PD and control subjects, but all were previously reported polymorphisms. Several secondary LHON mutations were found, as well as a number of novel missense mutations, but all were rare and did not differ between PD and control subjects. Finally, PD and control subjects did not differ in the total number of all mutations, nor the total number of missense mutations. Thus, mtDNA involvement in PD, if any, is likely to be complex and should be reconsidered carefully., (©2002 Elsevier Science (USA).)

Background: Some patients presenting with isolated lifelong exercise intolerance and ragged-red fibres, harbour skeletal-muscle restricted mutations in their mitochondrial DNA., Aim: To identify the molecular defect in a patient presenting with lifelong exercise intolerance, ragged-red fibres and deficiencies of complexes III and IV in skeletal muscle., Methods: The muscle biopsy was studied for activities of the respiratory chain, histochemical stains, and sequencing the tRNA genes of mitochondrial DNA., Results: The patient had a heteroplasmic mutation in the tRNA(Leu (CUN)) gene of mitochondrial DNA (G12334A). Clinical and morphological data as well as restriction fragment length polymorphism (RFLP) and single-fibre polymerase chain reaction (PCR) analyses strongly indicate that this molecular defect is the primary cause of the myopathy., Conclusion: Mutations in any mitochondrial gene should be considered in the differential diagnosis of patients with lifelong exercise intolerance, even when the neurological examination is normal.

Impairment of haemoglobin synthesis occurs in the genetic diseases known as thalassaemia. The consequent chronic anaemia leads to increased dietary iron absorption which results in iron overload. Treatment through regular blood transfusions increases oxygen capacity, but also adds iron from haemoglobin. An essential treatment, in parallel with transfusions, is the use of chelating agents to remove the excess iron. Thalassaemia patients are particularly at risk of free radical damage. Human lymphocytes from normal individuals can be investigated in vitro as a model system in the presence of free radicals in the Comet assay. This assay measures DNA damage, particularly DNA strand breakage. We examined cells from an Australian thalassaemic patient (sickle/beta thal double heterozygote-sickle phenotype) who had not yet received chelation therapy to determine if the cells were more sensitive to simulated iron overload and to haemosiderins. Lymphocytes from the patient were received as frozen samples after 28 h on dry ice and then placed in liquid nitrogen. Normal lymphocytes frozen under the same conditions and normal nonfrozen lymphocytes were compared. The lymphocytes from a normal female did not respond in vitro to ferric chloride (FeCl(3)) or haemosiderin but did to ferrous chloride (FeCl(2)) and ferrous sulphate (FeSO(4)). Deferoxamine appeared to reduce the response to FeCl(2) and FeSO(4) but deferiprone did not. When the lymphocytes from the nonchelated patient were treated with FeSO(4) and hydrogen peroxide, deferoxamine and deferiprone both reduced the response. Over the same dose range of iron salt (FeSO(4)), the lymphocytes from the thalassaemic patient were more sensitive, with much higher background levels of damage and induced damage. When deferiprone and deferoxamine were compared over a nontoxic range, deferiprone appeared to produce a greater reduction of damage in lymphocytes of the thalassaemia patient. Ferritin iron appears to be more available than haemosiderin iron in reactions leading to DNA damage. Haemosiderin containing higher amounts of the goethite-like (alpha-FeOOH) iron oxide phase leads to lower levels of DNA damage.

Thalassaemia is a group of genetic diseases where haemoglobin synthesis is impaired. This chronic anaemia leads to increased dietary iron absorption, which develops into iron overload pathology. Treatment through regular transfusions increases oxygen capacity but also provides iron through the red cells' haemoglobin. An essential treatment, in parallel with transfusions, is the use of chelating agents to remove the excess iron deposited in tissues. These deposits are found in the liver, spleen, heart, and pancreas and are associated with cardiac failure and diabetes. The deposits in these tissues of patients have been isolated as haemosiderin. Thalassaemia patients are particularly at risk of free radical induced damage. Thus, the present study has investigated, as a model system, human cells in vitro in the Comet assay in the presence of free radicals. This assay measures DNA damage, particularly DNA strand breakage. The effects of iron overload on cells oxidatively stressed with hydrogen peroxide (H(2)O(2)) have been determined as well as the effect of the chelating agent, deferoxamine. Iron overload was simulated with ferric (FeCl(3)) and ferrous chloride (FeCl(2)), ferrous sulphate (FeSO(4)) and haemosiderins. Both human lymphocytes from a male and a female donor and human adenocarcinoma colonic cells showed an increase in DNA damage in the Comet assay after treatment with H(2)O(2). Ferric chloride produced an increase in DNA damage in human colonic cells, but little or no damage in human lymphocytes. Ferrous chloride also produced weak DNA damage in human lymphocytes, but ferrous sulphate produced a dose-related response. Deferoxamine produced no DNA damage. When H(2)O(2) was combined with FeCl(3), FeCl(2), or FeSO(4), the DNA damage produced was as least as great as or slightly greater than with H(2)O(2) alone. When deferoxamine was combined with H(2)O(2) and FeSO(4) there was a consistent decrease in response. There was little or no decrease in response when deferoxamine was combined with H(2)O(2) and FeCl(3) or FeCl(2), but at high (100-300microm) doses there were changes in the appearance of cellular DNA from Comet tails to dense centres surrounded by a diffuse area. This was probably as a consequence of chelation processes. Haemosiderin produced no damage. The three fractions of haemosiderin examined were of three different densities and from a Thai patient where the oxyhydroxide phase is the ferrihydrite. The colour change was similar to that for FeCl(3), but the level of the ferric ion in the haemosiderin was possibly too low in the sample to produce a response. The next stage is to examine peripheral lymphocytes from thalassaemic patients, with and without chelation therapy, whose cells may be more sensitive to simulated iron overload and to lower levels of haemosiderin. Teratogenesis Carcinog. Mutagen. 20:11-26, 2000., (Copyright 2000 Wiley-Liss, Inc.)

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64 x 10(-6). The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P < 0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.