Deoxyguanosine kinase deficiency: natural history and liver transplant outcome.

Autosomal recessive pathogenetic variants in the DGUOK gene cause deficiency of deoxyguanosine kinase activity and mitochondrial deoxynucleotides pool imbalance, consequently, leading to quantitative and/or qualitative impairment of mitochondrial DNA synthesis. Typically, patients present early-onset liver failure with or without neurological involvement and a clinical course rapidly progressing to death. This is an international multicentre study aiming to provide a retrospective natural history of deoxyguanosine kinase deficient patients. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centres or clinicians all around the world caring for previously reported patients were contacted to provide followup information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of deoxyguanosine kinase deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from a systematic literature review, were analyzed. Patients had a neonatal onset (≤ 1 month) in 55.7% of cases, infantile (>1 month and ≤ 1 year) in 32.3%, pediatric (>1 year and ≤18 years) in 2.5% and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed statistically different survival rates (P < 0.0001) among the four age groups with the highest mortality for neonatal onset. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype-phenotype correlation was identified. Liver transplant significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after liver transplant. In conclusion, deoxyguanosine kinase deficiency is a disease spectrum with a prevalent liver and brain tissue specificity in neonatal and infantile-onset patients and muscle tissue specificity in adult-onset cases. Our study provides clinical, molecular genetics and biochemical data for early diagnosis, clinical trial planning and immediate intervention with liver transplant and/or nucleoside supplementation.

Exploring the impact of the PNPLA3 I148M variant on primary human hepatic stellate cells using 3D extracellular matrix models.

BACKGROUND & AIMS: The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSC biology using transcriptomic data and validated findings in 3D-culture models. METHODS: RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of individuals with obesity, genotyped for the PNPLA3 I148M variant. Data were validated in wild-type (WT) or PNPLA3 I148M variant-carrying HSCs cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic livers, with/without TGFB1 or cytosporone B (Csn-B) treatment. RESULTS: Transcriptomic analyses of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in WT/PNPLA3-I148M HSCs cultured in 3D liver scaffolds. Mitochondrial dysfunction in PNPLA3-I148M cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in PNPLA3-I148M HSCs, while reactive oxygen species secretion was increased in PNPLA3-I148M HSCs and higher in bioengineered cirrhotic vs. healthy scaffolds. TGFB1 signalling followed the same trend. In PNPLA3-I148M cells, expression and activation of the endogenous TGFB1 inhibitor NR4A1 were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in PNPLA3-WT HSCs and to Erk signalling in PNPLA3-I148M HSCs. CONCLUSION: HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant responses, and increased TGFB1 signalling, which dampens antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs requires further investigation. Here, by using comparative transcriptomics and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs' behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.

Huntington disease affects mitochondrial network dynamics predisposing to pathogenic mtDNA mutations.

Huntington disease (HD) predominantly affects the brain causing a mixed movement disorder, cognitive decline and behavioural abnormalities. It also causes a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been reported in tissues of HD models, including skeletal muscle, and lymphoblasts and fibroblasts cultures from HD patients. Mutant huntingtin protein (mutHTT) expression can impair mitochondrial quality control and accelerate mitochondrial ageing. Here we obtained fresh human skeletal muscle, a post-mitotic tissue expressing the mutated HTT allele at physiological levels since birth, and primary cell lines from HTT CAG repeat expansion mutation carriers and matched healthy volunteers to examine whether such a mitochondrial phenotype exists in human HD. Using ultra-deep mitochondrial DNA (mtDNA) sequencing, we show an accumulation of mtDNA mutations affecting oxidative phosphorylation. Tissue proteomics indicate impairments in mtDNA maintenance with increased mitochondrial biogenesis of less efficient oxidative phosphorylation (lower complex I and IV activity). In full-length mutHTT expressing primary human cell lines, fission inducing mitochondrial stress resulted in normal mitophagy. In contrast, expression of high levels of N-terminal mutHTT fragments promoted mitochondrial fission and resulted in slower, less dynamic mitophagy. Expression of high levels of mutHTT fragments due to somatic nuclear HTT CAG instability can thus affect mitochondrial network dynamics and mitophagy leading to pathogenic mtDNA mutations. We show that life-long expression of mutant HTT causes a mitochondrial phenotype indicative of mtDNA instability in fresh post-mitotic human skeletal muscle. Thus, genomic instability may not be limited to nuclear DNA where it results in somatic expansion of HTT CAG repeat length in particularly vulnerable cells, such as striatal neurons. In addition to efforts targeting the causative mutation promoting mitochondrial health may be a complementary strategy in treating diseases with DNA instability, such as HD.

Remodelling of the Mitochondrial Bioenergetic Pathways in Human Cultured Fibroblasts with Carbohydrates.

Mitochondrial oxidative phosphorylation defects underlie many neurological and neuromuscular diseases. Patients' primary dermal fibroblasts are one of the most commonly used in vitro models to study mitochondrial pathologies. However, fibroblasts tend to rely more on glycolysis than oxidative phosphorylation for their energy when cultivated in standard high-glucose medium, rendering it difficult to expose mitochondrial dysfunctions. This study aimed to systematically investigate to which extent the use of galactose- or fructose-based medium switches the fibroblasts' energy metabolism to a more oxidative state. Highly proliferative cells depend more on glycolysis than less proliferative cells. Therefore, we investigated two primary dermal fibroblast cultures from healthy subjects: a highly proliferative neonatal culture and a slower-growing adult culture. Cells were cultured with 25 mM glucose, galactose or fructose, and 4 mM glutamine as carbon sources. Compared to glucose, both galactose and fructose reduce the cellular proliferation rate, but the galactose-induced drop in proliferation is much more profound than the one observed in cells cultivated in fructose. Both galactose and fructose result in a modest increase in mitochondrial content, including mitochondrial DNA, and a disproportionate increase in protein levels, assembly, and activity of the oxidative phosphorylation enzyme complexes. Galactose- and fructose-based media induce a switch of the prevalent biochemical pathway in cultured fibroblasts, enhancing aerobic metabolism when compared to glucose-based medium. While both galactose and fructose stimulate oxidative phosphorylation to a comparable degree, galactose decreases the cellular proliferation rate more than fructose, suggesting that a fructose-based medium is a better choice when studying partial oxidative phosphorylation defects in patients' fibroblasts.

Prostacyclin mimetics inhibit DRP1-mediated pro-proliferative mitochondrial fragmentation in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disorder, involving the remodelling of the small pulmonary arteries. Underlying this remodelling is the hyper-proliferation of pulmonary arterial smooth muscle cells within the medial layers of these arteries and their encroachment on the lumen. Previous studies have demonstrated an association between excessive mitochondrial fragmentation, a consequence of increased expression and post-translational activation of the mitochondrial fission protein dynamin-related protein 1 (DRP1), and pathological proliferation in PASMCs derived from PAH patients. However, the impact of prostacyclin mimetics, widely used in the treatment of PAH, on this pathological mitochondrial fragmentation remains unexplored. We hypothesise that these agents, which are known to attenuate the proliferative phenotype of PAH PASMCs, do so in part by inhibiting mitochondrial fragmentation. In this study, we confirmed the previously reported increase in DRP1-mediated mitochondrial hyper-fragmentation in PAH PASMCs. We then showed that the prostacyclin mimetic treprostinil signals via either the Gs-coupled IP or EP2 receptor to inhibit mitochondrial fragmentation and the associated hyper-proliferation in a manner analogous to the DRP1 inhibitor Mdivi-1. We also showed that treprostinil recruits either the IP or EP2 receptor to activate PKA and induce the phosphorylation of DRP1 at the inhibitory residue S637 and inhibit that at the stimulatory residue S616, both of which are suggestive of reduced DRP1 fission activity. Like treprostinil, MRE-269, an IP receptor agonist, and butaprost, an EP2 receptor agonist, attenuated DRP1-mediated mitochondrial fragmentation through PKA. We conclude that prostacyclin mimetics produce their anti-proliferative effects on PAH PASMCs in part by inhibiting DRP1-mediated mitochondrial fragmentation.

Exploring metabolism in scleroderma reveals opportunities for pharmacological intervention for therapy in fibrosis.

Background: Recent evidence has indicated that alterations in energy metabolism play a critical role in the pathogenesis of fibrotic diseases. Studies have suggested that 'metabolic reprogramming' involving the glycolysis and oxidative phosphorylation (OXPHOS) in cells lead to an enhanced generation of energy and biosynthesis. The aim of this study was to assess the molecular basis of changes in fibrotic metabolism in systemic sclerosis (Scleroderma; SSc) and highlight the most appropriate targets for anti-fibrotic therapies. Materials and methods: Dermal fibroblasts were isolated from five SSc patients and five healthy donors. Cells were cultured in medium with/without TGF-ß1 and with/without ALK5, pan-PIM or ATM kinase inhibitors. Extracellular flux analyses were performed to evaluate glycolytic and mitochondrial respiratory function. The mitochondrial network in TMRM-stained cells was visualized by confocal laser-scanning microscopy, followed by semi-automatic analysis on the ImageJ platform. Protein expression of ECM and fibroblast components, glycolytic enzymes, subunits of the five OXPHOS complexes, and dynamin-related GTPases and receptors involved in mitochondrial fission/fusion were assessed by western blotting. Results: Enhanced mitochondrial respiration coupled to ATP production was observed in SSc fibroblasts at the expense of spare respiratory capacity. Although no difference was found in glycolysis when comparing SSc with healthy control fibroblasts, levels of phophofructokinase-1 isoform PFKM were significantly lower in SSc fibroblasts (P<0.05). Our results suggest that the number of respirasomes is decreased in the SSc mitochondria; however, the organelles formed a hyperfused network, which is thought to increase mitochondrial ATP production through complementation. The increased mitochondrial fusion correlated with a change in expression levels of regulators of mitochondrial morphology, including decreased levels of DRP1, increased levels of MIEF2 and changes in OPA1 isoform ratios. TGF-ß1 treatment strongly stimulated glycolysis and mitochondrial respiration and induced the expression of fibrotic markers. The pan-PIM kinase inhibitor had no effect, whereas both ALK5 and ATM kinase inhibition abrogated TGF-ß1-mediated fibroblast activation, and upregulation of glycolysis and respiration. Conclusions: Our data provide evidence for a novel mechanism(s) by which SSc fibroblasts exhibit altered metabolic programs and highlight changes in respiration and dysregulated mitochondrial morphology and function, which can be selectively targeted by small molecule kinase inhibitors.

Ambroxol reverses tau and α-synuclein accumulation in a cholinergic N370S GBA1 mutation model.

Cognitive impairment is a common non-motor complication of Parkinson's disease (PD). Glucocerebrosidase gene (GBA1) variants are found in 10-15% of PD cases and are numerically the most important risk factor for PD and dementia with Lewy bodies. Accumulation of α-synuclein and tau pathology is thought to underlie cognitive impairment in PD and likely involves cholinergic as well as dopaminergic neurons. Neural crest stem cells were isolated from both PD patients with the common heterozygous N370S GBA1 mutation and normal subjects without GBA1 mutations. The stem cells were used to generate a cholinergic neuronal cell model. The effects of the GBA1 variant on glucocerebrosidase (GCase) protein and activity, and cathepsin D, tau and α-synuclein protein levels in cholinergic neurons were examined. Ambroxol, a GCase chaperone, was used to investigate whether GCase enhancement was able to reverse the effects of the GBA1 variant on cholinergic neurons. Significant reductions in GCase protein and activity, as well as in cathepsin D levels, were found in GBA1 mutant (N370S/WT) cholinergic neurons. Both tau and α-synuclein levels were significantly increased in GBA1 mutant (N370S/WT) cholinergic neurons. Ambroxol significantly enhanced GCase activity and decreased both tau and α-synuclein levels in cholinergic neurons. GBA1 mutations interfere with the metabolism of α-synuclein and tau proteins and induce higher levels of α-synuclein and tau proteins in cholinergic neurons. The GCase pathway provides a potential therapeutic target for neurodegenerative disorders related to pathological α-synuclein or tau accumulation.

The PINK1-Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells.

Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in PINK1 -Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential mitophagy defects. We aimed to characterise the expression of the PINK1 -Parkin mitophagy machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein expression levels were characterised with reverse transcriptase quantitative PCR and western blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable and PINK1 mRNA levels were remarkably low in control PBMCs. Our findings suggest that the PINK1 -Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable biosample for analysis of mitophagy function in Parkinson disease patients.

Mitochondrial respiratory chain and Krebs cycle enzyme function in human donor livers subjected to end-ischaemic hypothermic machine perfusion.

INTRODUCTION: Marginal human donor livers are highly susceptible to ischaemia reperfusion injury and mitochondrial dysfunction. Oxygenation during hypothermic machine perfusion (HMP) was proposed to protect the mitochondria but the mechanism is unclear. Additionally, the distribution and uptake of perfusate oxygen during HMP are unknown. This study aimed to examine the feasibility of mitochondrial function analysis during end-ischaemic HMP, assess potential mitochondrial viability biomarkers, and record oxygenation kinetics. METHODS: This was a randomised pilot study using human livers retrieved for transplant but not utilised. Livers (n = 38) were randomised at stage 1 into static cold storage (n = 6), hepatic artery HMP (n = 7), and non-oxygen supplemented portal vein HMP (n = 7) and at stage 2 into oxygen supplemented and non-oxygen supplemented portal vein HMP (n = 11 and 7, respectively). Mitochondrial parameters were compared between the groups and between low- and high-risk marginal livers based on donor history, organ steatosis and preservation period. The oxygen delivery efficiency was assessed in additional 6 livers using real-time measurements of perfusate and parenchymal oxygen. RESULTS: The change in mitochondrial respiratory chain (complex I, II, III, IV) and Krebs cycle enzyme activity (aconitase, citrate synthase) before and after 4-hour preservation was not different between groups in both study stages (p > 0.05). Low-risk livers that could have been used clinically (n = 8) had lower complex II-III activities after 4-hour perfusion, compared with high-risk livers (73 nmol/mg/min vs. 113 nmol/mg/min, p = 0.01). Parenchymal pO2 was consistently lower than perfusate pO2 (p ≤ 0.001), stabilised in 28 minutes compared to 3 minutes in perfusate (p = 0.003), and decreased faster upon oxygen cessation (75 vs. 36 minutes, p = 0.003). CONCLUSIONS: Actively oxygenated and air-equilibrated end-ischaemic HMP did not induce oxidative damage of aconitase, and respiratory chain complexes remained intact. Mitochondria likely respond to variable perfusate oxygen levels by adapting their respiratory function during end-ischaemic HMP. Complex II-III activities should be further investigated as viability biomarkers.

Sirtuin 5 depletion impairs mitochondrial function in human proximal tubular epithelial cells.

Ischemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD+-dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia.

Mitochondria as target to inhibit proliferation and induce apoptosis of cancer cells: the effects of doxycycline and gemcitabine.

Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells were not affected by doxycycline. Key effects of doxycycline observed in A549 cells, such as the decrease of mitochondrial-encoded proteins and surviving cells were also seen in the cancer cell lines COLO357 and HT29. Our results suggest that doxycycline suppresses cancer cell proliferation and primes cells for apoptosis by gemcitabine.

Huntingtin Aggregates and Mitochondrial Pathology in Skeletal Muscle but not Heart of Late-Stage R6/2 Mice.

BACKGROUND: Cell or tissue specific background may influence the consequences of expressing the Huntington's disease (HD) mutation. Aggregate formation is known to occur in skeletal muscle, but not heart of the R6/2 fragment HD model. OBJECTIVE: We asked whether aggregate formation and the expression and subcellular localization of huntingtin species was associated with mitochondrial dysfunction. METHODS: We analyzed levels of soluble HTT and HTT aggregates, as well as important fission and fusion proteins and mitochondrial respiratory chain activities, in quadriceps and heart of the R6/2 N-terminal fragment mouse model (12 weeks, 160±10 CAG repeats). RESULTS: Soluble mutant HTT was present in both tissues with expression higher in cytoplasmic/mitochondrial than nuclear fractions. HTT aggregates were only detectable in R6/2 quadriceps, in association with increased levels of the pro-fission factor DRP1 and its phosphorylated active form, and decreased levels of the pro-fusion factor MFN2. In addition, respiratory chain complex activities were decreased. In heart that was without detectable HTT aggregates, we found no evidence for mitochondrial dysfunction. CONCLUSION: Tissue specific factors may exist that protect the R6/2 heart from HTT aggregate formation and mitochondrial pathology.

Correction: The clinical spectrum and natural history of early-onset diseases due to DNA polymerase gamma mutations.

Since the online publication of the article, the authors have noted errors with Table 2; this has now been corrected in both the HTML and the PDF.

Mitochondria as oncotarget: a comparison between the tetracycline analogs doxycycline and COL-3.

Tetracyclines have anticancer properties in addition to their well-known antibacterial properties. It has been proposed that tetracyclines slow metastasis and angiogenesis through inhibition of matrix metalloproteinases. However, we believe that the anticancer effect of tetracyclines is due to their inhibition of mitochondrial protein synthesis, resulting in a decrease of the mitochondrial energy generating capacity. Several groups have developed analogs that are void of antibacterial action. An example is COL-3, which is currently tested for its anticancer effects in clinical trials. We have undertaken a comparative study of the tetracycline analogs COL-3 and doxycycline, which has an antibacterial function, to further investigate the role of the mitochondrial energy generating capacity in the anticancer mechanism and, thereby, evaluate the usefulness of mitochondria as an oncotarget. Our experiments with cultures of the human A549, COLO357 and HT29 cancer cells and fibroblasts indicated that COL-3 is significantly more cytotoxic than doxycycline. Mitochondrial translation assays demonstrated that COL-3 has retained its inhibitory effect on mitochondrial protein synthesis. Both drugs caused a severe decrease in the levels of mitochondrially encoded cytochrome-c oxidase subunits and cytochrome-c oxidase activity. In addition, COL-3 produced a marked drop in the level of nuclear-encoded succinate dehydrogenase subunit A and citrate synthase activity, indicating that COL-3 has multiple inhibitory effects. Contrary to COL-3, the anticancer action of doxycycline appears to be based specifically on inhibition of mitochondrial protein synthesis, which is thought to affect rapidly proliferating cancer cells more than healthy tissue. Doxycycline is likely to cause less side effects that COL-3.

Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains.

The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.10.1093/brain/awy157_video1awy157media15813519976001.

NDUFA4 (Renamed COXFA4) Is a Cytochrome-c Oxidase Subunit.

Groundbreaking work by Kadenbach and colleagues in the 1980s revealed the presence of 13 subunits in the mammalian mitochondrial cytochrome-c oxidase (COX; Complex IV). This observation stood the test of time until 2012 when it was demonstrated that NDUFA4, a polypeptide previously attributed to mitochondrial Complex I, was a 14th subunit of COX. In his recent opinion article, Kadenbach argued that NDUFA4 is not a subunit of COX. However, based on the findings that NDUFA4 deficiency results in a severe loss of COX activity and that NDUFA4 represents a stoichiometric component of the individual COX complex, we reason that NDUFA4 is a bona fide COX subunit and propose renaming it as COX subunit FA4 (COXFA4).

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease.

OBJECTIVE: Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and establish clinically meaningful genotype-phenotype correlations. METHODS: RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evidence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously reported patients harboring RNASEH1 mutations was subsequently undertaken. RESULTS: Pathogenic c.424G>A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 genetic analysis was undertaken in 50 additional Indian probands with variable clinical presentations associated with multiple mtDNA deletions, but no further RNASEH1 mutations were confirmed. RNASEH1-related mitochondrial disease was characterized by PEO (100%), cerebellar ataxia (57%), and dysphagia (50%). The ataxia neuropathy spectrum phenotype was observed in 1 patient. Although the c.424G>A p.Val142Ile mutation underpins all reported RNASEH1-related mitochondrial disease, haplotype analysis suggested an independent origin, rather than a founder event, for the variant in our families. CONCLUSIONS: In our cohort, RNASEH1 mutations represent the fourth most common cause of adult mendelian PEO associated with multiple mtDNA deletions, following mutations in POLG, RRM2B, and TWNK. RNASEH1 genetic analysis should also be considered in all patients with POLG-negative ataxia neuropathy spectrum. The pathophysiologic mechanisms by which the c.424G>A p.Val142Ile mutation impairs human RNase H1 warrant further investigation.

The clinical spectrum and natural history of early-onset diseases due to DNA polymerase gamma mutations.

PurposeMutations in POLG, the most common single-gene cause of inherited mitochondrial disease, are diagnostically challenging owing to clinical heterogeneity and overlap between syndromes. We aimed to improve the clinical recognition of POLG-related disorders in the pediatric population.MethodsWe performed a multinational, phenotype: genotype study using patients from three centers, two Norwegian and one from the United Kingdom. Patients with age at onset <12 years and confirmed pathogenic biallelic POLG mutations were considered eligible.ResultsA total of 27 patients were identified with a median age at onset of 11 months (range 0.6-80.4). The majority presented with global developmental delay (n=24/24, 100%), hypotonia (n=22/23, 96%) and faltering growth (n=24/27, 89%). Epilepsy was common, but notably absent in patients with the myocerebrohepatopathy spectrum phenotype. We identified two novel POLG gene mutations.ConclusionOur data suggest that POLG-related disease should be suspected in any child presenting with diffuse neurological symptoms. Full POLG sequencing is recommended since targeted screening may miss mutations. Finally, we simplify the classification of POLG-related disease in children using epilepsy as the crucial defining element; we show that Alpers and myocerebrohepatopathy spectrum follow different outcomes and that they manifest different degrees of respiratory chain dysfunction.

A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers.

Numerically the most important risk factor for the development of Parkinson's disease (PD) is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase) activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.