A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders.

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.

A novel mouse model of mitochondrial disease exhibits juvenile-onset severe neurological impairment due to parvalbumin cell mitochondrial dysfunction.

Mitochondrial diseases comprise a common group of neurometabolic disorders resulting from OXPHOS defects, that may manifest with neurological impairments, for which there are currently no disease-modifying therapies. Previous studies suggest inhibitory interneuron susceptibility to mitochondrial impairment, especially of parvalbumin-expressing interneurons (PV+). We have developed a mouse model of mitochondrial dysfunction specifically in PV+ cells via conditional Tfam knockout, that exhibited a juvenile-onset progressive phenotype characterised by cognitive deficits, anxiety-like behaviour, head-nodding, stargazing, ataxia, and reduced lifespan. A brain region-dependent decrease of OXPHOS complexes I and IV in PV+ neurons was detected, with Purkinje neurons being most affected. We validated these findings in a neuropathological study of patients with pathogenic mtDNA and POLG variants showing PV+ interneuron loss and deficiencies in complexes I and IV. This mouse model offers a drug screening platform to propel the discovery of therapeutics to treat severe neurological impairment due to mitochondrial dysfunction.

Uncoupling of energy production.

Mitochondrial genetic diseases are a very diverse group of conditions. A recent report by Mootha and colleagues in NEJM describes the underlying genetic defect and clinical findings in monozygotic twins with uncoupling of ATP production.

Dynamics of the most common pathogenic mtDNA variant m.3243A > G demonstrate frequency-dependency in blood and positive selection in the germline.

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A > G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A > G levels decrease in blood with age, and an age correction representing ~ 2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate that the dynamics of m.3243A > G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate 'on average', creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A > G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A > G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A > G. Germline selection of m.3243A > G shows an 'arching' profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

Endocrine Manifestations and New Developments in Mitochondrial Disease.

Mitochondrial diseases are a group of common inherited diseases causing disruption of oxidative phosphorylation. Some patients with mitochondrial disease have endocrine manifestations, with diabetes mellitus being predominant but also include hypogonadism, hypoadrenalism, and hypoparathyroidism. There have been major developments in mitochondrial disease over the past decade that have major implications for all patients. The collection of large cohorts of patients has better defined the phenotype of mitochondrial diseases and the majority of patients with endocrine abnormalities have involvement of several other systems. This means that patients with mitochondrial disease and endocrine manifestations need specialist follow-up because some of the other manifestations, such as stroke-like episodes and cardiomyopathy, are potentially life threatening. Also, the development and follow-up of large cohorts of patients means that there are clinical guidelines for the management of patients with mitochondrial disease. There is also considerable research activity to identify novel therapies for the treatment of mitochondrial disease. The revolution in genetics, with the introduction of next-generation sequencing, has made genetic testing more available and establishing a precise genetic diagnosis is important because it will affect the risk for involvement for different organ systems. Establishing a genetic diagnosis is also crucial because important reproductive options have been developed that will prevent the transmission of mitochondrial disease because of mitochondrial DNA variants to the next generation.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue.

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

Arrhythmia prevalence and sudden death risk in adults with the m.3243A>G mitochondrial disorder.

AIMS: To define the prevalence of non-sustained tachyarrhythmias and bradyarrhythmias in patients with the m.3243A>G mitochondrial genotype and a previously defined, profile, associated with 'high sudden-death risk'. METHODS AND RESULTS: Patients at high risk of sudden death because of combinations of ventricular hypertrophy, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes family phenotype, epilepsy or high mutation load, due to the m.3243A>G mutation, were identified from a mitochondrial cohort of 209 patients. All recruited had serial ECG and echo assessments previously according to schedule, had an ECG-loop recorder implanted and were followed for as long as the device allowed. Devices were programmed to detect non-sustained brady- or tachy-arrhythmias. This provided comprehensive rhythm surveillance and automatic downloads of all detections to a monitoring station for cardiology interpretation. Those with sinus tachycardia were treated with beta-blockers and those with ventricular hypertrophy received a beta-blocker and ACE-inhibitor combination.Nine consecutive patients, approached (37.2±3.9 years, seven males) and consented, were recruited. None died and no arrhythmias longer than 30s duration occurred during 3-year follow-up. Three patients reported palpitations but ECGs correlated with sinus rhythm. One manifest physiological, sinus pauses >3.5 s during sleep and another had one asymptomatic episode of non-sustained ventricular tachycardia. CONCLUSIONS: Despite 'high-risk' features for sudden death, those studied had negligible prevalence of arrhythmias over prolonged follow-up. By implication, the myocardium in this genotype is not primarily arrhythmogenic. Arrhythmias may not explain sudden death in patients without Wolff-Parkinson-White or abnormal atrioventricular conduction or, it must require a confluence of other, dynamic, proarrhythmic factors to trigger them.

Mitochondrial complex I subunit deficiency promotes pancreatic α-cell proliferation.

OBJECTIVE: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition. METHODS: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgAmut/mut) and control C57BL/6 mice at 12 and 44 weeks of age. RESULTS: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgAmut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with ß-cells in islets from 44 week PolgAmut/mut mice. Islet cell composition was normal in 12 week PolgAmut/mut mice, but the ß: α cell ratio was decreased in islets from 44 week PolgAmut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation. CONCLUSION: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.

A subcellular cookie cutter for spatial genomics in human tissue.

Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of  >20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

Detecting respiratory chain defects in osteoblasts from osteoarthritic patients using imaging mass cytometry.

Osteoporosis is a skeletal disease which is characterised by reduced bone mass and microarchitecture, with a subsequent loss of strength that predisposes to fragility and risk of fractures. The pathogenesis of falling bone mineral density, ultimately leading to a diagnosis of osteoporosis is incompletely understood but the disease is currently thought to be multifactorial. Humans are known to accumulate mitochondrial mutations and respiratory chain deficiency with age and mounting evidence suggests that this may indeed be the overarching cause intrinsic to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of about 30 years onwards. Mitochondria contain their own genome which encodes 13 essential mitochondrial proteins and accumulates somatic variants at up to 10 times the rate of the nuclear genome. Once the concentration of any pathogenic mitochondrial genome variant exceeds a threshold, respiratory chain deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model is a knock-in mutant that expresses a proof-reading-deficient version of PolgA, a nuclear encoded subunit of mtDNA polymerase. These mice are a useful model of age-related accumulation of mtDNA mutations in humans since their defective proof-reading mechanism leads to a mitochondrial DNA mutation rate 3-5 times higher than in wild-type mice. These mice showed enhanced levels of age-related osteoporosis along with respiratory chain deficiency in osteoblasts. To explore whether respiratory chain deficiency is also seen in human osteoblasts, we developed a protocol and analysis framework for imaging mass cytometry in bone tissue sections to analyse osteoblasts in situ. By comparing bone tissue sampled at one timepoint from femoral neck of 10 older healthy volunteers aged 40-85 with samples from young patients aged 1-19, we have identified complex I defect in osteoblasts from 6 out of 10 older volunteers, complex II defect in 2 out of 10 older volunteers, complex IV defect in 1 out of 10 older volunteers and complex V defect in 4 out of 10 older volunteers. These observations are consistent with findings from the PolgD257A/D257A mouse model and suggest that respiratory chain deficiency, as a consequence of the accumulation of age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

Natural History of Leigh Syndrome: A Study of Disease Burden and Progression.

OBJECTIVE: This observational cohort study aims to quantify disease burden over time, establish disease progression rates, and identify factors that may determine the disease course of Leigh syndrome. METHODS: Seventy-two Leigh syndrome children who completed the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) at baseline at 3.7 years (interquartile range [IQR] = 2.0-7.6) and follow-up assessments at 7.5 years (IQR = 3.7-11.0) in clinics were enrolled. Eighty-two percent of this cohort had a confirmed genetic diagnosis, with pathogenic variants in the MT-ATP6 and SURF1 genes being the most common cause. The total NPMDS scores denoted mild (0-14), moderate (15-25), and severe (>25) disease burden. Detailed clinical, neuroradiological, and molecular genetic findings were also analyzed. RESULTS: The median total NPMDS scores rose significantly (Z = -6.9, p < 0.001), and the percentage of children with severe disease burden doubled (22% → 42%) over 2.6 years of follow-up. Poor function (especially mobility, self-care, communication, feeding, and education) and extrapyramidal features contributed significantly to the disease burden (τb  ≈ 0.45-0.68, p < 0.001). These children also deteriorated to wheelchair dependence (31% → 57%), exclusive enteral feeding (22% → 46%), and one-to-one assistance for self-care (25% → 43%) during the study period. Twelve children (17%) died after their last NPMDS scores were recorded. These children had higher follow-up NPMDS scores (disease burden; p < 0.001) and steeper increase in NPMDS score per annum (disease progression; p < 0.001). Other predictors of poor outcomes include SURF1 gene variants (p < 0.001) and bilateral caudate changes on neuroimaging (p < 0.01). INTERPRETATION: This study has objectively defined the disease burden and progression of Leigh syndrome. Our analysis has also uncovered potential influences on the trajectory of this neurodegenerative condition. ANN NEUROL 2022;91:117-130.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy.

CONTEXT: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). OBJECTIVE: This work sought a role for mitochondria in OAT adipogenesis in GO. METHODS: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. RESULTS: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. CONCLUSION: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

Mitochondrial DNA disorders: from pathogenic variants to preventing transmission.

Mitochondrial DNA (mtDNA) disorders are recognized as one of the most common causes of inherited metabolic disorders. The mitochondrial genome occurs in multiple copies resulting in both homoplasmic and heteroplasmic pathogenic mtDNA variants. A biochemical defect arises when the pathogenic variant level reaches a threshold, which differs between variants. Moreover, variants can segregate, clonally expand, or be lost from cellular populations resulting in a dynamic and tissue-specific mosaic pattern of oxidative deficiency. MtDNA is maternally inherited but transmission patterns of heteroplasmic pathogenic variants are complex. During oogenesis, a mitochondrial bottleneck results in offspring with widely differing variant levels to their mother, whilst highly deleterious variants, such as deletions, are not transmitted. Complemented by a complex interplay between mitochondrial and nuclear genomes, these peculiar genetics produce marked phenotypic variation, posing challenges to the diagnosis and clinical management of patients. Novel therapeutic compounds and several genetic therapies are currently under investigation, but proven disease-modifying therapies remain elusive. Women who carry pathogenic mtDNA variants require bespoke genetic counselling to determine their reproductive options. Recent advances in in vitro fertilization techniques, have greatly improved reproductive choices, but are not without their challenges. Since the first pathogenic mtDNA variants were identified over 30 years ago, there has been remarkable progress in our understanding of these diseases. However, many questions remain unanswered and future studies are required to investigate the mechanisms of disease progression and to identify new disease-specific therapeutic targets.

Mitochondrial disease in adults: recent advances and future promise.

Mitochondrial diseases are some of the most common inherited neurometabolic disorders, and major progress has been made in our understanding, diagnosis, and treatment of these conditions in the past 5 years. Development of national mitochondrial disease cohorts and international collaborations has changed our knowledge of the spectrum of clinical phenotypes and natural history of mitochondrial diseases. Advances in high-throughput sequencing technologies have altered the diagnostic algorithm for mitochondrial diseases by increasingly using a genetics-first approach, with more than 350 disease-causing genes identified to date. While the current management strategy for mitochondrial disease focuses on surveillance for multisystem involvement and effective symptomatic treatment, new endeavours are underway to find better treatments, including repurposing current drugs, use of novel small molecules, and gene therapies. Developments made in reproductive technology offer women the opportunity to prevent transmission of DNA-related mitochondrial disease to their children.

Imaging mass cytometry reveals generalised deficiency in OXPHOS complexes in Parkinson's disease.

Here we report the application of a mass spectrometry-based technology, imaging mass cytometry, to perform in-depth proteomic profiling of mitochondrial complexes in single neurons, using metal-conjugated antibodies to label post-mortem human midbrain sections. Mitochondrial dysfunction, particularly deficiency in complex I has previously been associated with the degeneration of dopaminergic neurons in Parkinson's disease. To further our understanding of the nature of this dysfunction, and to identify Parkinson's disease specific changes, we validated a panel of antibodies targeting subunits of all five mitochondrial oxidative phosphorylation complexes in dopaminergic neurons from Parkinson's disease, mitochondrial disease, and control cases. Detailed analysis of the expression profile of these proteins, highlighted heterogeneity between individuals. There is a widespread decrease in expression of all complexes in Parkinson's neurons, although more severe in mitochondrial disease neurons, however, the combination of affected complexes varies between the two groups. We also provide evidence of a potential neuronal response to mitochondrial dysfunction through a compensatory increase in mitochondrial mass. This study highlights the use of imaging mass cytometry in the assessment and analysis of expression of oxidative phosphorylation proteins, revealing the complexity of deficiencies of these proteins within individual neurons which may contribute to and drive neurodegeneration in Parkinson's disease.

Neuromuscular Junction Abnormalities in Mitochondrial Disease: An Observational Cohort Study.

OBJECTIVE: To determine the prevalence of neuromuscular junction (NMJ) abnormalities in patients with mitochondrial disease. METHODS: Eighty patients with genetically proven mitochondrial disease were recruited from a national center for mitochondrial disease in the United Kingdom. Participants underwent detailed clinical and neurophysiologic testing including single-fiber electromyography. RESULTS: The overall prevalence of neuromuscular transmission defects was 25.6%. The highest prevalence was in patients with pathogenic dominant RRM2B variants (50%), but abnormalities were found in a wide range of mitochondrial genotypes. The presence of NMJ abnormalities was strongly associated with coexistent myopathy, but not with neuropathy. Furthermore, 15% of patients with NMJ abnormality had no evidence of either myopathy or neuropathy. CONCLUSIONS: NMJ transmission defects are common in mitochondrial disease. In some patients, NMJ dysfunction occurs in the absence of obvious pre- or post-synaptic pathology, suggesting that the NMJ may be specifically affected.

Age-associated mitochondrial complex I deficiency is linked to increased stem cell proliferation rates in the mouse colon.

One of the hallmarks of aging is an accumulation of cells with defects in oxidative phosphorylation (OXPHOS) due to mutations of mitochondrial DNA (mtDNA). Rapidly dividing tissues maintained by stem cells, such as the colonic epithelium, are particularly susceptible to accumulation of OXPHOS defects over time; however, the effects on the stem cells are unknown. We have crossed a mouse model in which intestinal stem cells are labelled with EGFP (Lgr5-EGFP-IRES-creERT2) with a model of accelerated mtDNA mutagenesis (PolgAmut/mut ) to investigate the effect of OXPHOS dysfunction on colonic stem cell proliferation. We show that a reduction in complex I protein levels is associated with an increased rate of stem cell cycle re-entry. These changes in stem cell homeostasis could have significant implications for age-associated intestinal pathogenesis.

Exercise Training and Neurodegeneration in Mitochondrial Disorders: Insights From the Harlequin Mouse.

AIM: Cerebellar neurodegeneration is a main phenotypic manifestation of mitochondrial disorders caused by apoptosis-inducing factor (AIF) deficiency. We assessed the effects of an exercise training intervention at the cerebellum and brain level in a mouse model (Harlequin, Hq) of AIF deficiency. METHODS: Male wild-type (WT) and Hq mice were assigned to an exercise (Ex) or control (sedentary [Sed]) group (n = 10-12/group). The intervention (aerobic and resistance exercises) was initiated upon the first symptoms of ataxia in Hq mice (∼3 months on average) and lasted 8 weeks. Histological and biochemical analyses of the cerebellum were performed at the end of the training program to assess indicators of mitochondrial deficiency, neuronal death, oxidative stress and neuroinflammation. In brain homogenates analysis of enzyme activities and levels of the oxidative phosphorylation system, oxidative stress and neuroinflammation were performed. RESULTS: The mean age of the mice at the end of the intervention period did not differ between groups: 5.2 ± 0.2 (WT-Sed), 5.2 ± 0.1 (WT-Ex), 5.3 ± 0.1 (Hq-Sed), and 5.3 ± 0.1 months (Hq-Ex) (p = 0.489). A significant group effect was found for most variables indicating cerebellar dysfunction in Hq mice compared with WT mice irrespective of training status. However, exercise intervention did not counteract the negative effects of the disease at the cerebellum level (i.e., no differences for Hq-Ex vs. Hq-Sed). On the contrary, in brain, the activity of complex V was higher in both Hq mice groups in comparison with WT animals (p < 0.001), and post hoc analysis also revealed differences between sedentary and trained Hq mice. CONCLUSION: A combined training program initiated when neurological symptoms and neuron death are already apparent is unlikely to promote neuroprotection in the cerebellum of Hq model of mitochondrial disorders, but it induces higher complex V activity in the brain.