Mitochondrial Abnormality Associates with Type-Specific Neuronal Loss and Cell Morphology Changes in the Pedunculopontine Nucleus in Parkinson Disease

Cholinergic neuronal loss in the pedunculopontine nucleus (PPN) associates with abnormal functions, including certain motor and nonmotor symptoms. This realization has led to low-frequency stimulation of the PPN for treating patients with Parkinson disease (PD) who are refractory to other treatment modalities. However, the molecular mechanisms underlying PPN neuronal loss and the therapeutic substrate for the clinical benefits following PPN stimulation remain poorly characterized, hampering progress toward designing more efficient therapies aimed at restoring the PPN's normal functions during progressive parkinsonism. Here, we investigated postmortem pathological changes in the PPN of PD cases. Our study detected a loss of neurons producing gamma-aminobutyric acid (GABA) as their output and glycinergic neurons, along with the pronounced loss of cholinergic neurons. These losses were accompanied by altered somatic cell size that affected the remaining neurons of all neuronal subtypes studied here. Because studies showed that mitochondrial dysfunction exists in sporadic PD and in PD animal models, we investigated whether altered mitochondrial composition exists in the PPN. A significant up-regulation of several mitochondrial proteins was seen in GABAergic and glycinergic neurons; however, cholinergic neurons indicated down-regulation of the same proteins. Our findings suggest an imbalance in the activity of key neuronal subgroups of the PPN in PD, potentially because of abnormal inhibitory activity and altered cholinergic outflow

Distal weakness with respiratory insufficiency caused by the m.8344A>G “MERRF” mutation

AbstractThe m.8344A>G mutation in the mt-tRNALys gene, first described in myoclonic epilepsy and ragged red fibers (MERRF), accounts for approximately 80% of mutations in individuals with MERRF syndrome. Although originally described in families with a classical syndrome of myoclonus, ataxia, epilepsy and ragged red fibers in muscle biopsy, the m.8344A>G mutation is increasingly recognised to exhibit marked phenotypic heterogeneity. This paper describes the clinical, morphological and laboratory features of an unusual phenotype in a patient harboring the m.8344A>G ‘MERRF’ mutation. We present the case of a middle-aged woman with distal weakness since childhood who also had ptosis and facial weakness and who developed mid-life respiratory insufficiency necessitating non-invasive nocturnal ventilator support. Neurophysiological and acetylcholine receptor antibody analyses excluded myasthenia gravis whilst molecular genetic testing excluded myotonic dystrophy, prompting a diagnostic needle muscle biopsy. Mitochondrial histochemical abnormalities including subsarcolemmal mitochondrial accumulation (ragged-red fibers) and in excess of 90% COX-deficient fibers, was seen leading to sequencing of the mitochondrial genome in muscle. This identified the m.8344A>G mutation commonly associated with the MERRF phenotype. This case extends the evolving phenotypic spectrum of the m.8344A>G mutation and emphasizes that it may cause indolent distal weakness with respiratory insufficiency, with marked histochemical defects in muscle. Our findings support consideration of screening of this gene in cases of indolent myopathy resembling distal limb-girdle muscular dystrophy in which screening of the common genes prove negative

Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study

Epilepsy in adults with mitochondrial disease: A cohort study.

OBJECTIVE: The aim of this work was to determine the prevalence and progression of epilepsy in adult patients with mitochondrial disease. METHODS: We prospectively recruited a cohort of 182 consecutive adult patients attending a specialized mitochondrial disease clinic in Newcastle upon Tyne between January 1, 2005 and January 1, 2008. We then followed this cohort over a 7-year period, recording primary outcome measures of occurrence of first seizure, status epilepticus, stroke-like episode, and death. RESULTS: Overall prevalence of epilepsy in the cohort was 23.1%. Mean age of epilepsy onset was 29.4 years. Prevalence varied widely between genotypes, with several genotypes having no cases of epilepsy, a prevalence of 34.9% in the most common genotype (m.3243A>G mutation), and 92.3% in the m.8344A>G mutation. Among the cohort as a whole, focal seizures, with or without progression to bilateral convulsive seizures, was the most common seizure type. Conversely, all of the patients with the m.8344A>G mutation and epilepsy experienced myoclonic seizures. Patients with the m.3243A>G mutation remain at high risk of developing stroke-like episodes (1.16% per year). However, although the standardized mortality ratio for the entire cohort was high (2.86), this ratio did not differ significantly between patients with epilepsy (2.96) and those without (2.83). INTERPRETATION: Epilepsy is a common manifestation of mitochondrial disease. It develops early in the disease and, in the case of the m.3243A>G mutation, often presents in the context of a stroke-like episode or status epilepticus. However, epilepsy does not itself appear to contribute to the increased mortality in mitochondrial disease

Neuromelanin, neurotransmitter status and brainstem location determine the differential vulnerability of catecholaminergic neurons to mitochondrial DNA deletions

<p>Abstract</p> <p>Background</p> <p>Deletions of the mitochondrial DNA (mtDNA) accumulate to high levels in dopaminergic neurons of the substantia nigra pars compacta (SNc) in normal aging and in patients with Parkinson's disease (PD). Human nigral neurons characteristically contain the pigment neuromelanin (NM), which is believed to alter the cellular redox-status. The impact of neuronal pigmentation, neurotransmitter status and brainstem location on the susceptibility to mtDNA damage remains unclear. We quantified mtDNA deletions (ΔmtDNA) in single pigmented and non-pigmented catecholaminergic, as well as non-catecholaminergic neurons of the human SNc, the ventral tegmental area (VTA) and the locus coeruleus (LC), using laser capture microdissection and single-cell real-time PCR.</p> <p>Results</p> <p>In healthy aged individuals, ΔmtDNA levels were highest in pigmented catecholaminergic neurons (25.2 ± 14.9%), followed by non-pigmented catecholamergic (18.0 ± 11.2%) and non-catecholaminergic neurons (12.3 ± 12.3%; p < 0.001). Within the catecholaminergic population, ΔmtDNA levels were highest in dopaminergic neurons of the SNc (33.9 ± 21.6%) followed by dopaminergic neurons of the VTA (21.9 ± 12.3%) and noradrenergic neurons of the LC (11.1 ± 11.4%; p < 0.001). In PD patients, there was a trend to an elevated mutation load in surviving non-pigmented nigral neurons (27.13 ± 16.73) compared to age-matched controls (19.15 ± 11.06; p = 0.052), but levels where similar in pigmented nigral neurons of PD patients (41.62 ± 19.61) and controls (41.80 ± 22.62).</p> <p>Conclusions</p> <p>Catecholaminergic brainstem neurons are differentially susceptible to mtDNA damage. Pigmented dopaminergic neurons of the SNc show the highest ΔmtDNA levels, possibly explaining the exceptional vulnerability of the nigro-striatal system in PD and aging. Although loss of pigmented noradrenergic LC neurons also is an early feature of PD pathology, mtDNA levels are not elevated in this nucleus in healthy controls. Thus, ΔmtDNA are neither an inevitable consequence of catecholamine metabolism nor a universal explanation for the regional vulnerability seen in PD.</p

Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal

Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.

OBJECTIVE: The prevalence of mitochondrial disease has proven difficult to establish, predominantly as a result of clinical and genetic heterogeneity. The phenotypic spectrum of mitochondrial disease has expanded significantly since the original reports that associated classic clinical syndromes with mitochondrial DNA (mtDNA) rearrangements and point mutations. The revolution in genetic technologies has allowed interrogation of the nuclear genome in a manner that has dramatically improved the diagnosis of mitochondrial disorders. We comprehensively assessed the prevalence of all forms of adult mitochondrial disease to include pathogenic mutations in both nuclear and mtDNA. METHODS: Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center from 1990 to 2014. For the midyear period of 2011, we evaluated the minimum prevalence of symptomatic nuclear DNA mutations and symptomatic and asymptomatic mtDNA mutations causing mitochondrial diseases. RESULTS: The minimum prevalence rate for mtDNA mutations was 1 in 5,000 (20 per 100,000), comparable with our previously published prevalence rates. In this population, nuclear mutations were responsible for clinically overt adult mitochondrial disease in 2.9 per 100,000 adults. INTERPRETATION: Combined, our data confirm that the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (≈1 in 4,300), is among the commonest adult forms of inherited neurological disorders. These figures hold important implications for the evaluation of interventions, provision of evidence-based health policies, and planning of future services

Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease

Mitochondrial disease can be attributed to both mitochondrial and nuclear gene mutations. It has a heterogeneous clinical and biochemical profile, which is compounded by diversity of the genetic background. Disease-based epidemiological information has expanded significantly in recent decades, but little information is known that clarifies the aetiology in African patients. The aim of this study was to investigate mitochondrial DNA variation and pathogenic mutations in muscle of diagnosed paediatric patients from South Africa. A cohort of 71 South African paediatric patients was included and a high-throughput nucleotide sequencing approach was used to sequence full-length muscle mtDNA. The average coverage of the mtDNA genome was 81 ± 26 per position. After assigning haplogroups, it was determined that although the nature of non-haplogroup defining variants was similar in African and non-African haplogroup patients, the number of substitutions were significantly higher in African patients. We describe previously reported disease-associated and novel variants in this cohort. We observed a general lack of commonly reported syndrome-associated mutations, which supports clinical observations and confirms general observations in African patients when using single mutation screening strategies based on (predominantly non-African) mtDNA disease-based information. It is finally concluded that this first extensive report on muscle mtDNA sequences in African paediatric patients highlights the need for a full length mtDNA sequencing strategy, which applies to all populations where specific mutations is not present. This, in addition to nuclear DNA gene mutation and pathogenicity evaluations, will be required to better unravel the aetiology of these disorders in African patients.http://www.nature.com/ejhg/index.htm