RESUMO
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.
Assuntos
Síndrome MELAS , Doenças Mitocondriais , Acidente Vascular Cerebral , Adulto , DNA Mitocondrial/genética , Humanos , Síndrome MELAS/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Mutação , Estudos Retrospectivos , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/genéticaRESUMO
AIMS: Alpers' syndrome is a severe neurodegenerative disease typically caused by bi-allelic variants in the mitochondrial DNA (mtDNA) polymerase gene, POLG, leading to mtDNA depletion. Intractable epilepsy, often with an occipital focus, and extensive neurodegeneration are prominent features of Alpers' syndrome. Mitochondrial oxidative phosphorylation (OXPHOS) is severely impaired with mtDNA depletion and is likely to be a major contributor to the epilepsy and neurodegeneration in Alpers' syndrome. We hypothesised that parvalbumin-positive(+) interneurons, a neuronal class critical for inhibitory regulation of physiological cortical rhythms, would be particularly vulnerable in Alpers' syndrome due to the excessive energy demands necessary to sustain their fast-spiking activity. METHODS: We performed a quantitative neuropathological investigation of inhibitory interneuron subtypes (parvalbumin+, calretinin+, calbindin+, somatostatin interneurons+) in postmortem neocortex from 14 Alpers' syndrome patients, five sudden unexpected death in epilepsy (SUDEP) patients (to control for effects of epilepsy) and nine controls. RESULTS: We identified a severe loss of parvalbumin+ interneurons and clear evidence of OXPHOS impairment in those that remained. Comparison of regional abundance of interneuron subtypes in control tissues demonstrated enrichment of parvalbumin+ interneurons in the occipital cortex, while other subtypes did not exhibit such topographic specificity. CONCLUSIONS: These findings suggest that the vulnerability of parvalbumin+ interneurons to OXPHOS deficits coupled with the high abundance of parvalbumin+ interneurons in the occipital cortex is a key factor in the aetiology of the occipital-predominant epilepsy that characterises Alpers' syndrome. These findings provide novel insights into Alpers' syndrome neuropathology, with important implications for the development of preclinical models and disease-modifying therapeutics.
Assuntos
Esclerose Cerebral Difusa de Schilder , Epilepsia , Doenças Neurodegenerativas , DNA Mitocondrial/genética , Esclerose Cerebral Difusa de Schilder/complicações , Epilepsia/patologia , Humanos , Interneurônios/patologia , Doenças Neurodegenerativas/complicações , Parvalbuminas/genéticaRESUMO
Approximately one-quarter of patients with mitochondrial disease experience epilepsy. Their epilepsy is often severe and resistant towards conventional antiepileptic drugs. Despite the severity of this epilepsy, there are currently no animal models available to provide a mechanistic understanding of mitochondrial epilepsy. We conducted neuropathological studies on patients with mitochondrial epilepsy and found the involvement of the astrocytic compartment. As a proof of concept, we developed a novel brain slice model of mitochondrial epilepsy by the application of an astrocytic-specific aconitase inhibitor, fluorocitrate, concomitant with mitochondrial respiratory inhibitors, rotenone and potassium cyanide. The model was robust and exhibited both face and predictive validity. We then used the model to assess the role that astrocytes play in seizure generation and demonstrated the involvement of the GABA-glutamate-glutamine cycle. Notably, glutamine appears to be an important intermediary molecule between the neuronal and astrocytic compartment in the regulation of GABAergic inhibitory tone. Finally, we found that a deficiency in glutamine synthetase is an important pathogenic process for seizure generation in both the brain slice model and the human neuropathological study. Our study describes the first model for mitochondrial epilepsy and provides a mechanistic insight into how astrocytes drive seizure generation in mitochondrial epilepsy.
Assuntos
Astrócitos/patologia , Astrócitos/fisiologia , Epilepsia do Lobo Temporal/patologia , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Convulsões/patologia , Adulto , Idoso , Animais , Epilepsia do Lobo Temporal/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Convulsões/metabolismo , Adulto JovemRESUMO
Mitochondria are double-membrane-bound organelles that are present in all nucleated eukaryotic cells and are responsible for the production of cellular energy in the form of ATP. Mitochondrial function is under dual genetic control - the 16.6-kb mitochondrial genome, with only 37 genes, and the nuclear genome, which encodes the remaining â¼1300 proteins of the mitoproteome. Mitochondrial dysfunction can arise because of defects in either mitochondrial DNA or nuclear mitochondrial genes, and can present in childhood or adulthood in association with vast clinical heterogeneity, with symptoms affecting a single organ or tissue, or multisystem involvement. There is no cure for mitochondrial disease for the vast majority of mitochondrial disease patients, and a genetic diagnosis is therefore crucial for genetic counselling and recurrence risk calculation, and can impact on the clinical management of affected patients. Next-generation sequencing strategies are proving pivotal in the discovery of new disease genes and the diagnosis of clinically affected patients; mutations in >250 genes have now been shown to cause mitochondrial disease, and the biochemical, histochemical, immunocytochemical and neuropathological characterization of these patients has led to improved diagnostic testing strategies and novel diagnostic techniques. This review focuses on the current genetic landscape associated with mitochondrial disease, before focusing on advances in studying associated mitochondrial pathology in two, clinically relevant organs - skeletal muscle and brain. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Encéfalo/patologia , DNA Mitocondrial/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Músculo Esquelético/patologia , Humanos , Mitocôndrias/metabolismo , Mutação/genéticaRESUMO
AIMS: To provide insight into the mechanism of sudden adult death syndrome (SADS) and to give new clinical guidelines for the cardiac management of patients with the most common mitochondrial DNA mutation, m.3243A>G. These studies were initiated after two young, asymptomatic adults harbouring the m.3243A>G mutation died suddenly and unexpectedly. The m.3243A>G mutation is present in â¼1 in 400 of the population, although the recognized incidence of mitochondrial DNA (mtDNA) disease is â¼1 in 5000. METHODS AND RESULTS: Pathological studies including histochemistry and molecular genetic analyses performed on various post-mortem samples including cardiac tissues (atrium and ventricles) showed marked respiratory chain deficiency and high levels of the m.3243A>G mutation. Systematic review of cause of death in our m.3243A>G patient cohort showed the person-time incidence rate of sudden adult death is 2.4 per 1000 person-years. A further six cases of sudden death among extended family members have been identified from interrogation of family pedigrees. CONCLUSION: Our findings suggest that SADS is an important cause of death in patients with m.3243A>G and likely to be due to widespread respiratory chain deficiency in cardiac muscle. The involvement of asymptomatic relatives highlights the importance of family tracing in patients with m.3243A>G and the need for specific cardiac arrhythmia surveillance in the management of this common genetic disease. In addition, these findings have prompted the derivation of cardiac guidelines specific to patients with m.3243A>G-related mitochondrial disease. Finally, due to the prevalence of this mtDNA point mutation, we recommend inclusion of testing for m.3243A>G mutations in the genetic autopsy of all unexplained cases of SADS.
Assuntos
Morte Súbita , Adulto , DNA Mitocondrial , Humanos , Mitocôndrias , Doenças Mitocondriais , MutaçãoRESUMO
AIMS: Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. METHODS: We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age-matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. RESULTS: Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. CONCLUSIONS: This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia.
Assuntos
Ataxia Cerebelar/etiologia , Complexo I de Transporte de Elétrons/deficiência , Doenças Mitocondriais/complicações , Células de Purkinje/enzimologia , Sinapses/enzimologia , Adulto , Ataxia Cerebelar/enzimologia , Ataxia Cerebelar/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Células de Purkinje/patologia , Sinapses/patologia , Adulto JovemRESUMO
AIMS: Mitochondrial disorders are among the most frequently inherited cause of neurological disease and arise due to mutations in mitochondrial or nuclear DNA. Currently, we do not understand the specific involvement of certain brain regions or selective neuronal vulnerability in mitochondrial disease. Recent studies suggest γ-aminobutyric acid (GABA)-ergic interneurones are particularly susceptible to respiratory chain dysfunction. In this neuropathological study, we assess the impact of mitochondrial DNA defects on inhibitory interneurones in patients with mitochondrial disease. METHODS: Histochemical, immunohistochemical and immunofluorescent assays were performed on post-mortem brain tissue from 10 patients and 10 age-matched control individuals. We applied a quantitative immunofluorescent method to interrogate complex I and IV protein expression in mitochondria within GABAergic interneurone populations in the frontal, temporal and occipital cortices. We also evaluated the density of inhibitory interneurones in serial sections to determine if cell loss was occurring. RESULTS: We observed significant, global reductions in complex I expression within GABAergic interneurones in frontal, temporal and occipital cortices in the majority of patients. While complex IV expression is more variable, there is reduced expression in patients harbouring m.8344A>G point mutations and POLG mutations. In addition to the severe respiratory chain deficiencies observed in remaining interneurones, quantification of GABAergic cell density showed a dramatic reduction in cell density suggesting interneurone loss. CONCLUSIONS: We propose that the combined loss of interneurones and severe respiratory deficiency in remaining interneurones contributes to impaired neuronal network oscillations and could underlie development of neurological deficits, such as cognitive impairment and epilepsy, in mitochondrial disease.
Assuntos
Encéfalo/fisiopatologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Neurônios GABAérgicos/patologia , Interneurônios/patologia , Doenças Mitocondriais/fisiopatologia , Adulto , Idoso , Autopsia , Feminino , Imunofluorescência , Neurônios GABAérgicos/metabolismo , Humanos , Imuno-Histoquímica , Interneurônios/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
DNA Mitocondrial/genética , Epilepsia/etiologia , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Acidente Vascular Cerebral/etiologia , Adolescente , Adulto , Idade de Início , Progressão da Doença , Epilepsia/epidemiologia , Epilepsia/mortalidade , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/epidemiologia , Doenças Mitocondriais/mortalidade , Mutação , Prevalência , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/mortalidade , Adulto JovemRESUMO
Neuropathological findings in mitochondrial DNA disease vary and are often dependent on the type of mitochondrial DNA defect. Many reports document neuronal cell loss, demyelination, gliosis and necrotic lesions in post-mortem material. However, previous studies highlight vascular abnormalities in patients harbouring mitochondrial DNA defects, particularly in those with the m.3243A>G mutation in whom stroke-like events are part of the mitochondrial encephalopathy lactic acidosis and stroke-like episodes syndrome. We investigated microangiopathic changes in the cerebellum of 16 genetically and clinically well-defined patients. Respiratory chain deficiency, high levels of mutated mitochondrial DNA and increased mitochondrial mass were present within the smooth muscle cells and endothelial cells comprising the vessel wall in patients. These changes were not limited to those harbouring the m.3243A>G mutation frequently associated with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes, but were documented in patients harbouring m.8344A>G and autosomal recessive polymerase (DNA directed), gamma (POLG) mutations. In 8 of the 16 patients, multiple ischaemic-like lesions occurred in the cerebellar cortex suggestive of vascular smooth muscle cell dysfunction. Indeed, changes in vascular smooth muscle and endothelium distribution and cell size are indicative of vascular cell loss. We found evidence of blood-brain barrier breakdown characterized by plasma protein extravasation following fibrinogen and IgG immunohistochemistry. Reduced immunofluorescence was also observed using markers for endothelial tight junctions providing further evidence in support of blood-brain barrier breakdown. Understanding the structural and functional changes occurring in central nervous system microvessels in patients harbouring mitochondrial DNA defects will provide an important insight into mechanisms of neurodegeneration in mitochondrial DNA disease. Since therapeutic strategies targeting the central nervous system are limited, modulating vascular function presents an exciting opportunity to lessen the burden of disease in these patients.
Assuntos
Cerebelo/patologia , Transtornos Cerebrovasculares/complicações , Microvasos/patologia , Doenças Mitocondriais/complicações , Actinas/metabolismo , Adulto , Estudos de Casos e Controles , Cerebelo/metabolismo , Colágeno Tipo IV/metabolismo , Análise Mutacional de DNA , DNA Polimerase gama , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Transportador de Glucose Tipo 1/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , NADH Desidrogenase/genética , Mutação Puntual/genética , Porinas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Adulto JovemRESUMO
Defects in the mitochondrial DNA replication enzyme, polymerase γ, are an important cause of mitochondrial disease with â¼25% of all adult diagnoses attributed to mutations in the POLG gene. Peripheral neuronopathy is often part of the clinical syndrome and can represent the most disabling feature. In spite of this, the molecular mechanisms underlying the neuronopathy remain to be elucidated and treatment strategies are limited. In the present study, we use a combined approach comprising clinical, electrophysiological, neuropathological and molecular genetic investigations to unravel the mechanisms underpinning peripheral neuronopathy in autosomal recessive polymerase γ-related disease. Electrophysiological assessments documented a dorsal root ganglionopathy in all 11 cases. Of the 11 cases, eight also showed changes consistent with motor fibre loss. Detailed neuropathological investigation of two patients confirmed the electrophysiological findings, revealing atrophy of posterior columns and striking neuronal cell loss from the dorsal root ganglia, which was accompanied by severe mitochondrial biochemical abnormalities involving respiratory chain complexes I and IV due to clonally-expanded mitochondrial DNA deletions and a significant reduction in mitochondrial DNA copy number in affected neurons. We propose that the respiratory chain defects, secondary to mitochondrial DNA deletion and depletion, are likely to be responsible for pathology observed in the dorsal root ganglion and the sensory ganglionopathy documented electrophysiologically.
Assuntos
DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Gânglios Espinais/fisiopatologia , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Adolescente , Adulto , DNA Polimerase gama , Eletrodiagnóstico , Feminino , Gânglios Espinais/patologia , Neuropatias Hereditárias Sensoriais e Autônomas/diagnóstico , Neuropatias Hereditárias Sensoriais e Autônomas/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Condução Nervosa/fisiologia , FenótipoRESUMO
Refractory epilepsy is the main neurological manifestation of Alpers' syndrome, a severe childhood-onset mitochondrial disease caused by bi-allelic pathogenic variants in the mitochondrial DNA (mtDNA) polymerase gamma gene (POLG). The pathophysiological mechanisms underpinning neuronal hyperexcitabilty leading to seizures in Alpers' syndrome remain unknown. However, pathological changes to reactive astrocytes are hypothesised to exacerbate neural dysfunction and seizure-associated cortical activity in POLG-related disease. Therefore, we sought to phenotypically characterise astrocytic pathology in Alpers' syndrome. We performed a detailed quantitative investigation of reactive astrocytes in post-mortem neocortical tissues from thirteen patients with Alpers' syndrome, eight neurologically normal controls and five sudden unexpected death in epilepsy (SUDEP) patients, to control for generalised epilepsy-associated astrocytic pathology. Immunohistochemistry to identify glial fibrillary acidic protein (GFAP)-reactive astrocytes revealed striking reactive astrogliosis localised to the primary visual cortex of Alpers' syndrome tissues, characterised by abnormal-appearing hypertrophic astrocytes. Phenotypic characterisation of individual GFAP-reactive astrocytes demonstrated decreased abundance of mitochondrial oxidative phosphorylation (OXPHOS) proteins and altered expression of key astrocytic proteins including Kir4.1 (subunit of the inwardly rectifying K+ ion channel), AQP4 (astrocytic water channel) and glutamine synthetase (enzyme that metabolises glutamate). These phenotypic astrocytic changes were typically different from the pathology observed in SUDEP tissues, suggesting alternative mechanisms of astrocytic dysfunction between these epilepsies. Crucially, our findings provide further evidence of occipital lobe involvement in Alpers' syndrome and support the involvement of reactive astrocytes in the pathogenesis of POLG-related disease.
Assuntos
Esclerose Cerebral Difusa de Schilder , Epilepsia , Morte Súbita Inesperada na Epilepsia , Humanos , Criança , Astrócitos/metabolismo , Esclerose Cerebral Difusa de Schilder/genética , Esclerose Cerebral Difusa de Schilder/metabolismo , Convulsões/genética , DNA Mitocondrial/genética , Epilepsia/metabolismo , Proteína Glial Fibrilar Ácida/metabolismoRESUMO
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.
Assuntos
Doenças Mitocondriais , Parvalbuminas , Camundongos , Animais , Humanos , Parvalbuminas/metabolismo , Neurônios/metabolismo , Interneurônios/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , MitocôndriasRESUMO
Mitochondrial DNA mutations are an important cause of neurological disease. The clinical presentation is very varied in terms of age of onset and different neurological signs and symptoms. The clinical course varies considerably but in many patients there is a progressive decline, and in some evidence of marked neurodegeneration. Our understanding of the mechanisms involved is limited due in part to limited availability of animal models of disease. However, studies on human post-mortem brains, combined with clinical and radiological studies, are giving important insights into specific neuronal involvement.
Assuntos
Encéfalo/patologia , Genoma Mitocondrial , Mitocôndrias/química , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Mutação , Degeneração Neural/patologia , Animais , Encéfalo/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/metabolismo , Degeneração Neural/metabolismoRESUMO
Alpers' syndrome is an early-onset neurodegenerative disorder often caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG) which is essential for mitochondrial DNA (mtDNA) replication. Alpers' syndrome is characterized by intractable epilepsy, developmental regression and liver failure which typically affects children aged 6 months-3 years. Although later onset variants are now recognized, they differ in that they are primarily an epileptic encephalopathy with ataxia. The disorder is progressive, without cure and inevitably leads to death from drug-resistant status epilepticus, often with concomitant liver failure. Since our understanding of the mechanisms contributing the neurological features in Alpers' syndrome is rudimentary, we performed a detailed and quantitative neuropathological study on 13 patients with clinically and histologically-defined Alpers' syndrome with ages ranging from 2 months to 18 years. Quantitative immunofluorescence showed severe respiratory chain deficiencies involving mitochondrial respiratory chain subunits of complex I and, to a lesser extent, complex IV in inhibitory interneurons and pyramidal neurons in the occipital cortex and in Purkinje cells of the cerebellum. Diminished densities of these neuronal populations were also observed. This study represents the largest cohort of post-mortem brains from patients with clinically defined Alpers' syndrome where we provide quantitative evidence of extensive complex I defects affecting interneurons and Purkinje cells for the first time. We believe interneuron and Purkinje cell pathology underpins the clinical development of seizures and ataxia seen in Alpers' syndrome. This study also further highlights the extensive involvement of GABAergic neurons in mitochondrial disease.
Assuntos
Esclerose Cerebral Difusa de Schilder/genética , Esclerose Cerebral Difusa de Schilder/patologia , Adolescente , Ataxia/genética , Encéfalo/patologia , Criança , Pré-Escolar , DNA Polimerase gama/genética , DNA Polimerase gama/fisiologia , DNA Mitocondrial/genética , Eletroencefalografia , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Doenças Mitocondriais , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Neuropatologia , Convulsões/genéticaRESUMO
OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Encefalomiopatias Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Proteínas Nucleares/genética , Fosforilação Oxidativa , Sequência de Aminoácidos , Animais , Sequência de Bases , Pré-Escolar , DNA Mitocondrial/genética , Drosophila , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Evolução Fatal , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lactente , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Neuroimagem , Proteínas Nucleares/química , LinhagemRESUMO
Mutations in the m.13094T>C MT-ND5 gene have been previously described in three cases of Leigh Syndrome (LS). In this retrospective, international cohort study we identified 20 clinically affected individuals (13 families) and four asymptomatic carriers. Ten patients were deceased at the time of analysis (median age of death was 10years (range: 5·4months-37years, IQR=17·9years). Nine patients manifested with LS, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and one with Leber hereditary optic neuropathy. The remaining nine patients presented with either overlapping syndromes or isolated neurological symptoms. Mitochondrial respiratory chain activity analysis was normal in five out of ten muscle biopsies. We confirmed maternal inheritance in six families, and demonstrated marked variability in tissue segregation, and phenotypic expression at relatively low blood mutant loads. Neuropathological studies of two patients manifesting with LS/MELAS showed prominent capillary proliferation, microvacuolation and severe neuronal cell loss in the brainstem and cerebellum, with conspicuous absence of basal ganglia involvement. These findings suggest that whole mtDNA genome sequencing should be considered in patients with suspected mitochondrial disease presenting with complex neurological manifestations, which would identify over 300 known pathogenic variants including the m.13094T>C.
Assuntos
Encéfalo/patologia , Complexo I de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Mutação/genética , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Criança , Estudos de Coortes , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Síndrome , Adulto JovemRESUMO
CLARITY enables immunofluorescent labelling and imaging of large volumes of tissue to provide a better insight into the three dimensional relationship between cellular morphology and spatial interactions between different cell types. In the current study, we optimise passive CLARITY and immunofluorescent labelling of neurons and mitochondrial proteins in mouse and human brain tissues to gain further insights into mechanisms of neurodegeneration occurring in mitochondrial disease. This is the first study to utilise human cerebellum fixed in paraformaldehyde and cryoprotected in conjunction with formalin-fixed tissues opening up further avenues for use of archived tissue. We optimised hydrogel-embedding and passive clearance of lipids from both mouse (n = 5) and human (n = 9) cerebellum as well as developing an immunofluorescent protocol that consistently labels different neuronal domains as well as blood vessels. In addition to visualising large structures, we were able to visualise mitochondrial proteins in passively cleared tissues to reveal respiratory chain deficiency associated with mitochondrial disease. We also demonstrate multiple use of tissues by stripping antibodies and re-probing the cerebellum. This technique allows interrogation of large volumes intact brain samples for better understanding of the complex pathological changes taking place in mitochondrial disease.
Assuntos
Vasos Sanguíneos/metabolismo , Cerebelo/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Fixação de Tecidos/métodos , Idoso , Animais , Vasos Sanguíneos/patologia , Cerebelo/patologia , Criopreservação , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/genética , Doenças Mitocondriais/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Neurônios/ultraestrutura , Coloração e RotulagemRESUMO
Increasingly in the realm of neurological disorders, particularly those involving neurodegeneration, mitochondrial dysfunction is emerging at the core of their pathogenic processes. Most of these diseases still lack effective treatment and are hampered by a shortfall in the development of novel medicines. Clearly new targets that translate well to the clinic are required. Physiological parameters in the form of neuronal network activity are increasingly being used as a therapeutic screening approach in drug development and disorders with mitochondrial dysfunction generally display neuronal network activity disturbance. However research directly linking the disturbances in neuronal network activity with mitochondrial dysfunction is only just starting to emerge. This review will summarize the breadth of knowledge linking neuronal network activity to mitochondrial dysfunction in neurodegenerative diseases and suggest potential avenues for exploration in respect to future drug development.
Assuntos
Mitocôndrias/fisiologia , Doenças Mitocondriais/fisiopatologia , Rede Nervosa/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/fisiologia , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Rede Nervosa/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/efeitos dos fármacosRESUMO
Dementia with Lewy bodies (DLB) patients frequently experience well formed recurrent complex visual hallucinations (RCVH). This is associated with reduced blood flow or hypometabolism on imaging of the primary visual cortex. To understand these associations in DLB we used pathological and biochemical analysis of the primary visual cortex to identify changes that could underpin RCVH. Alpha-synuclein or neurofibrillary tangle pathology in primary visual cortex was essentially absent. Neurone density or volume within the primary visual cortex in DLB was also unchanged using unbiased stereology. Microarray analysis, however, demonstrated changes in neuropeptide gene expression and other markers, indicating altered GABAergic neuronal function. Calcium binding protein and GAD65/67 immunohistochemistry showed preserved interneurone populations indicating possible interneurone dysfunction. This was demonstrated by loss of post synaptic GABA receptor markers including gephyrin, GABARAP, and Kif5A, indicating reduced GABAergic synaptic activity. Glutamatergic neuronal signalling was also altered with vesicular glutamate transporter protein and PSD-95 expression being reduced. Changes to the primary visual cortex in DLB indicate that reduced GABAergic transmission may contribute to RCVH in DLB and treatment using targeted GABAergic modulation or similar approaches using glutamatergic modification may be beneficial.