Delineating selective vulnerability of inhibitory interneurons in Alpers' syndrome.

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.

Simplifying the clinical classification of polymerase gamma (POLG) disease based on age of onset; studies using a cohort of 155 cases.

BACKGROUND: Variants in POLG are one of the most common causes of inherited mitochondrial disease. Phenotypic classification of POLG disease has evolved haphazardly making it complicated and difficult to implement in everyday clinical practise. The aim of our study was to simplify the classification and facilitate better clinical recognition. METHODS: A multinational, retrospective study using data from 155 patients with POLG variants recruited from seven European countries. RESULTS: We describe the spectrum of clinical features associated with POLG variants in the largest known cohort of patients. While clinical features clearly form a continuum, stratifying patients simply according to age of onset-onset prior to age 12 years; onset between 12 and 40 years and onset after the age of 40 years, permitted us to identify clear phenotypic and prognostic differences. Prior to 12 years of age, liver involvement (87%), seizures (84%), and feeding difficulties (84%) were the major features. For those with onset between 12 and 40 years, ataxia (90%), peripheral neuropathy (84%), and seizures (71%) predominated, while for those with onset over 40 years, ptosis (95%), progressive external ophthalmoplegia (89%), and ataxia (58%) were the major clinical features. The earlier the onset the worse the prognosis. Patients with epilepsy and those with compound heterozygous variants carried significantly worse prognosis. CONCLUSION: Based on our data, we propose a simplified POLG disease classification, which can be used to guide diagnostic investigations and predict disease course.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder.

BACKGROUND: Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. METHODS AND RESULTS: We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNA(Cys) was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. CONCLUSIONS: Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy.

Mitochondrial disorders and epilepsy.

INTRODUCTION: Mitochondrial respiratory chain defects (RCD) often exhibit multiorgan involvement, affecting mainly tissues with high-energy requirements such as the brain. Epilepsy is frequent during the evolution of mitochondrial disorders (30%) with different presentation in childhood and adulthood in term of type of epilepsy, of efficacy of treatment and also in term of prognosis. STATE OF ART: Mitochondrial disorders can begin at any age but the diseases with early onset during childhood have generally severe or fatal outcome in few years. Four age-related epileptic phenotypes could be identified in infancy: infantile spasms, refractory or recurrent status epilepticus, epilepsia partialis continua and myoclonic epilepsy. Except for infantile spasms, epilepsy is difficult to control in most cases (95%). In pediatric patients, mitochondrial epilepsy is more frequent due to mutations in nDNA-located than mtDNA-located genes and vice versa in adults. Ketogenic diet could be an interesting alternative treatment in case of recurrent status epilepticus or pharmacoresistant epilepsy. CONCLUSION: Epileptic seizures increase the energy requirements of the metabolically already compromised neurons establishing a vicious cycle resulting in worsening energy failure and neuronal death.

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20 µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200 µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7 days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50 µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7 days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

Astrocytic pathology in Alpers' syndrome.

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.

A de novo GABRB2 variant associated with myoclonic status epilepticus and rhythmic high-amplitude delta with superimposed (poly) spikes (RHADS).

We report a child who developed myoclonic status epilepticus (MSE) at four months of age, associated with rhythmic high-amplitude delta and superimposed (poly) spikes (RHADS), harbouring a GABRB2 (ß2 subunit of the GABA A receptor) variant. The patient was treated under a presumptive diagnosis of neonatal-onset Alpers syndrome (AS) and underwent targeted sequence analysis for POLG1 (polymerase gamma 1) and subsequent whole-exome sequence analysis (WES). The patient is currently a 10-year, eight-month-old boy, suffering from daily MSE associated with RHADS and severe global developmental delay from early infancy. Although POLG1 mutation was negative, WES revealed a de novo missense variant of GABRB2 (NM_021911.2: c.784G>T, p.[Val262Phe]). Based on a review of case series with GABRB2 variants, we found that five of the 18 cases shared the clinical and EEG characteristics associated with our patient. In summary, this de novo GABRB2 variant was associated with an AS phenotype, characterized by treatment-resistant MSE and RHADS, and may represent an alternative aetiology for neonatal-onset AS without POLG1 mutation [Published with video sequence].

Dissecting the neuronal vulnerability underpinning Alpers' syndrome: a clinical and neuropathological study.

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.

Lethal NARS2-Related Disorder Associated With Rapidly Progressive Intractable Epilepsy and Global Brain Atrophy.

BACKGROUND: Infantile epileptic encephalopathy is a heterogeneous condition that has been associated with variants in more than 200 genes. The variability in findings and prognosis creates challenges to making the correct diagnosis and initiating the appropriate therapy. Biallelic variants in NARS2, a mitochondrial aminoacyl-tRNA synthetase gene, were recently associated with neurodegenerative disorders that include epilepsy. METHODS: We describe two infant brothers who presented with focal status epilepticus that progressed to lethal epileptic encephalopathy. We compared the cost of diagnostic laboratory evaluation for each child. Detailed NARS2 protein analysis was performed using a sequence-to-structure-to-function workflow, merging multiple homologous structures, to suggest biologic impact of the NARS2 variants. RESULTS: Brain magnetic resonance imaging showed rapid progression to generalized atrophy. Extensive metabolic, infectious, chromosomal and genetic testing of the first infant failed to reach a specific diagnosis. The younger brother presented similarly. Rapid whole exome sequencing was performed revealing novel biallelic variants in NARS2. The variants c.167A>G (p.Gln56Arg) and c.631T>A (p.Phe211Ile) were confirmed in a reserved sample from the older brother. Management was then redirected toward palliative care and the child died at age nine months. CONCLUSIONS: NARS2-related disorder should be considered in infants presenting with refractory seizures and rapid brain atrophy. Metabolic screening tests may be normal or yield nonspecific findings. Rapid whole exome sequencing in children with fulminant onset intractable epilepsy may minimize extensive diagnostic evaluation and aid in prognosis and medical management.

Myocerebrohepatopathy spectrum disorder due to POLG mutations: A clinicopathological report.

We report on the clinical, neuropathological, and genetic findings of a Japanese case with myocerebrohepatopathy spectrum (MCHS) disorder due to polymerase gamma (POLG) mutations. A girl manifested poor sucking and failure to thrive since 4 months of age and had frequent vomiting and developmental regression at 5 months of age. She showed significant hypotonia and hepatomegaly. Laboratory tests showed hepatocellular dysfunction and elevated protein and lactate levels in the cerebrospinal fluid. Her liver function and neurologic condition exacerbated, and she died at 8 months of age. At autopsy, fatty degeneration and fibrosis were observed in the liver. Neuropathological examination revealed white matter-predominant spongy changes with Alzheimer type II glia and loss of myelin. Enzyme activities of the respiratory chain complex I, III, and IV relative to citrate synthase in the muscle were normal in the biopsied muscle tissue, but they were reduced in the liver to 0%, 10%, and 14% of normal values, respectively. In the liver, the copy number of mitochondrial DNA compared to nuclear DNA was reduced to 3.3% of normal values as evaluated by quantitative polymerase chain reaction. Genetic analysis revealed compound heterozygous mutations for POLG (I1185T/A957V). This case represents the differential involvement of multiple organs and phenotype-specific distribution of brain lesions in mitochondrial DNA depletion disorders.

Whole exome sequencing reveals mutations in NARS2 and PARS2, encoding the mitochondrial asparaginyl-tRNA synthetase and prolyl-tRNA synthetase, in patients with Alpers syndrome.

Alpers syndrome is a progressive neurodegenerative disorder that presents in infancy or early childhood and is characterized by diffuse degeneration of cerebral gray matter. While mutations in POLG1, the gene encoding the gamma subunit of the mitochondrial DNA polymerase, have been associated with Alpers syndrome with liver failure (Alpers-Huttenlocher syndrome), the genetic cause of Alpers syndrome in most patients remains unidentified. With whole exome sequencing we have identified mutations in NARS2 and PARS2, the genes encoding the mitochondrial asparaginyl-and prolyl-tRNA synthetases, in two patients with Alpers syndrome. One of the patients was homozygous for a missense mutation (c.641C>T, p.P214L) in NARS2. The affected residue is predicted to be located in the stem of a loop that participates in dimer interaction. The other patient was compound heterozygous for a one base insertion (c.1130dupC, p.K378 fs*1) that creates a premature stop codon and a missense mutation (c.836C>T, p.S279L) located in a conserved motif of unknown function in PARS2. This report links for the first time mutations in these genes to human disease in general and to Alpers syndrome in particular.

Defects of mitochondrial DNA replication.

Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease.