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Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5–9 (Δ5–9) and 2–3 (Δ2–3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5–9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2–3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

Oxidative stress and protein aggregation are biochemical hallmarks of Parkinson’s disease (PD), a frequent sporadic late-onset degenerative disorder particularly of dopaminergic neurons in the substantia nigra, resulting in impaired spontaneous movement. PARK6 is a rare autosomal-recessively inherited disorder, mimicking the clinical picture of PD with earlier onset and slower progression. Genetic data demonstrated PARK6 to be caused by mutations in the protein PINK1, which is localized to mitochondria and has a serine–threonine kinase domain. To study the effect of PINK1 mutations on oxidative stress, we used primary fibroblasts and immortalized lymphoblasts from three patients homozygous for G309D-PINK1. Oxidative stress was evident from increases in lipid peroxidation and in antioxidant defenses by mitochondrial superoxide dismutase and glutathione. Elevated levels of glutathione reductase and glutathione-S-transferase were also observed. As a putative cause of oxidation, a mild decrease in complex I activity and a trend to superoxide elevation were detectable. These data indicate that PINK1 function is critical to prevent oxidative damage and that peripheral cells may be useful for studies of progression and therapy of PARK6.

Children with hereditary severe hyperhomocysteinemia present with a variety of neurological impairment, and mild hyperhomocysteinemia has been associated with neurodegeneration in the elderly. The link of hyperhomocysteinemia to neurological dysfunction is unknown. We investigated mitochondrial mechanisms of homocysteine (HCys) neurotoxicity in rat dopaminergic pheochromocytoma cells, human neuroblastoma cells and primary rat cerebellar granule neurons. HCys dose dependently impaired cytochrome c oxidase (COX) activity as well as stability and induced reactive oxygen species and apoptotic cell death. We found that HCys binds the COX cofactor Cu2+, and Cu2+ supplementation prior to HCys treatment preserved COX activity and prevented cell death. The Cu2+ chelating action of HCys and impairement of COX activity represent novel mechanisms of HCys neurotoxicity, which might be preventable by supplementation of Cu2+.

Background: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2., Methods: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families., Results: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency., Conclusions: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene., Competing Interests: Conflict of interest There is no conflict of interest by any author., (Copyright © 2023 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

In past decades, the identification of genes involved in epileptic disorders has grown exponentially. The pace of gene identification in epileptic disorders began to accelerate in the late 2000s, driven by new technologies such as molecular cytogenetics and next-generation sequencing (NGS). These technologies have also been applied to genetic diagnostics, with different configurations, such as gene panels, whole-exome sequencing and whole-genome sequencing. The clinician must be aware that any technology has its limitations and complementary techniques must still be used to establish a diagnosis for specific diseases. In addition, increasing the amount of genetic information available in a larger patient sample also increases the need for rigorous interpretation steps, when taking into account the clinical, electroclinical, and when available, functional data. Local, multidisciplinary discussions have proven valuable in difficult diagnostic situations, especially in cases where precision medicine is being considered. They also serve to improve genetic counseling in complex situations. In this article, we will briefly review the genetic basis of rare and common epilepsies, the current strategies used for molecular diagnosis, including their limitations, and some pitfalls for data interpretation, in the context of etiological diagnosis and genetic counseling., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca 2+ -activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Cannabis is one of the most abused drugs worldwide, with more than 20 million users in the United States (US). As access to cannabis products increases with expanding US legislation and decriminalization of marijuana, emergency physicians must be adept in recognizing unintentional cannabis toxicity in young children, which can range from altered mental status to encephalopathy and coma., Case Report: We report the case of a 13-month-old female presenting with self-limiting altered mental status and lethargy, with a subsequent diagnosis of tetrahydrocannabinol exposure on confirmatory urine gas chromatography-mass spectrometry. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Considering caretakers rarely report possible cannabis exposure, history-taking must review caretakers' medicinal and recreational drug exposures to prevent inadvertently missing the diagnosis. In the young child with altered mental status, prompt urine screening for cannabinoid detection can prevent further invasive and costly diagnostic investigations, such as brain imaging and lumbar puncture., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oxyphil cell transformation of epithelial cells due to the accumulation of mitochondria occurs often during cellular aging. To understand the pathogenic mechanisms, we studied mitochondrial DNA (mtDNA) alterations in the three cell types of the parathyroids using multiplex real-time PCR and next-generation sequencing. mtDNA was analyzed from cytochrome c oxidase (COX)-positive and COX-negative areas of 19 parathyroids. Mitochondria-rich pre-oxyphil/oxyphil cells were more prone to develop COX defects than the mitochondria-poor clear chief cells (P < 0.001). mtDNA increased approximately 2.5-fold from clear chief to oxyphil cells. In COX deficiency, the increase was even more pronounced, and COX-negative oxyphil cells had approximately two times more mtDNA than COX-positive oxyphil cells (P < 0.001), illustrating the influence of COX deficiency on mtDNA biosynthesis, probably as a consequence of insufficient ATP synthesis. Next-generation sequencing revealed a broad spectrum of putative pathogenic mtDNA point mutations affecting NADH dehydrogenase and COX genes as well as regulatory elements of mtDNA. NADH dehydrogenase gene mutations preferentially accumulated in COX-positive pre-oxyphil/oxyphil cells and, therefore, could be essential for inducing oxyphil cell transformation by increasing mtDNA/mitochondrial biogenesis. In contrast, COX-negative cells predominantly harbored mutations in the MT-CO1 and MT-CO3 genes and in regulatory mtDNA elements, but only rarely NADH dehydrogenase mutations. Thus, multiple hits in NADH dehydrogenase and COX activity-impairing genes represent the molecular basis of oxyphil cell transformation in the parathyroids.

Purpose: The group of the rare progressive myoclonic epilepsies (PME) include a wide spectrum of mitochondrial and metabolic diseases. In juvenile and adult ages, MERRF (myoclonic epilepsy with ragged red fibres) is the most common form. The underlying genetic defect in most patients with the syndrome of MERRF is a mutation in the tRNALys gene, but mutations were also detected in the tRNAPhe gene., Method: Here, we describe a 40 year old patient with prominent myoclonic seizures since 39 years of age without a mutation in the known genes who underwent intensive clinical, genetic and functional workup., Results: The patient had a slight mental retardation and a severe progressive hearing loss based on a defect of the inner ear on both sides. Ictal electroencephalography (EEG) showed bilateral occipital and generalized spikes and polyspikes induced and aggravated by photostimulation. A cranial magnetic resonance imaging (cMRI) detected a global cortical atrophy of the brain and mild periventricular white matter lesions. The electromyography (EMG) was normal but the muscle biopsy showed abundant ragged red fibres. Sequencing of the mitochondrial DNA from the skeletal muscle biopsy revealed a novel heteroplasmic mutation (m.4279A>G) in the tRNAIle gene which was functionally relevant as tested in single skeletal muscle fibre investigations., Conclusion: Mutations in tRNAIle were described in patients with chronic progressive external ophthalmoplegia (CPEO), prominent deafness or cardiomyopathy but, up to now, not in patients with myoclonic epilepsy. The degree of heteroplasmy of this novel mitochondrial DNA mutation was 70% in skeletal muscle but only 15% in blood, pointing to the diagnostic importance of a skeletal muscle biopsy also in patients with myoclonic epilepsy., (Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Background: The human amygdala plays a crucial role in processing social signals, such as face expressions, particularly fearful ones, and facilitates responses to them in face-sensitive cortical regions. This contributes to social competence and individual amygdala size correlates with that of social networks. While rare patients with focal bilateral amygdala lesion typically show impaired recognition of fearful faces, this deficit is variable, and an intriguing possibility is that other brain regions can compensate to support fear and social signal processing., Methods: To investigate the brain's functional compensation of selective bilateral amygdala damage, we performed a series of behavioral, psychophysiological, and functional magnetic resonance imaging experiments in two adult female monozygotic twins (patient 1 and patient 2) with equivalent, extensive bilateral amygdala pathology as a sequela of lipoid proteinosis due to Urbach-Wiethe disease., Results: Patient 1, but not patient 2, showed preserved recognition of fearful faces, intact modulation of acoustic startle responses by fear-eliciting scenes, and a normal-sized social network. Functional magnetic resonance imaging revealed that patient 1 showed potentiated responses to fearful faces in her left premotor cortex face area and bilaterally in the inferior parietal lobule., Conclusions: The premotor cortex face area and inferior parietal lobule are both implicated in the cortical mirror-neuron system, which mediates learning of observed actions and may thereby promote both imitation and empathy. Taken together, our findings suggest that despite the pre-eminent role of the amygdala in processing social information, the cortical mirror-neuron system may sometimes adaptively compensate for its pathology., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Button batteries within the gastrointestinal system are dangerous and must be suspected after any foreign body ingestion. Common complications include esophageal perforation, fistula formation, and esophageal scarring., Objectives: Spondylodiscitis resulting from button battery ingestion is extremely rare and, to our knowledge, has been described in the literature only once to date., Case Report: We will describe a case in which a 14-month-old girl developed spondylodiscitis of T1/T2 after an uncomplicated clinical course involving the ingestion and removal of an esophageal button battery. Discussion will include mechanisms in which button batteries cause harm and notable differences between the previously reported case and ours., Conclusions: We present this case to increase awareness of spondylodiscitis in patients with neck pain or stiffness and a history of button battery ingestion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Genetic studies have implicated the evolutionary novel, primates-specific gene locus G72/G30 in schizophrenia, bipolar and panic disorders. It encodes for a protein LG72 whose function has been controversially discussed as putative regulator of the peroxisomal enzyme D-amino-acid-oxidase (DAO), or as a mitochondrial protein, which promotes robust mitochondrial fragmentation in mammalian cell lines including human and rat primary neurons. Because of this conserved function we here have generated "humanized" BAC transgenic mice (G72Tg) expressing alternatively spliced G72 and G30 transcripts, and the LG72 protein. G72 expression is prominent in granular cells of the cerebellum, the hippocampus, the cortex and the olfactory bulb. Most strikingly, G72Tg mice displayed deficits in sensorimotor gating which could be reversed with haloperidol, increased sensitivity to PCP, motor-coordination deficits, increased compulsive behaviors and deficits in smell identification. These results demonstrate that expression of the human G72/G30 gene locus in mice produces behavioral phenotypes that are relevant to psychiatric disorders.

In search of tumor-specific mitochondrial DNA (mtDNA) mutations in head and neck squamous cell cancer, we found heteroplasmy in the blood of two individuals, i.e., these individuals carried two alleles of mtDNA. In both cases, the tumor was found to be homoplasmic, i.e., it contained only one of the two mtDNA alleles present in blood. More interestingly, in one case the tumor had acquired the wild-type allele, while in the other case it contained the mutant allele only. Sequencing of the whole 16.5 kb mtDNA showed that the observed heteroplasmic positions in the D-loop region, nucleotides 152 and 16187, respectively, were the only differences between tumor and blood mtDNA genotypes in these individuals. Our findings thus strongly support the hypothesis that accumulation of mtDNA mutations in solid tumors occurs by clonal and random expansion of pre-existing alleles and is not necessary for the metabolic changes generally associated with tumor formation, the Warburg effect.

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.

As the prevalence of type 2 diabetes mellitus is increasing at an alarming rate, effective nutritional and exercise strategies for the prevention of this disease are required. Specific dietary components with antidiabetic efficacy could be one aspect of these strategies. This study investigated the antidiabetic effects of the most abundant green tea catechin, epigallocatechin gallate (EGCG, TEAVIGO), in rodent models of type 2 diabetes mellitus and H4IIE rat hepatoma cells. We assessed glucose and insulin tolerance in db/db mice and ZDF rats after they ingested EGCG. Using gene microarray and real-time quantitative RT-PCR we investigated the effect of EGCG on gene expression in H4IIE rat hepatoma cells as well as in liver and adipose tissue of db/db mice. EGCG improved oral glucose tolerance and blood glucose in food-deprived rats in a dose-dependent manner. Plasma concentrations of triacylglycerol were reduced and glucose-stimulated insulin secretion was enhanced. In H4IIE cells, EGCG downregulated genes involved in gluconeogenesis and the synthesis of fatty acids, triacylgycerol, and cholesterol. EGCG decreased the mRNA expression of phosphoenolpyruvate carboxykinase in H4IIE cells as well as in liver and adipose tissue of db/db mice. Glucokinase mRNA expression was upregulated in the liver of db/db mice in a dose-dependent manner. This study shows that EGCG beneficially modifies glucose and lipid metabolism in H4IIE cells and markedly enhances glucose tolerance in diabetic rodents. Dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus.

Several lines of evidence suggest an impairment of mitochondrial function in the brain of patients with Parkinson's disease (PD). However, the presence of a detectable mitochondrial defect in extracerebral tissue of these patients remains a matter of dispute. Therefore, we investigated mitochondrial function in fibroblasts of 18 PD patients applying biochemical micromethods. Putative beneficial effects of coenzyme Q(10) (CoQ(10)), a potent antioxidant, on the mitochondrial function of skin fibroblast cultures were evaluated. Applying inhibitor titrations of the mitochondrial respiration to calculate flux control coefficients of respiratory chain complexes I and IV, we observed deficiencies of both complexes in cultivated skin fibroblasts of PD patients. Cultivation of fibroblasts in the presence of 5 microM CoQ(10) restored the activity of impaired respiratory chain complexes in the fibroblast cultures of 9 out of 18 PD patients. Our data support the presence of a generalised mitochondrial defect in at least a subgroup of patients with PD that can be partially ameliorated in vitro by coenzyme Q(10) treatment.

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.