Alzheimer's-Associated Upregulation of Mitochondria-Associated ER Membranes After Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer's disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-ß as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes ("MAM" domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.

Time to harmonize mitochondrial syndrome nomenclature and classification: A consensus from the North American Mitochondrial Disease Consortium (NAMDC).

OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

Cardiomyopathy and altered integrin-actin signaling in Fhl1 mutant female mice.

X-linked scapuloperoneal myopathy (X-SM), one of Four-and-a-half LIM 1 (FHL1) related diseases, is an adult-onset slowly progressive myopathy, often associated with cardiomyopathy. We previously generated a knock-in mouse model that has the same mutation (c.365 G > C, p.W122S) as human X-SM patients. The mutant male mouse developed late-onset slowly progressive myopathy without cardiomyopathy. In this study, we observed that heterozygous (Het) and homozygous (Homo) female mice did not show alterations of skeletal muscle function or histology. In contrast, 20-month-old mutant female mice showed signs of cardiomyopathy on echocardiograms with increased systolic diameter [wild-type (WT): 2.74 ± 0.22 mm, mean ± standard deviation (SD); Het: 3.13 ± 0.11 mm, P < 0.01; Homo: 3.08 ± 0.37 mm, P < 0.05) and lower fractional shortening (WT: 31.1 ± 4.4%, mean ± SD; Het: 22.7 ± 2.5%, P < 0.01; Homo: 22.4 ± 6.9%, P < 0.01]. Histological analysis of cardiac muscle revealed frequent extraordinarily large rectangular nuclei in mutant female mice that were also observed in human cardiac muscle from X-SM patients. Western blot demonstrated decreased Fhl1 protein levels in cardiac muscle, but not in skeletal muscle, of Homo mutant female mice. Proteomic analysis of cardiac muscle from 20-month-old Homo mutant female mice indicated abnormalities of the integrin signaling pathway (ISP) in association with cardiac dysfunction. The ISP dysregulation was further supported by altered levels of a subunit of the ISP downstream effectors Arpc1a in Fhl1 mutant mice and ARPC1A in X-SM patient muscles. This study reveals the first mouse model of FHL1-related cardiomyopathy and implicates ISP dysregulation in the pathogenesis of FHL1 myopathy.

Regulatory environment for novel therapeutic development in mitochondrial diseases.

At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability.

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Advances in primary mitochondrial myopathies.

PURPOSE OF REVIEW: Although mitochondrial diseases impose a significant functional limitation in the lives of patients, treatment of these conditions has been limited to dietary supplements, exercise, and physical therapy. In the past few years, however, translational medicine has identified potential therapies for these patients. RECENT FINDINGS: For patients with primary mitochondrial myopathies, preliminary phase I and II multicenter clinical trials of elamipretide indicate safety and suggest improvement in 6-min walk test (6MWT) performance and fatigue scales. In addition, for thymidine kinase 2-deficient (TK2d) myopathy, compassionate-use oral administration of pyrimidine deoxynucleosides have shown preliminary evidence of safety and efficacy in survival of early onset patients and motor functions relative to historical TK2d controls. SUMMARY: The prospects of effective therapies that improve the quality of life for patients with mitochondrial myopathy underscore the necessity for definitive diagnoses natural history studies for better understanding of the diseases.

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants.

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or ß-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.

A novel mouse model that recapitulates adult-onset glycogenosis type 4.

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen-branching enzyme (GBE). The diagnostic hallmark of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age at onset. Complete loss of enzyme activity is lethal in utero or in infancy and affects primarily the muscle and the liver. However, residual enzyme activity as low as 5-20% leads to juvenile or adult onset of a disorder that primarily affects the central and peripheral nervous system and muscles and in the latter is termed adult polyglucosan body disease (APBD). Here, we describe a mouse model of GSD IV that reflects this spectrum of disease. Homologous recombination was used to knock in the most common GBE1 mutation p.Y329S c.986A > C found in APBD patients of Ashkenazi Jewish decent. Mice homozygous for this allele (Gbe1(ys/ys)) exhibit a phenotype similar to APBD, with widespread accumulation of PG. Adult mice exhibit progressive neuromuscular dysfunction and die prematurely. While the onset of symptoms is limited to adult mice, PG accumulates in tissues of newborn mice but is initially absent from the cerebral cortex and heart muscle. Thus, PG is well tolerated in most tissues, but the eventual accumulation in neurons and their axons causes neuropathy that leads to hind limb spasticity and premature death. This mouse model mimics the pathology and pathophysiologic features of human adult-onset branching enzyme deficiency.

Fhl1 W122S causes loss of protein function and late-onset mild myopathy.

A member of the four-and-a-half-LIM (FHL) domain protein family, FHL1, is highly expressed in human adult skeletal and cardiac muscle. Mutations in FHL1 have been associated with diverse X-linked muscle diseases: scapuloperoneal (SP) myopathy, reducing body myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS) and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and investigated the effects of this mutation at three time points (3-5 months, 7-10 months and 18-20 months) in hemizygous male and heterozygous female mice. Survival was comparable in mutant and wild-type animals. We observed decreased forelimb strength and exercise capacity in adult hemizygous male mice starting from 7 to 10 months of age. Western blot analysis showed absence of Fhl1 in muscle at later stages. Thus, adult hemizygous male, but not heterozygous female, mice showed a slowly progressive phenotype similar to human patients with late-onset muscle weakness. In contrast to SP myopathy patients with the FHL1 W122S mutation, mutant mice did not manifest cytoplasmic inclusions (reducing bodies) in muscle. Because muscle weakness was evident prior to loss of Fhl1 protein and without reducing bodies, our findings indicate that loss of function is responsible for the myopathy in the Fhl1 W122S knock-in mice.

Infantile encephaloneuromyopathy and defective mitochondrial translation are due to a homozygous RMND1 mutation.

Defects of mitochondrial protein synthesis are clinically and genetically heterogeneous. We previously described a male infant who was born to consanguineous parents and who presented with severe congenital encephalopathy, peripheral neuropathy, myopathy, and lactic acidosis associated with deficiencies of multiple mitochondrial respiratory-chain enzymes and defective mitochondrial translation. In this work, we have characterized four additional affected family members, performed homozygosity mapping, and identified a homozygous splicing mutation in the splice donor site of exon 2 (c.504+1G>A) of RMND1 (required for meiotic nuclear division-1) in the affected individuals. Fibroblasts from affected individuals expressed two aberrant transcripts and had decreased wild-type mRNA and deficiencies of mitochondrial respiratory-chain enzymes. The RMND1 mutation caused haploinsufficiency that was rescued by overexpression of the wild-type transcript in mutant fibroblasts; this overexpression increased the levels and activities of mitochondrial respiratory-chain proteins. Knockdown of RMND1 via shRNA recapitulated the biochemical defect of the mutant fibroblasts, further supporting a loss-of-function pathomechanism in this disease. RMND1 belongs to the sif2 family, an evolutionary conserved group of proteins that share the DUF155 domain, have unknown function, and have never been associated with human disease. We documented that the protein localizes to mitochondria in mammalian and yeast cells. Further studies are necessary for understanding the function of this protein in mitochondrial protein translation.

Metabolic Myoglobinuria.

One large group of hereditary myopathies characterized by recurrent myoglobinuria, almost invariably triggered by exercise, comprises metabolic disorders of two main fuels, glycogen and long-chain fatty acids, or mitochondrial diseases of the respiratory chain. Differential diagnosis is required to distinguish the three conditions, although all cause a crisis of muscle energy. Muscle biopsy may be useful when performed well after the episode of rhabdomyolysis. Molecular genetics is increasingly the diagnostic test of choice to discover the underlying genetic basis.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice.

Primary human CoQ(10) deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ(10) biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ(10) of control cells, revealed reduced activity of CoQ(10)-dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30-50% CoQ(10) of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ(10) deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2(kd/kd) mice at presymptomatic, phenotypic-onset, and end-stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ(9) deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney-specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2(kd/kd) mice.

Whole-Exome Sequencing Study of Fibroblasts Derived From Patients With Cerebellar Ataxia Referred to Investigate CoQ10 Deficiency.

Background and Objectives: Coenzyme Q10 (CoQ10)-deficient cerebellar ataxia can be due to pathogenic variants in genes encoding for CoQ10 biosynthetic proteins or associated with defects in protein unrelated to its biosynthesis. Diagnosis is crucial because patients may respond favorably to CoQ10 supplementation. The aim of this study was to identify through whole-exome sequencing (WES) the pathogenic variants, and assess CoQ10 levels, in fibroblasts from patients with undiagnosed cerebellar ataxia referred to investigate CoQ10 deficiency. Methods: WES was performed on genomic DNA extracted from 16 patients. Sequencing data were filtered using a virtual panel of genes associated with CoQ10 deficiency and/or cerebellar ataxia. CoQ10 levels were measured by high-performance liquid chromatography in 14 patient-derived fibroblasts. Results: A definite genetic etiology was identified in 8 samples of 16 (diagnostic yield = 50%). The identified genetic causes were pathogenic variants of the genes COQ8A (ADCK3) (n = 3 samples), ATP1A3 (n = 2), PLA2G6 (n = 1), SPG7 (n = 1), and MFSD8 (n = 1). Five novel mutations were found (COQ8A n = 3, PLA2G6 n = 1, and MFSD8 n = 1). CoQ10 levels were significantly decreased in 3/14 fibroblast samples (21.4%), 1 carrying compound heterozygous COQ8A pathogenic variants, 1 harboring a homozygous pathogenic SPG7 variant, and 1 with an unknown molecular defect. Discussion: This work confirms the importance of COQ8A gene mutations as a frequent genetic cause of cerebellar ataxia and CoQ10 deficiency and suggests SPG7 mutations as a novel cause of secondary CoQ10 deficiency.

MERRF and Kearns-Sayre overlap syndrome due to the mitochondrial DNA m.3291T>C mutation.

A 48-year-old man presented with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) syndrome and Kearns-Sayre syndrome (KSS), which included progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, bifascicular heart block, and ragged-red fibers. The m.3291T>C mutation in the tRNA(Leu(UUR)) gene was found with 92% heteroplasmy in muscle. This mutation has been reported with MELAS, myopathy, and deafness with cognitive impairment. This is the first description with a MERRF/KSS syndrome.

Leber hereditary optic neuropathy plus dystonia, and transverse myelitis due to double mutations in MT-ND4 and MT-ND6.

Leber hereditary optic neuropathy (LHON) typically presents as painless central or centrocecal scotoma and is due to maternally inherited mitochondrial DNA (mtDNA) mutations. Over 95% of LHON cases are caused by one of three mtDNA "common" point mutations: m.3460G>A, m.11778G>A, or m.14484T>C, which are all in genes encoding structural subunits of complex I of the respiratory chain. Intriguing features of LHON include: incomplete penetrance, tissue specificity, and male predominance, indicating that additional genetic or environmental factors are modulating the phenotypic expression of the pathogenic mtDNA mutations. However, since its original description as a purely ophthalmological disorder, LHON has also been linked to multisystemic conditions with variable neurological, cardiac, and skeletal abnormalities. Although double "common" mutations have been reported to cause LHON and LHON-plus, they are extremely rare. Here, we present a patient with an unusual double point mutation (m.11778 G>A and m.14484T>C) with a multisystemic LHON-plus phenotype characterized by: optic neuropathy, ptosis, ataxia, dystonia, dysarthria, and recurrent extensive transverse myelitis.

Mitochondrial diseases in North America: An analysis of the NAMDC Registry.

OBJECTIVE: To describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry. METHODS: This cross-sectional, multicenter, retrospective database analysis evaluates the phenotypic and molecular characteristics of participants enrolled in the NAMDC Registry from September 2011 to December 2018. The NAMDC is a network of 17 centers with expertise in MtDs and includes both adult and pediatric specialists. RESULTS: One thousand four hundred ten of 1,553 participants had sufficient clinical data for analysis. For this study, we included only participants with molecular genetic diagnoses (n = 666). Age at onset ranged from infancy to adulthood. The most common diagnosis was multisystemic disorder (113 participants), and only a minority of participants were diagnosed with a classical mitochondrial syndrome. The most frequent classical syndromes were Leigh syndrome (97 individuals) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (71 individuals). Pathogenic variants in the mitochondrial DNA were more frequently observed (414 participants) than pathogenic nuclear gene variants (252 participants). Pathogenic variants in 65 nuclear genes were identified, with POLG1 and PDHA1 being the most commonly affected. Pathogenic variants in 38 genes were reported only in single participants. CONCLUSIONS: The NAMDC Registry data confirm the high variability of clinical, biochemical, and genetic features of participants with MtDs. This study serves as an important resource for future enhancement of MtD research and clinical care by providing the first comprehensive description of participant with MtD in North America.

Toll-free telephone medical information on nocturnal enuresis: the first Italian experience over a two-year period.

PURPOSE: The aim of the study is to promote, through this toll-free number (TFN) service, a health communication program providing information on nocturnal enuresis (NE) and related problems by a subspecialty-trained physician and to collect the callers' characteristics too. All phone calls were scheduled to data collections. METHODS: The telephone service operated as follows: the TFN was available from March 1 to May 31, 2000, and from April 1 to June 30, 2001. People called the free telephone line and received information needs. RESULTS: A total of 12,806 calls were received by the help line during the two study periods (7,046 in 2000 and 5,760 in 2001). Of the calls, 61% came from subjects with NE without pharmacological or non-pharmacological treatment, 16% (2000) and 13% (2001) came from subjects >12 years old. CONCLUSIONS: A TFN for NE can be both accessible and effective in order to provide information on NE and related problems. Finally, such a service should be included in a national program to improve health and well-being.

Anti-Oxidant Drugs: Novelties and Clinical Implications in Cerebellar Ataxias.

BACKGROUND: Hereditary cerebellar ataxias are a group of disorders characterized by heterogeneous clinical manifestations, progressive clinical course, and diverse genetic causes. No disease modifying treatments are yet available for many of these disorders. Oxidative stress has been recurrently identified in different progressive cerebellar diseases, and it represents a widely investigated target for treatment. OBJECTIVE: To review the main aspects and new perspectives of antioxidant therapy in cerebellar ataxias ranging from bench to bedside. METHOD: This article is a summary of the state-of-the-art on the use of antioxidant molecules in cerebellar ataxia treatments. It also briefly summarizes aspects of oxidative stress production and general characteristics of antioxidant compounds. RESULTS: Antioxidants represent a vast category of compounds; old drugs have been extensively studied and modified in order to achieve better biological effects. Despite the vast body of literature present on the use of antioxidants in cerebellar ataxias, for the majority of these disorders conclusive results on the efficacy are still missing. CONCLUSION: Antioxidant therapy in cerebellar ataxias is a promising field of investigations. To achieve the success in identifying the correct treatment more work needs to be done. In particular, a combined effort is needed by basic scientists in developing more efficient molecules, and by clinical researchers together with patients communities, to run clinical trials in order to identify conclusive treatments strategies.