Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

Normal FGF-21-Serum Levels in Patients with Carnitine Palmitoyltransferase II (CPT II) Deficiency.

Fibroblast growth factor 21 (FGF-21) is known to be a biomarker for mitochondrial disorders. An upregulation of FGF-21 in serum and muscle of carnitine palmitoyltransferase I (CPT I) and carnitine palmitoyltransferase II (CPT II) knock-out mice has been reported. In human CPT II deficiency, enzyme activity and protein content are normal, but the enzyme is abnormally regulated by malonyl-CoA and is abnormally thermolabile. Citrate synthase (CS) activity is increased in patients with CPT II deficiency. This may indicate a compensatory response to an impaired function of CPT II. In this study, FGF-21 serum levels in patients with CPT II deficiency during attack free intervals and in healthy controls were measured by enzyme linked immunosorbent assay (ELISA). The data showed no significant difference between FGF-21 concentration in the serum of patients with CPT II deficiency and that in the healthy controls. The results of the present work support the hypothesis that in muscle CPT II deficiency, in contrast to the mouse knockout model, mitochondrial fatty acid utilization is not persistently reduced. Thus, FGF-21 does not seem to be a useful biomarker in the diagnosis of CPT II deficiency.

FGF-21 as a Potential Biomarker for Mitochondrial Diseases.

The diagnosis of mitochondrial diseases is still challenging due to clinical and genetical heterogeneity. The development of advanced technologies including Whole-Exome- Sequencing (WES) and Whole-Genome-Sequencing (WGS) has led to improvements in genetic diagnosis. However, a reliable biomarker in serum could enhance and ease the diagnosis and indeed reduce the need for muscle biopsy. Several studies suggest Fibroblast growth factor 21 (FGF-21) as a biomarker for diagnosis in mitochondrial disorders. It is known, that in patients with mitochondrial disorders, the expression of FGF-21 gets elevated in an effort to counteract the underlying metabolic deficiency. The growth and differentiation factor 15 (GDF-15) has been described as a potential biomarker for mitochondrial diseases, too. In the present review, a literature research, using PubMed database about the reliability of FGF-21 as a biomarker for mitochondrial disorders and its comparison with GDF-15 has been performed.

Overview of Approaches to Mitochondrial Disease Therapy

Abstract Mitochondrial respiratory chain diseases are the most prevalent group of inherited neurometabolic disorders and are clinically, biochemically, and genetically heterogeneous. They may present at any stage of life and often manifest with severe multisystem disease, although single organ involvement is characteristic of some conditions such as Leber hereditary optic neuropathy. As a result of these complexities, the diagnostic pathway is often challenging, so too is further advice, guidance, and therapy following diagnosis. Difficulties also occur with regard to genetic counseling, prognostic assessment, and treatment; there is still no cure or even agreed standards of treatment available for these debilitating diseases. Limited therapeutic options and a lack of curative treatment have led to physicians prescribing individual "trials of therapy" for which no evidence-based recommendations are available. However, new therapeutic options are the focus of active molecular genetic, biochemical, and clinical research, and some medicinal compounds have achieved international governmental approval. In this chapter, we summarize these advances and provide a broad overview of the treatment and novel approaches to preventing transmission of mitochondrial disease.

Muscle Carnitine Palmitoyltransferase II Deficiency: A Review of Enzymatic Controversy and Clinical Features.

CPT (carnitine palmitoyltransferase) II muscle deficiency is the most common form of muscle fatty acid metabolism disorders. In contrast to carnitine deficiency, it is clinically characterized by attacks of myalgia and rhabdomyolysis without persistent muscle weakness and lipid accumulation in muscle fibers. The biochemical consequences of the disease-causing mutations are still discussed controversially. CPT activity in muscles of patients with CPT II deficiency ranged from not detectable to reduced to normal. Based on the observation that in patients, total CPT is completely inhibited by malony-CoA, a deficiency of malonyl-CoA-insensitive CPT II has been suggested. In contrast, it has also been shown that in muscle CPT II deficiency, CPT II protein is present in normal concentrations with normal enzymatic activity. However, CPT II in patients is abnormally sensitive to inhibition by malonyl-CoA, Triton X-100 and fatty acid metabolites. A recent study on human recombinant CPT II enzymes (His6-N-hCPT2 and His6-N-hCPT2/S113L) revealed that the wild-type and the S113L variants showed the same enzymatic activity. However, the mutated enzyme showed an abnormal thermal destabilization at 40 and 45 °C and an abnormal sensitivity to inhibition by malony-CoA. The thermolability of the mutant enzyme might explain why symptoms in muscle CPT II deficiency mainly occur during prolonged exercise, infections and exposure to cold. In addition, the abnormally regulated enzyme might be mostly inhibited when the fatty acid metabolism is stressed.

Peripheral neuropathy in patients with CPEO associated with single and multiple mtDNA deletions.

OBJECTIVE: To characterize peripheral nerve involvement in patients with chronic progressive external ophthalmoplegia (CPEO) with single and multiple mitochondrial DNA (mtDNA) deletions, based on clinical scores and detailed nerve conduction studies. METHODS: Peripheral nerve involvement was prospectively investigated in 33 participants with CPEO (single deletions n = 18 and multiple deletions n = 15). Clinically, a modified Total Neuropathy Score (mTNS) and a modified International Cooperative Ataxia Rating Scale (mICARS) were used. Nerve conduction studies included Nn. suralis, superficialis radialis, tibialis, and peroneus mot. Early somatosensory evoked potentials were obtained by N. tibialis stimulation. RESULTS: Participants with multiple deletions had higher mTNS and mICARS scores than those with single deletions. Electrophysiologically in both sensory nerves (N. suralis and N. radialis superficialis), compound action potential (CAP) amplitudes and nerve conduction velocities were lower and mostly abnormal in multiple deletions than those in single deletions. Early somatosensory evoked potentials of N. tibialis revealed increased P40 latencies and decreased N35-P40 amplitudes in multiple deletions. Both sensory nerves had higher areas under the receiver operating characteristic curves for the decreased CAP amplitudes than the 2 motor nerves. The N. suralis had the best Youden index, indicating a sensitivity of 93.3% and a specificity of 72.2% to detect multiple deletions. CONCLUSIONS: Peripheral nerve involvement in participants with multiple mtDNA deletions is an axonal type of predominant sensory neuropathy. This is clinically consistent with higher mTNS and mICARS scores. Sensory nerve involvement in participants with multiple deletions was not correlated with age at onset and duration of disease.

Expanded phenotypic spectrum of the m.8344A>G "MERRF" mutation: data from the German mitoNET registry.

The m.8344A>G mutation in the MTTK gene, which encodes the mitochondrial transfer RNA for lysine, is traditionally associated with myoclonic epilepsy and ragged-red fibres (MERRF), a multisystemic mitochondrial disease that is characterised by myoclonus, seizures, cerebellar ataxia, and mitochondrial myopathy with ragged-red fibres. We studied the clinical and paraclinical phenotype of 34 patients with the m.8344A>G mutation, mainly derived from the nationwide mitoREGISTER, the multicentric registry of the German network for mitochondrial disorders (mitoNET). Mean age at symptom onset was 24.5 years ±10.9 (6-48 years) with adult onset in 75 % of the patients. In our cohort, the canonical features seizures, myoclonus, cerebellar ataxia and ragged-red fibres that are traditionally associated with MERRF, occurred in only 61, 59, 70, and 63 % of the patients, respectively. In contrast, other features such as hearing impairment were even more frequently present (72 %). Other common features in our cohort were migraine (52 %), psychiatric disorders (54 %), respiratory dysfunction (45 %), gastrointestinal symptoms (38 %), dysarthria (36 %), and dysphagia (35 %). Brain MRI revealed cerebral and/or cerebellar atrophy in 43 % of our patients. There was no correlation between the heteroplasmy level in blood and age at onset or clinical phenotype. Our findings further broaden the clinical spectrum of the m.8344A>G mutation, document the large clinical variability between carriers of the same mutation, even within families and indicate an overlap of the phenotype with other mitochondrial DNA-associated syndromes.

Pathogenic mitochondrial mt-tRNA(Ala) variants are uniquely associated with isolated myopathy.

Pathogenic mitochondrial DNA (mtDNA) point mutations are associated with a wide range of clinical phenotypes, often involving multiple organ systems. We report two patients with isolated myopathy owing to novel mt-tRNA(Ala) variants. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for both mutations (m.5631G>A and m.5610G>A) whilst single-muscle fibre segregation studies (revealing statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres), hierarchical mutation segregation within patient tissues and decreased steady-state mt-tRNA(Ala) levels all provide compelling evidence of pathogenicity. Interestingly, both patients showed very high-mutation levels in all tissues, inferring that the threshold for impairment of oxidative phosphorylation, as evidenced by COX deficiency, appears to be extremely high for these mt-tRNA(Ala) variants. Previously described mt-tRNA(Ala) mutations are also associated with a pure myopathic phenotype and demonstrate very high mtDNA heteroplasmy thresholds, inferring at least some genotype:phenotype correlation for mutations within this particular mt-tRNA gene.

A novel m.7539C>T point mutation in the mt-tRNA(Asp) gene associated with multisystemic mitochondrial disease.

Mitochondrial transfer RNA (mt-tRNA) mutations are the commonest sub-type of mitochondrial (mtDNA) mutations associated with human disease. We report a patient with multisytemic disease characterised by myopathy, spinal ataxia, sensorineural hearing loss, cataract and cognitive impairment in whom a novel m.7539C>T mt-tRNA(Asp) transition was identified. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for the mutation whilst single muscle fibre segregation studies revealed statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres. Absence from control databases, hierarchical mt-tRNA mutation segregation within tissues, and occurrence at conserved sequence positions, further confirm this novel mt-tRNA mutation to be pathogenic. To date only three mt-tRNA(Asp) gene mutations have been described with clear evidence of pathogenicity. The novel m.7539C>T mt-tRNA(Asp) gene mutation extends the spectrum of pathogenic mutations in this gene, further supporting the notion that mt-tRNA(Asp) gene mutations are associated with multisystemic disease presentations.

Normal protein content but abnormally inhibited enzyme activity in muscle carnitine palmitoyltransferase II deficiency.

The biochemical consequences of the disease causing mutations of muscle carnitine palmitoyltransferase II (CPT II) deficiency are still enigmatic. Therefore, CPT II was characterized in muscle biopsies of nine patients with genetically proven muscle CPT II deficiency. Total CPT activity (CPT I+CPT II) of patients was not significantly different from that of controls. Remaining activities upon inhibition by malonyl-CoA and Triton X-100 were significantly reduced in patients. Immunohistochemically CPT II protein was predominantly expressed in type-I-fibers with the same intensity in patients as in controls. Western blot showed the same CPT II staining intensity ratio in patients and controls. CPT I and CPT II protein concentrations estimated by ELISA were not significantly different in patients and in controls. Citrate synthase activity in patients was significantly increased. Total CPT activity significantly correlated with both CPT I and CPT II protein concentrations in patients and controls. This implies (i) that normal total CPT activity in patients with muscle CPT II deficiency is not due to compensatory increase of CPT I activity and that (ii) the mutant CPT II is enzymatically active. The data further support the notion that in muscle CPT II deficiency enzyme activity and protein content are not reduced, but rather abnormally inhibited when fatty acid metabolism is stressed.

Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation.

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

Differential effects of octreotide and pasireotide on somatostatin receptor internalization and trafficking in vitro.

OBJECTIVE: The clinically used somatostatin analogs, octreotide and lanreotide, act primarily by binding to somatostatin receptor 2 (sst2). In contrast, the novel multireceptor ligand pasireotide (SOM230) binds with high affinity to somatostatin receptor subtypes sst1, sst2, sst3, and sst5. SOM230 is currently under clinical evaluation for treatment of acromegaly, Cushing's disease, and octreotide-resistant carcinoid tumors. However, the effects of SOM230 on internalization and postendosomal sorting of individual human somatostatin receptor subtypes have not been determined so far. RESULTS: Here we show that SOM230 was less potent than octreotide in inducing internalization and signaling of sst2 receptors expressed in human embryonic kidney cells. In contrast, SOM230 was more potent than octreotide in inducing internalization and signaling of sst3 and sst5 receptors. Both SOM230 and octreotide stimulated a rapid down-regulation of sst3 but not of sst2 or sst5 receptors. SOM230 and octreotide profoundly differed in their patterns of sst2-stimulated beta-arrestin mobilization. Whereas octreotide-mediated receptor activation led to the formation of stable complexes facilitating the internalization of sst2 and beta-arrestin-2 into the same endocytic vesicles, SOM230-mediated receptor activation led to the formation of unstable complexes that dissociated at or near the plasma membrane. Consequently, sst2 receptors recycled rapidly to the plasma membrane after endocytosis in SOM230-treated cells, but not in octreotide-treated cells. CONCLUSION: We show that SOM230 modulates somatostatin receptor trafficking in a manner clearly distinct from octreotide and somatostatin. These findings may provide an explanation for the differential regulation of somatostatin receptor responsiveness during long-term administration of stable somatostatin analogs.