Search

Abstract Objective COASY, the gene encoding the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of cellular de novo coenzyme A (CoA) biosynthesis, has been linked to two exceedingly rare autosomal recessive disorders, such as COASY protein‐associated neurodegeneration (CoPAN), a form of neurodegeneration with brain iron accumulation (NBIA), and pontocerebellar hypoplasia type 12 (PCH12). We aimed to expand the phenotypic spectrum and gain insights into the pathogenesis of COASY‐related disorders. Methods Patients were identified through targeted or exome sequencing. To unravel the molecular mechanisms of disease, RNA sequencing, bioenergetic analysis, and quantification of critical proteins were performed on fibroblasts. Results We identified five new individuals harboring novel COASY variants. While one case exhibited classical CoPAN features, the others displayed atypical symptoms such as deafness, language and autism spectrum disorders, brain atrophy, and microcephaly. All patients experienced epilepsy, highlighting its potential frequency in COASY‐related disorders. Fibroblast transcriptomic profiling unveiled dysregulated expression in genes associated with mitochondrial respiration, responses to oxidative stress, transmembrane transport, various cellular signaling pathways, and protein translation, modification, and trafficking. Bioenergetic analysis revealed impaired mitochondrial oxygen consumption in COASY fibroblasts. Despite comparable total CoA levels to control cells, the amounts of mitochondrial 4′‐phosphopantetheinylated proteins were significantly reduced in COASY patients. Interpretation These results not only extend the clinical phenotype associated with COASY variants but also suggest a continuum between CoPAN and PCH12. The intricate interplay of altered cellular processes and signaling pathways provides valuable insights for further research into the pathogenesis of COASY‐associated diseases.

Coenzyme A (CoA), which is widely distributed and vital for cellular metabolism, is a critical molecule essential in both synthesizing and breaking down key energy sources in the body. Inborn errors of metabolism in the cellular de novo biosynthetic pathway of CoA have been linked to human genetic disorders, emphasizing the importance of this pathway. The COASY gene encodes the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of the CoA biosynthetic pathway and serves as one of the rate-limiting components of the pathway. Recessive variants of this gene cause an exceptionally rare and devastating disease called COASY protein-associated neurodegeneration (CoPAN) while complete loss-of-function variants in COASY have been identified in fetuses/neonates with Pontocerebellar Hypoplasia type 12 (PCH 12). Understanding why the different symptoms emerge in these disorders and what determines the development of one syndrome over the other is still not achieved. To shed light on the pathogenesis, we generated a new conditional animal model in which Coasy was deleted under the control of the human GFAP promoter. We used this mouse model to investigate how defects in the CoA biosynthetic pathway affect brain development. This model showed a broad spectrum of severity of the in vivo phenotype, ranging from very short survival (less than 2 weeks) to normal life expectancy in some animals. Surviving mice displayed a behavioral phenotype with sensorimotor defects. Ex vivo histological analysis revealed variable but consistent cerebral and cerebellar cortical hypoplasia, in parallel with a broad astrocytic hyper-proliferation in the cerebral cortex. In addition, primary astrocytes derived from this model exhibited lipid peroxidation, iron dyshomeostasis, and impaired mitochondrial respiration. Notably, Coasy ablation in radial glia and astrocytic lineage triggers abnormal neuronal development and chronic neuroinflammation, offering new insights into disease mechanisms.

Summary: Here, we present a protocol describing the quantification of oxygen consumption rate (OCR) and maximal respiration rate (MRR) in living induced pluripotent stem cell (iPSC)-derived neurons using the Seahorse analyzer. We guide you through the whole process: culture amplification and seeding of neural progenitor cells (NPCs), their differentiation into neurons, and normalization of the results to cell number in the analytical phase. The assessment of cellular mitochondrial function, by analyzing mitochondrial respiration, could be useful in various diseases as well as in drug screening.For complete details on the use and execution of this protocol, please refer to Aleo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Leber hereditary optic neuropathy (LHON) is one of the most common mitochondrial illness, causing retinal ganglion cell degeneration and central vision loss. It stems from point mutations in mitochondrial DNA (mtDNA), with key mutations being m.3460G > A, m.11778G > A, and m.14484 T > C. Fibroblasts from identical twins, sharing m.14484 T > C and m.10680G > A variants each with 70 % heteroplasmy, were used to generate iPSC lines. Remarkably, one twin, a LHON patient, displayed symptoms, while the other, a carrier, remained asymptomatic. These iPSCs offer a valuable tool for studying factors influencing disease penetrance and unravelling the role of m.10680G > A, which is still debated.

Mitochondrial membrane Protein-Associated Neurodegeneration (MPAN) is a lethal neurodegenerative disorder caused by mutations in the human gene C19orf12. The molecular mechanisms underlying the disorder are still unclear, and no established therapy is available. Here, we describe the generation and characterization of two human induced pluripotent stem cell (iPSC) lines derived from skin fibroblasts of two MPAN patients carrying homozygous recessive mutations in C19orf12. These iPSC lines represent a useful resource for future investigations on the pathology of MPAN, as well as for the development of successful treatments.

Abstract Neurodegeneration associated with defective pantothenate kinase-2 (PKAN) is an early-onset monogenic autosomal-recessive disorder. The hallmark of the disease is the massive accumulation of iron in the globus pallidus brain region of patients. PKAN is caused by mutations in the PANK2 gene encoding the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway. To date, the way in which this alteration leads to brain iron accumulation has not been elucidated. Starting from previously obtained hiPS clones, we set up a differentiation protocol able to generate inhibitory neurons. We obtained striatal-like medium spiny neurons composed of approximately 70–80% GABAergic neurons and 10–20% glial cells. Within this mixed population, we detected iron deposition in both PKAN cell types, however, the viability of PKAN GABAergic neurons was strongly affected. CoA treatment was able to reduce cell death and, notably, iron overload. Further differentiation of hiPS clones in a pure population of astrocytes showed particularly evident iron accumulation, with approximately 50% of cells positive for Perls staining. The analysis of these PKAN astrocytes indicated alterations in iron metabolism, mitochondrial morphology, respiratory activity, and oxidative status. Moreover, PKAN astrocytes showed signs of ferroptosis and were prone to developing a stellate phenotype, thus gaining neurotoxic features. This characteristic was confirmed in iPS-derived astrocyte and glutamatergic neuron cocultures, in which PKAN glutamatergic neurons were less viable in the presence of PKAN astrocytes. This newly generated astrocyte model is the first in vitro disease model recapitulating the human phenotype and can be exploited to deeply clarify the pathogenetic mechanisms underlying the disease.

PKAN disease is caused by mutations in the PANK2 gene, encoding the mitochondrial enzyme pantothenate kinase 2, catalyzing the first and key reaction in Coenzyme A (CoA) biosynthetic process. This disorder is characterized by progressive neurodegeneration and excessive iron deposition in the brain. The pathogenic mechanisms of PKAN are still unclear, and the available therapies are only symptomatic. Although iron accumulation is a hallmark of PKAN, its relationship with CoA dysfunction is not clear. We have previously developed hiPS-derived astrocytes from PKAN patients showing iron overload, thus recapitulating the human phenotype. In this work, we demonstrated that PKAN astrocytes presented an increase in transferrin uptake, a key route for cellular iron intake via transferrin receptor-mediated endocytosis of transferrin-bound iron. Investigation of constitutive exo-endocytosis and vesicular dynamics, exploiting the activity-enriching biosensor SynaptoZip, led to the finding of a general impairment in the constitutive endosomal trafficking in PKAN astrocytes. CoA and 4-phenylbutyric acid treatments were found to be effective in partially rescuing the aberrant vesicular behavior and iron intake. Our results demonstrate that the impairment of CoA biosynthesis could interfere with pivotal intracellular mechanisms involved in membrane fusions and vesicular trafficking, leading to an aberrant transferrin receptor-mediated iron uptake.

Coenzyme A (CoA) is a vital and ubiquitous cofactor required in a vast number of enzymatic reactions and cellular processes. To date, four rare human inborn errors of CoA biosynthesis have been described. These disorders have distinct symptoms, although all stem from variants in genes that encode enzymes involved in the same metabolic process. The first and last enzymes catalyzing the CoA biosynthetic pathway are associated with two neurological conditions, namely pantothenate kinase-associated neurodegeneration (PKAN) and COASY protein-associated neurodegeneration (CoPAN), which belong to the heterogeneous group of neurodegenerations with brain iron accumulation (NBIA), while the second and third enzymes are linked to a rapidly fatal dilated cardiomyopathy. There is still limited information about the pathogenesis of these diseases, and the knowledge gaps need to be resolved in order to develop potential therapeutic approaches. This review aims to provide a summary of CoA metabolism and functions, and a comprehensive overview of what is currently known about disorders associated with its biosynthesis, including available preclinical models, proposed pathomechanisms, and potential therapeutic approaches.

Mitochondrial diseases (MDs) are inherited genetic conditions characterized by pathogenic mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Current therapies are still far from being fully effective and from covering the broad spectrum of mutations in mtDNA. For example, unlike heteroplasmic conditions, MDs caused by homoplasmic mtDNA mutations do not yet benefit from advances in molecular approaches. An attractive method of providing dysfunctional cells and/or tissues with healthy mitochondria is mitochondrial transplantation. In this review, we discuss what is known about intercellular transfer of mitochondria and the methods used to transfer mitochondria both in vitro and in vivo, and we provide an outlook on future therapeutic applications. Overall, the transfer of healthy mitochondria containing wild-type mtDNA copies could induce a heteroplasmic shift even when homoplasmic mtDNA variants are present, with the aim of attenuating or preventing the progression of pathological clinical phenotypes. In summary, mitochondrial transplantation is a challenging but potentially ground-breaking option for the treatment of various mitochondrial pathologies, although several questions remain to be addressed before its application in mitochondrial medicine.

The novel brain-penetrant peroxisome proliferator-activated receptor gamma agonist leriglitazone, previously validated for other rare neurodegenerative diseases, is a small molecule that acts as a regulator of mitochondrial function and exerts neuroprotective, anti-oxidative and anti-inflammatory effects. Herein, we tested whether leriglitazone can be effective in ameliorating the mitochondrial defects that characterize an hiPS-derived model of Pantothenate kinase-2 associated Neurodegeneration (PKAN). PKAN is caused by a genetic alteration in the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway, and for which no effective cure is available. The PKAN hiPS-derived astrocytes are characterized by mitochondrial dysfunction, cytosolic iron deposition, oxidative stress and neurotoxicity. We monitored the effect of leriglitazone in comparison with CoA on hiPS-derived astrocytes from three healthy subjects and three PKAN patients. The treatment with leriglitazone did not affect the differentiation of the neuronal precursor cells into astrocytes, and it improved the viability of PKAN cells and their respiratory activity, while diminishing the iron accumulation similarly or even better than CoA. The data suggest that leriglitazone is well tolerated in this cellular model and could be considered a beneficial therapeutic approach in the treatment of PKAN.

More than 30 years after discovering Leber's hereditary optic neuropathy (LHON) as the first maternally inherited disease associated with homoplasmic mtDNA mutations, we still struggle to achieve effective therapies. LHON is characterized by selective degeneration of retinal ganglion cells (RGCs) and is the most frequent mitochondrial disease, which leads young people to blindness, in particular males. Despite that causative mutations are present in all tissues, only a specific cell type is affected. Our deep understanding of the pathogenic mechanisms in LHON is hampered by the lack of appropriate models since investigations have been traditionally performed in non-neuronal cells. Effective in-vitro models of LHON are now emerging, casting promise to speed our understanding of pathophysiology and test therapeutic strategies to accelerate translation into clinic. We here review the potentials of these new models and their impact on the future of LHON patients.

Abstract Background Complex I (CI or NADH:ubiquinone oxidoreductase) deficiency is the most frequent cause of mitochondrial respiratory chain defect. Successful attempts to rescue CI function by introducing an exogenous NADH dehydrogenase, such as the NDI1 from Saccharomyces cerevisiae (ScNDI1), have been reported although with drawbacks related to competition with CI. In contrast to ScNDI1, which is permanently active in yeast naturally devoid of CI, plant alternative NADH dehydrogenases (NDH-2) support the oxidation of NADH only when the CI is metabolically inactive and conceivably when the concentration of matrix NADH exceeds a certain threshold. We therefore explored the feasibility of CI rescue by NDH-2 from Arabidopsis thaliana (At) in human CI defective fibroblasts. Results We showed that, other than ScNDI1, two different NDH-2 (AtNDA2 and AtNDB4) targeted to the mitochondria were able to rescue CI deficiency and decrease oxidative stress as indicated by a normalization of SOD activity in human CI-defective fibroblasts. We further demonstrated that when expressed in human control fibroblasts, AtNDA2 shows an affinity for NADH oxidation similar to that of CI, thus competing with CI for the oxidation of NADH as opposed to our initial hypothesis. This competition reduced the amount of ATP produced per oxygen atom reduced to water by half in control cells. Conclusions In conclusion, despite their promising potential to rescue CI defects, due to a possible competition with remaining CI activity, plant NDH-2 should be regarded with caution as potential therapeutic tools for human mitochondrial diseases.

Mitochondrial diseases (MDs) are a group of severe genetic disorders caused by mutations in the nuclear or mitochondrial genome encoding proteins involved in the oxidative phosphorylation (OXPHOS) system. MDs have a wide range of symptoms, ranging from organ-specific to multisystemic dysfunctions, with different clinical outcomes. The lack of natural history information, the limits of currently available preclinical models, and the wide range of phenotypic presentations seen in MD patients have all hampered the development of effective therapies. The growing number of pre-clinical and clinical trials over the last decade has shown that gene therapy is a viable precision medicine option for treating MD. However, several obstacles must be overcome, including vector design, targeted tissue tropism and efficient delivery, transgene expression, and immunotoxicity. This manuscript offers a comprehensive overview of the state of the art of gene therapy in MD, addressing the main challenges, the most feasible solutions, and the future perspectives of the field.

Pearson marrow pancreas syndrome (PMPS) is a sporadic mitochondrial disease, resulting from the clonal expansion of a mutated mitochondrial DNA (mtDNA) molecule bearing a macro-deletion, and therefore missing essential genetic information. PMPS is characterized by the presence of deleted (Δ) mtDNA that co-exist with the presence of a variable amount of wild-type mtDNA, a condition termed heteroplasmy. All tissues of the affected individual, including the haemopoietic system and the post-mitotic, highly specialized tissues (brain, skeletal muscle, and heart) contain the large-scale mtDNA deletion in variable amount. We generated human induced pluripotent stem cells (hiPSCs) from two PMPS patients, carrying different type of large-scale deletion.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1 protein, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. EE is usually a fatal disease with a severe clinical course mainly associated with developmental delay and regression, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhoea. Treatment includes antioxidants, antibiotics that lower H2S levels and antispastic medications, which are not curative. The mutations causing absence of the ETHE1 protein, as is the case for the described patient, usually entail a severe fatal phenotype. Although there are rare reported cases with mild clinical findings, the mechanism leading to these milder cases is also unclear. Here, we describe an 11-year-old boy with an ETHE1 gene mutation who has no neurocognitive impairment but chronic diarrhoea, which is controlled by oral medical treatment, and progressive spastic paraparesis that responded to Achilles tendon lengthening.

Leber’s Hereditary Optic Neuropathy (LHON) is a maternally inherited disorder caused by homoplasmic mutations of mitochondrial DNA (mtDNA). LHON is characterized by the selective degeneration of the retinal ganglion cells (RGC). Almost all LHON maternal lineages are homoplasmic mutant (100% mtDNA copies are mutant) for one of three frequent mtDNA mutations now found in over 90% of patients worldwide (m.11778G > A/MT-ND4, m.3460G > A/MT-ND1, m.14484 T > C/MT-ND6). Human induced pluripotent stem cells (hiPSCs) were generated from a patient carrying the homoplasmic m.3460G > A/MT-ND1 mutation using the Sendai virus non-integrating virus.

Summary: Dysfunctions in mitochondrial dynamics and metabolism are common pathological processes associated with Parkinson’s disease (PD). It was recently shown that an inherited form of PD and dementia is caused by mutations in the OPA1 gene, which encodes for a key player in mitochondrial fusion and structure. iPSC-derived neural cells from these patients exhibited severe mitochondrial fragmentation, respiration impairment, ATP deficits, and heightened oxidative stress. Reconstitution of normal levels of OPA1 in PD-derived neural cells normalized mitochondria morphology and function. OPA1-mutated neuronal cultures showed reduced survival in vitro. Intriguingly, selective inhibition of necroptosis effectively rescued this survival deficit. Additionally, dampening necroptosis in MPTP-treated mice protected from DA neuronal cell loss. This human iPSC-based model captures both early pathological events in OPA1 mutant neural cells and the beneficial effects of blocking necroptosis, highlighting this cell death process as a potential therapeutic target for PD.

Genome sequencing alone fails to provide a genetic diagnosis for many Mendelian disorder patients. Here, the authors utilize RNA sequencing to complement genotyping of patients with a rare mitochondrial disease by detecting aberrant RNA expression, splicing and allele-specific expression.

The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

COASY protein-associated neurodegeneration (CoPAN) is a rare but devastating genetic autosomal recessive disorder of inborn error of CoA metabolism, which shares with pantothenate kinase-associated neurodegeneration (PKAN) similar features, such as dystonia, parkinsonian traits, cognitive impairment, axonal neuropathy, and brain iron accumulation. These two disorders are part of the big group of neurodegenerations with brain iron accumulation (NBIA) for which no effective treatment is available at the moment. To date, the lack of a mammalian model, fully recapitulating the human disorder, has prevented the elucidation of pathogenesis and the development of therapeutic approaches. To gain new insights into the mechanisms linking CoA metabolism, iron dyshomeostasis, and neurodegeneration, we generated and characterized the first CoPAN disease mammalian model. Since CoA is a crucial metabolite, constitutive ablation of the Coasy gene is incompatible with life. On the contrary, a conditional neuronal-specific Coasy knock-out mouse model consistently developed a severe early onset neurological phenotype characterized by sensorimotor defects and dystonia-like movements, leading to premature death. For the first time, we highlighted defective brain iron homeostasis, elevation of iron, calcium, and magnesium, together with mitochondrial dysfunction. Surprisingly, total brain CoA levels were unchanged, and no signs of neurodegeneration were present.

Abstract Coenzyme Q (CoQ) is an electron acceptor for sulfide‐quinone reductase (SQR), the first enzyme of the hydrogen sulfide oxidation pathway. Here, we show that lack of CoQ in human skin fibroblasts causes impairment of hydrogen sulfide oxidation, proportional to the residual levels of CoQ. Biochemical and molecular abnormalities are rescued by CoQ supplementation in vitro and recapitulated by pharmacological inhibition of CoQ biosynthesis in skin fibroblasts and ADCK3 depletion in HeLa cells. Kidneys of Pdss2kd/kd mice, which only have ~15% residual CoQ concentrations and are clinically affected, showed (i) reduced protein levels of SQR and downstream enzymes, (ii) accumulation of hydrogen sulfides, and (iii) glutathione depletion. These abnormalities were not present in brain, which maintains ~30% residual CoQ and is clinically unaffected. In Pdss2kd/kd mice, we also observed low levels of plasma and urine thiosulfate and increased blood C4‐C6 acylcarnitines. We propose that impairment of the sulfide oxidation pathway induced by decreased levels of CoQ causes accumulation of sulfides and consequent inhibition of short‐chain acyl‐CoA dehydrogenase and glutathione depletion, which contributes to increased oxidative stress and kidney failure.

Abstract Pantothenate kinase‐associated neurodegeneration (PKAN) is an early onset and severely disabling neurodegenerative disease for which no therapy is available. PKAN is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyze the first limiting step of Coenzyme A (CoA) biosynthesis. We generated induced pluripotent stem cells from PKAN patients and showed that their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions—including impairment of mitochondrial iron‐dependent biosynthesis—and major membrane excitability defects. CoA supplementation prevented neuronal death and ROS formation by restoring mitochondrial and neuronal functionality. Our findings provide direct evidence that PANK2 malfunctioning is responsible for abnormal phenotypes in human neuronal cells and indicate CoA treatment as a possible therapeutic intervention.

Karin Tuschl, Philippa Mills and colleagues report mutations in the manganese (Mn) transporter gene SLC39A14in childhood-onset parkinsonism-dystonia. Using functional recapitulation, the authors also show that slc39A14 loss-of-function in zebrafish can lead to Mn dysregulation and locomotor impairment.

Primary mitochondrial diseases (PMD) refer to a group of severe, often inherited genetic conditions due to mutations in the mitochondrial genome or in the nuclear genes encoding for proteins involved in oxidative phosphorylation (OXPHOS). The mutations hamper the last step of aerobic metabolism, affecting the primary source of cellular ATP synthesis. Mitochondrial diseases are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. The limited information of the natural history, the limitations of currently available preclinical models, coupled with the large variability of phenotypical presentations of PMD patients, have strongly penalized the development of effective therapies. However, new therapeutic strategies have been emerging, often with promising preclinical and clinical results. Here we review the state of the art on experimental treatments for mitochondrial diseases, presenting “one-size-fits-all” approaches and precision medicine strategies. Finally, we propose novel perspective therapeutic plans, either based on preclinical studies or currently used for other genetic or metabolic diseases that could be transferred to PMD.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Pantothenate kinase-associated neurodegeneration is an early onset autosomal recessive movement disorder caused by mutation of the pantothenate kinase-2 gene, which encodes a mitochondrial enzyme involved in coenzyme A synthesis. The disorder is characterised by high iron levels in the brain, although the pathological mechanism leading to this accumulation is unknown. To address this question, we tested primary skin fibroblasts from three patients and three healthy subjects, as well as neurons induced by direct fibroblast reprogramming, for oxidative status, mitochondrial functionality and iron parameters. The patients' fibroblasts showed altered oxidative status, reduced antioxidant defence, and impaired cytosolic and mitochondrial aconitase activities compared to control cells. Mitochondrial iron homeostasis and functionality analysis of patient fibroblasts indicated increased labile iron pool content and reactive oxygen species development, altered mitochondrial shape, decreased membrane potential and reduced ATP levels. Furthermore, analysis of induced neurons, performed at a single cell level, confirmed some of the results obtained in fibroblasts, indicating an altered oxidative status and signs of mitochondrial dysfunction, possibly due to iron mishandling. Thus, for the first time, altered biological processes have been identified in vitro in live diseased neurons. Moreover, the obtained induced neurons can be considered a suitable human neuronal model for the identification of candidate therapeutic compounds for this disease.

Abstract Mitochondrial disorders are a group of highly invalidating human conditions for which effective treatment is currently unavailable and characterized by faulty energy supply due to defective oxidative phosphorylation (OXPHOS). Given the complexity of mitochondrial genetics and biochemistry, mitochondrial inherited diseases may present with a vast range of symptoms, organ involvement, severity, age of onset, and outcome. Despite the wide spectrum of clinical signs and biochemical underpinnings of this group of dis‐orders, some common traits can be identified, based on both pathogenic mechanisms and potential therapeutic approaches. Here, we will review two peculiar mitochondrial disorders, ethylmalonic encephalopathy (EE) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), caused by mutations in the ETHE1 and TYMP nuclear genes, respectively. ETHE1 encodes for a mitochondrial enzyme involved in sulfide detoxification and TYMP for a cytosolic enzyme involved in the thymidine/deoxyuridine catabolic pathway. We will discuss these two clinical entities as a paradigm of mitochondrial diseases caused by the accumulation of compounds normally present in traces, which exerts a toxic and inhibitory effect on the OXPHOS system.

Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA), namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases.

Neurodegeneration with brain iron accumulation (NBIA) is a set of neurodegenerative disorders, which includes very rare monogenetic diseases. They are heterogeneous in regard to the onset and the clinical symptoms, while the have in common a specific brain iron deposition in the region of the basal ganglia that can be visualized by radiological and histopathological examinations. Nowadays, 15 genes have been identified as causative for NBIA, of which only two code for iron-proteins, while all the other causative genes codify for proteins not involved in iron management. Thus, how iron participates to the pathogenetic mechanism of most NBIA remains unclear, essentially for the lack of experimental models that fully recapitulate the human phenotype. In this review we reported the recent data on new models of these disorders aimed at highlight the still scarce knowledge of the pathogenesis of iron deposition.

ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.

Mitochondrial respiratory chain (RC) disorders are a group of genetically and clinically heterogeneous diseases. This is because protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis and maintenance of mitochondria, including mitochondrial DNA (mtDNA) replication, transcription, and translation, require nuclear-encoded genes. In the past decade, a growing number of syndromes associated with dysfunction of mtDNA translation have been reported. This paper reviews the current knowledge of mutations affecting mitochondrial aminoacyl tRNAs synthetases and their role in the pathogenic mechanisms underlying the different clinical presentations.

Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nDNA. Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. To investigate how the cellular transcriptome is modified in response to the absence of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene expression profile of two human cell lines, 143BTK(-) and A549, which had been entirely depleted of mtDNA (rho(o) cells), and compared it with that of corresponding undepleted parental cells (rho(+) cells).Our data indicate that absence of mtDNA is associated with: i) a down-regulation of cell cycle control genes and a reduction of cell replication rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the respiratory chain and iii) a down-regulation of a gene described as the human homolog of ELAC2 of E. coli, which encodes a protein that we show to also target to the mitochondrial compartment.Our results indicate a strong correlation between mitochondrial biogenesis and cell cycle control and suggest that some proteins could have a double role: for instance in controlling both cell cycle progression and mitochondrial functions. In addition, the finding that ELAC2 and maybe other transcripts that are located into mitochondria, are down-regulated in rho(o) cells, make them good candidates for human disorders associated with defective replication and expression of mtDNA.