Novel phosphopantothenoylcysteine synthetase (PPCS) mutations with prominent neuromuscular features: Expanding the phenotypical spectrum of PPCS-related disorders.

Biallelic pathogenic variants in phosphopantothenoylcysteine synthetase, PPCS, are a rare cause of a severe early-onset dilated cardiomyopathy with high morbidity and mortality. To date, only five individuals with PPCS-mutations have been reported. Here, we report a female infant who presented in the neonatal period with hypotonia, a necrotizing myopathy with intermittent rhabdomyolysis and other extracardiac manifestations before developing a progressive and ultimately fatal dilated cardiomyopathy. Gene agnostic trio genome sequencing revealed two rare variants in the PPCS [MIM: 609853] in trans, a previously reported pathogenic c.320_334del p. (Pro107_Ala111del) variant, and a c.613-3C>G intronic variant of uncertain significance. Functional studies confirmed the likely pathogenicity of this variant. Our case provides clinical and histopathological evidence for an associated neuromuscular phenotype not previously recognized and expands the evolving phenotypic spectrum of PPCS-related disorders. We also performed a literature search of all previously published cases and summarize the common features.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

A comprehensive phenotypic characterization of a whole-body Wdr45 knock-out mouse.

Pathogenic variants in the WDR45 (OMIM: 300,526) gene on chromosome Xp11 are the genetic cause of a rare neurological disorder characterized by increased iron deposition in the basal ganglia. As WDR45 encodes a beta-propeller scaffold protein with a putative role in autophagy, the disease has been named Beta-Propeller Protein-Associated Neurodegeneration (BPAN). BPAN represents one of the four most common forms of Neurodegeneration with Brain Iron Accumulation (NBIA). In the current study, we generated and characterized a whole-body Wdr45 knock-out (KO) mouse model. The model, developed using TALENs, presents a 20-bp deletion in exon 2 of Wdr45. Homozygous females and hemizygous males are viable, proving that systemic depletion of Wdr45 does not impair viability and male fertility in mice. The in-depth phenotypic characterization of the mouse model revealed neuropathology signs at four months of age, neurodegeneration progressing with ageing, hearing and visual impairment, specific haematological alterations, but no brain iron accumulation. Biochemically, Wdr45 KO mice presented with decreased complex I (CI) activity in the brain, suggesting that mitochondrial dysfunction accompanies Wdr45 deficiency. Overall, the systemic Wdr45 KO described here complements the two mouse models previously reported in the literature (PMIDs: 26,000,824, 31,204,559) and represents an additional robust model to investigate the pathophysiology of BPAN and to test therapeutic strategies for the disease.

Loss-of-Function Variants in HOPS Complex Genes VPS16 and VPS41 Cause Early Onset Dystonia Associated with Lysosomal Abnormalities.

OBJECTIVES: The majority of people with suspected genetic dystonia remain undiagnosed after maximal investigation, implying that a number of causative genes have not yet been recognized. We aimed to investigate this paucity of diagnoses. METHODS: We undertook weighted burden analysis of whole-exome sequencing (WES) data from 138 individuals with unresolved generalized dystonia of suspected genetic etiology, followed by additional case-finding from international databases, first for the gene implicated by the burden analysis (VPS16), and then for other functionally related genes. Electron microscopy was performed on patient-derived cells. RESULTS: Analysis revealed a significant burden for VPS16 (Fisher's exact test p value, 6.9 × 109 ). VPS16 encodes a subunit of the homotypic fusion and vacuole protein sorting (HOPS) complex, which plays a key role in autophagosome-lysosome fusion. A total of 18 individuals harboring heterozygous loss-of-function VPS16 variants, and one with a microdeletion, were identified. These individuals experienced early onset progressive dystonia with predominant cervical, bulbar, orofacial, and upper limb involvement. Some patients had a more complex phenotype with additional neuropsychiatric and/or developmental comorbidities. We also identified biallelic loss-of-function variants in VPS41, another HOPS-complex encoding gene, in an individual with infantile-onset generalized dystonia. Electron microscopy of patient-derived lymphocytes and fibroblasts from both patients with VPS16 and VPS41 showed vacuolar abnormalities suggestive of impaired lysosomal function. INTERPRETATION: Our study strongly supports a role for HOPS complex dysfunction in the pathogenesis of dystonia, although variants in different subunits display different phenotypic and inheritance characteristics. ANN NEUROL 2020;88:867-877.

Bi-allelic Truncating Mutations in TANGO2 Cause Infancy-Onset Recurrent Metabolic Crises with Encephalocardiomyopathy.

Molecular diagnosis of mitochondrial disorders is challenging because of extreme clinical and genetic heterogeneity. By exome sequencing, we identified three different bi-allelic truncating mutations in TANGO2 in three unrelated individuals with infancy-onset episodic metabolic crises characterized by encephalopathy, hypoglycemia, rhabdomyolysis, arrhythmias, and laboratory findings suggestive of a defect in mitochondrial fatty acid oxidation. Over the course of the disease, all individuals developed global brain atrophy with cognitive impairment and pyramidal signs. TANGO2 (transport and Golgi organization 2) encodes a protein with a putative function in redistribution of Golgi membranes into the endoplasmic reticulum in Drosophila and a mitochondrial localization has been confirmed in mice. Investigation of palmitate-dependent respiration in mutant fibroblasts showed evidence of a functional defect in mitochondrial ß-oxidation. Our results establish TANGO2 deficiency as a clinically recognizable cause of pediatric disease with multi-organ involvement.

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy.

SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases.

Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts.

BACKGROUND: The combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be challenging. OBJECTIVE: The objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented clinically with a similar phenotype that included neurodevelopmental delay, febrile illness-induced encephalopathy and episodes of rhabdomyolysis, followed by developmental arrest, epilepsy and tetraplegia. METHODS: Whole exome sequencing was used to identify pathogenic variants in the two individuals. Biochemical and cell biological analyses were performed on fibroblasts from these individuals and a yeast two-hybrid analysis was used to assess protein-protein interactions. RESULTS: Probands shared a homozygous TRAPPC2L variant (c.109G>T) resulting in a p.Asp37Tyr missense variant. TRAPPC2L is a component of transport protein particle (TRAPP), a group of multisubunit complexes that function in membrane traffic and autophagy. Studies in patient fibroblasts as well as in a yeast system showed that the p.Asp37Tyr protein was present but not functional and resulted in specific membrane trafficking delays. The human missense mutation and the analogous mutation in the yeast homologue Tca17 ablated the interaction between TRAPPC2L and TRAPPC10/Trs130, a component of the TRAPP II complex. Since TRAPP II activates the GTPase RAB11, we examined the activation state of this protein and found increased levels of the active RAB, correlating with changes in its cellular morphology. CONCLUSIONS: Our study implicates a RAB11 pathway in the aetiology of the TRAPPC2L disorder and has implications for other TRAPP-related disorders with similar phenotypes.

Rare causes of early-onset dystonia-parkinsonism with cognitive impairment: a de novo PSEN-1 mutation.

Mutations in PSEN1 are responsible for familial Alzheimer's disease (FAD) inherited as autosomal dominant trait, but also de novo mutations have been rarely reported in sporadic early-onset dementia cases. Parkinsonism in FAD has been mainly described in advanced disease stages. We characterized a patient presenting with early-onset dystonia-parkinsonism later complicated by dementia and myoclonus. Brain MRI showed signs of iron accumulation in the basal ganglia mimicking neurodegeneration with brain iron accumulation (NBIA) as well as fronto-temporal atrophy. Whole exome sequencing revealed a novel PSEN1 mutation and segregation within the family demonstrated the mutation arose de novo.We suggest considering PSEN1 mutations in cases of dystonia-parkinsonism with positive DAT-Scan, later complicated by progressive cognitive decline and cortical myoclonus even without a dominant family history.

Disturbed mitochondrial and peroxisomal dynamics due to loss of MFF causes Leigh-like encephalopathy, optic atrophy and peripheral neuropathy.

BACKGROUND: Mitochondria are dynamic organelles which undergo continuous fission and fusion to maintain their diverse cellular functions. Components of the fission machinery are partly shared between mitochondria and peroxisomes, and inherited defects in two such components (dynamin-related protein (DRP1) and ganglioside-induced differentiation-associated protein 1 (GDAP1)) have been associated with human disease. Deficiency of a third component (mitochondrial fission factor, MFF) was recently reported in one index patient, rendering MFF another candidate disease gene within the expanding field of mitochondrial and peroxisomal dynamics. Here we investigated three new patients from two families with pathogenic mutations in MFF. METHODS: The patients underwent clinical examination, brain MRI, and biochemical, cytological and molecular analyses, including exome sequencing. RESULTS: The patients became symptomatic within the first year of life, exhibiting seizures, developmental delay and acquired microcephaly. Dysphagia, spasticity and optic and peripheral neuropathy developed subsequently. Brain MRI showed Leigh-like patterns with bilateral changes of the basal ganglia and subthalamic nucleus, suggestive of impaired mitochondrial energy metabolism. However, activities of mitochondrial respiratory chain complexes were found to be normal in skeletal muscle. Exome sequencing revealed three different biallelic loss-of-function variants in MFF in both index cases. Western blot studies of patient-derived fibroblasts indicated normal content of mitochondria and peroxisomes, whereas immunofluorescence staining revealed elongated mitochondria and peroxisomes. Furthermore, increased mitochondrial branching and an abnormal distribution of fission-mediating DRP1 were observed. CONCLUSIONS: Our findings establish MFF loss of function as a cause of disturbed mitochondrial and peroxisomal dynamics associated with early-onset Leigh-like basal ganglia disease. We suggest that, even if laboratory findings are not indicative of mitochondrial or peroxisomal dysfunction, the co-occurrence of optic and/or peripheral neuropathy with seizures warrants genetic testing for MFF mutations.

Absence of an orphan mitochondrial protein, c19orf12, causes a distinct clinical subtype of neurodegeneration with brain iron accumulation.

The disease classification neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive neurodegenerative disorders characterized by brain iron deposits in the basal ganglia. For about half of the cases, the molecular basis is currently unknown. We used homozygosity mapping followed by candidate gene sequencing to identify a homozygous 11 bp deletion in the orphan gene C19orf12. Mutation screening of 23 ideopathic NBIA index cases revealed two mutated alleles in 18 of them, and one loss-of-function mutation is the most prevalent. We also identified compound heterozygous missense mutations in a case initially diagnosed with Parkinson disease at age 49. Psychiatric signs, optic atrophy, and motor axonal neuropathy were common findings. Compared to the most prevalent NBIA subtype, pantothenate kinase associated neurodegeneration (PKAN), individuals with two C19orf12 mutations were older at age of onset and the disease progressed more slowly. A polyclonal antibody against the predicted membrane spanning protein showed a mitochondrial localization. A histopathological examination in a single autopsy case detected Lewy bodies, tangles, spheroids, and tau pathology. The mitochondrial localization together with the immunohistopathological findings suggests a pathomechanistic overlap with common forms of neurodegenerative disorders.

A spatiotemporal proteomic map of human adipogenesis.

White adipocytes function as major energy reservoirs in humans by storing substantial amounts of triglycerides, and their dysfunction is associated with metabolic disorders; however, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, we generate a spatiotemporal proteomic atlas of human adipogenesis, which elucidates cellular remodelling as well as the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipocyte formation. We identify compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogramme branched-chain amino acids and one-carbon metabolism to provide building blocks and reduction equivalents. Additionally, we identify C19orf12 as a differentiation-induced adipocyte lipid droplet protein that interacts with the translocase of the outer membrane complex of lipid droplet-associated mitochondria and regulates adipocyte lipid storage by determining the capacity of mitochondria to metabolize fatty acids. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field.

Emerging variants, unique phenotypes, and transcriptomic signatures: an integrated study of COASY-associated diseases.

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.

Mutation screening of 75 candidate genes in 152 complex I deficiency cases identifies pathogenic variants in 16 genes including NDUFB9.

BACKGROUND: Mitochondrial complex I deficiency is the most common cause of mitochondrial disease in childhood. Identification of the molecular basis is difficult given the clinical and genetic heterogeneity. Most patients lack a molecular definition in routine diagnostics. METHODS: A large-scale mutation screen of 75 candidate genes in 152 patients with complex I deficiency was performed by high-resolution melting curve analysis and Sanger sequencing. The causal role of a new disease allele was confirmed by functional complementation assays. The clinical phenotype of patients carrying mutations was documented using a standardised questionnaire. RESULTS: Causative mutations were detected in 16 genes, 15 of which had previously been associated with complex I deficiency: three mitochondrial DNA genes encoding complex I subunits, two mitochondrial tRNA genes and nuclear DNA genes encoding six complex I subunits and four assembly factors. For the first time, a causal mutation is described in NDUFB9, coding for a complex I subunit, resulting in reduction in NDUFB9 protein and both amount and activity of complex I. These features were rescued by expression of wild-type NDUFB9 in patient-derived fibroblasts. CONCLUSION: Mutant NDUFB9 is a new cause of complex I deficiency. A molecular diagnosis related to complex I deficiency was established in 18% of patients. However, most patients are likely to carry mutations in genes so far not associated with complex I function. The authors conclude that the high degree of genetic heterogeneity in complex I disorders warrants the implementation of unbiased genome-wide strategies for the complete molecular dissection of mitochondrial complex I deficiency.

Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing.

BACKGROUND: Next generation sequencing has become the core technology for gene discovery in rare inherited disorders. However, the interpretation of the numerous sequence variants identified remains challenging. We assessed the application of exome sequencing for diagnostics in complex I deficiency, a disease with vast genetic heterogeneity. METHODS: Ten unrelated individuals with complex I deficiency were selected for exome sequencing and sequential bioinformatic filtering. Cellular rescue experiments were performed to verify pathogenicity of novel disease alleles. RESULTS: The first filter criterion was 'Presence of known pathogenic complex I deficiency variants'. This revealed homozygous mutations in NDUFS3 and ACAD9 in two individuals. A second criterion was 'Presence of two novel potentially pathogenic variants in a structural gene of complex I', which discovered rare variants in NDUFS8 in two unrelated individuals and in NDUFB3 in a third. Expression of wild-type cDNA in mutant cell lines rescued complex I activity and assembly, thus providing a functional validation of their pathogenicity. Using the third criterion 'Presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein', loss-of-function mutations in MTFMT were discovered in two patients. In three patients the molecular genetic correlate remained unclear and follow-up analysis is ongoing. CONCLUSION: Appropriate in silico filtering of exome sequencing data, coupled with functional validation of new disease alleles, is effective in rapidly identifying disease-causative variants in known and new complex I associated disease genes.

Generation of two human iPSC lines, HMGUi004-A and FINCBi004-A, from fibroblasts of MPAN patients carrying pathogenic recessive mutations in the gene C19orf12.

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.

A burning question from the first international BPAN symposium: is restoration of autophagy a promising therapeutic strategy for BPAN?

Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurodegenerative disease associated with severe cognitive and motor deficits. BPAN pathophysiology and phenotypic spectrum are still emerging due to the fact that mutations in the WDR45 (WD repeat domain 45) gene, a regulator of macroautophagy/autophagy, were only identified a decade ago. In the first international symposium dedicated to BPAN, which was held in Lyon, France, a panel of international speakers, including several researchers from the autophagy community, presented their work on human patients, cellular and animal models, carrying WDR45 mutations and their homologs. Autophagy researchers found an opportunity to explore the defective function of autophagy mechanisms associated with WDR45 mutations, which underlie neuronal dysfunction and early death. Importantly, BPAN is one of the few human monogenic neurological diseases targeting a regulator of autophagy, which raises the possibility that it is a relevant model to directly assess the roles of autophagy in neurodegeneration and to develop autophagy restorative therapeutic strategies for more common disorders.Abbreviations: ATG: autophagy related; BPAN: beta-propeller protein-associated neurodegeneration; ER: endoplasmic reticulum; KO: knockout; NBIA: neurodegeneration with brain iron accumulation; PtdIns3P: phosphatidylinositol-3-phosphate; ULK1: unc-51 like autophagy activating kinase 1; WDR45: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting.

Identification of Autophagy as a Functional Target Suitable for the Pharmacological Treatment of Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN) In Vitro.

Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a relentlessly progressive neurodegenerative disorder caused by mutations in the C19orf12 gene. C19orf12 has been implicated in playing a role in lipid metabolism, mitochondrial function, and autophagy, however, the precise functions remain unknown. To identify new robust cellular targets for small compound treatments, we evaluated reported mitochondrial function alterations, cellular signaling, and autophagy in a large cohort of MPAN patients and control fibroblasts. We found no consistent alteration of mitochondrial functions or cellular signaling messengers in MPAN fibroblasts. In contrast, we found that autophagy initiation is consistently impaired in MPAN fibroblasts and show that C19orf12 expression correlates with the amount of LC3 puncta, an autophagy marker. Finally, we screened 14 different autophagy modulators to test which can restore this autophagy defect. Amongst these compounds, carbamazepine, ABT-737, LY294002, oridonin, and paroxetine could restore LC3 puncta in the MPAN fibroblasts, identifying them as novel potential therapeutic compounds to treat MPAN. In summary, our study confirms a role for C19orf12 in autophagy, proposes LC3 puncta as a functionally robust and consistent readout for testing compounds, and pinpoints potential therapeutic compounds for MPAN.

Generation of two human iPSC lines, HMGUi003-A and MRIi028-A, carrying pathogenic biallelic variants in the PPCS gene.

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.

Cellular rescue-assay aids verification of causative DNA-variants in mitochondrial complex I deficiency.

Mitochondrial complex I deficiency is a frequent biochemical condition, causing about one third of respiratory chain disorders. Partly due to the large number of genes necessary for its assembly and function only a small proportion of complex I deficiencies are yet confirmed at the molecular genetic level. Now, next generation sequencing approaches are applied to close the gap between biochemical definition and molecular diagnosis. Nevertheless such approaches result in a long list of novel rare single nucleotide variants. Identifying the causative mutations still remains challenging. Here we describe the identification and functional confirmation of novel NDUFS1 mutations using a cellular rescue-assay. Patient-derived complex I-defective fibroblast cell lines were transduced with wild type and mutant NDUFS1-cDNA and subsequently analyzed on the functional and protein level. We established the pathogenic nature of identified rare variants in four out of five disease alleles. This approach is a valuable add-on in disease genetics and it allows the analysis of the functional consequences of genetic variants in metabolic disorders.

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import.

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.