Adriana P. Rebelo

PNPLA6 mutations cause Boucher-Neuhäuser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum

Boucher-Neuhäuser and Gordon Holmes syndromes are clinical syndromes defined by early-onset ataxia and hypogonadism plus chorioretinal dystrophy (Boucher-Neuhäuser syndrome) or brisk reflexes (Gordon Holmes syndrome). Here we uncover the genetic basis of these two syndromes, demonstrating that both clinically distinct entities are allelic for recessive mutations in the gene PNPLA6. In five of seven Boucher-Neuhäuser syndrome/Gordon Holmes syndrome families, we identified nine rare conserved and damaging mutations by applying whole exome sequencing. Further, by dissecting the complex clinical presentation of Boucher-Neuhäuser syndrome and Gordon Holmes syndrome into its neurological system components, we set out to analyse an additional 538 exomes from families with ataxia (with and without hypogonadism), pure and complex hereditary spastic paraplegia, and Charcot-Marie-Tooth disease type 2. We identified four additional PNPLA6 mutations in spastic ataxia and hereditary spastic paraplegia families, revealing that Boucher-Neuhäuser and Gordon Holmes syndromes in fact represent phenotypic clusters on a spectrum of neurodegenerative diseases caused by mutations in PNPLA6. Structural analysis indicates that the majority of mutations falls in the C-terminal phospholipid esterase domain and likely inhibits the catalytic activity of PNPLA6, which provides the precursor for biosynthesis of the neurotransmitter acetylcholine. Our findings show that PNPLA6 influences a manifold of neuronal systems, from the retina to the cerebellum, upper and lower motor neurons and the neuroendocrine system, with damage of this protein causing an extraordinarily broad continuous spectrum of associated neurodegenerative disease.

Mutations in the ER-shaping protein reticulon 2 cause the axon-degenerative disorder hereditary spastic paraplegia type 12

Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative conditions. They are characterized by progressive spastic paralysis of the legs as a result of selective, length-dependent degeneration of the axons of the corticospinal tract. Mutations in 3 genes encoding proteins that work together to shape the ER into sheets and tubules - receptor accessory protein 1 (REEP1), atlastin-1 (ATL1), and spastin (SPAST) - have been found to underlie many cases of HSP in Northern Europe and North America. Applying Sanger and exome sequencing, we have now identified 3 mutations in reticulon 2 (RTN2), which encodes a member of the reticulon family of prototypic ER-shaping proteins, in families with spastic paraplegia 12 (SPG12). These autosomal dominant mutations included a complete deletion of RTN2 and a frameshift mutation predicted to produce a highly truncated protein. Wild-type reticulon 2, but not the truncated protein potentially encoded by the frameshift allele, localized to the ER. RTN2 interacted with spastin, and this interaction required a hydrophobic region in spastin that is involved in ER localization and that is predicted to form a curvature-inducing/sensing hairpin loop domain. Our results directly implicate a reticulon protein in axonopathy, show that this protein participates in a network of interactions among HSP proteins involved in ER shaping, and further support the hypothesis that abnormal ER morphogenesis is a pathogenic mechanism in HSP.

In vivo methylation of mtDNA reveals the dynamics of protein–mtDNA interactions

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.

The role of PGC-1 coactivators in aging skeletal muscle and heart

Aging is the progressive decline in cellular, tissue, and organ function. This complex process often manifests as loss of muscular strength, cardiovascular function, and cognitive ability. Mitochondrial dysfunction and decreased mitochondrial biogenesis are believed to participate in metabolic abnormalities and loss of organ function, which will eventually contribute to aging and decreased lifespan. In this review, we discuss what is currently known about mitochondrial dysfunction in the aging skeletal muscle and heart. We focused our discussion on the role of PGC‐1 coactivators in the regulation of mitochondrial biogenesis and function and possible therapeutic benefits of increased mitochondrial biogenesis in compensating for mitochondrial dysfunction and circumventing aging and aging‐related diseases.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

Mitochondrial DNA transcription regulation and nucleoid organization

Mitochondrial biogenesis is a complex process depending on both nuclear and mitochondrial DNA (mtDNA) transcription regulation to tightly coordinate mitochondrial levels and the cell’s energy demand. The energy requirements for a cell to support its metabolic function can change in response to varying physiological conditions, such as, proliferation and differentiation. Therefore, mitochondrial transcription regulation is constantly being modulated in order to establish efficient mitochondrial oxidative metabolism and proper cellular function. The aim of this article is to review the function of major protein factors that are directly involved in the process of mtDNA transcription regulation, as well as, the importance of mitochondrial nucleoid structure and its influence on mtDNA segregation and transcription regulation. Here, we discuss the current knowledge on the molecular mode of action of transcription factors comprising the mitochondrial transcriptional machinery, as well as the action of nuclear receptors on regulatory regions of the mtDNA.

Pathophysiology and fate of hepatocytes in a mouse model of mitochondrial hepatopathies

Background: Although oxidative phosphorylation defects can affect the liver, these conditions are poorly understood, partially because of the lack of animal models. Aims: To create and characterise the pathophysiology of mitochondrial hepatopathies in a mouse model. Methods: A mouse model of mitochondrial hepatopathies was created by the conditional liver knockout (KO) of the COX10 gene, which is required for cytochrome c oxidase (COX) function. The onset and progression of biochemical, molecular and clinical phenotypes were analysed in several groups of animals, mostly at postnatal days 23, 56, 78 and 155. Results: Biochemical and histochemical analysis of liver samples from 23–56-day-old KO mice showed liver dysfunction, a severe COX deficiency, marked mitochondrial proliferation and lipid accumulation. Despite these defects, the COX-deficient hepatocytes were not immediately eliminated, and apoptosis followed by liver regeneration could be observed only at age 78 days. Hepatocytes from 56–78-day-old KO mice survived despite very low COX activity but showed a progressive depletion of glycogen stores. In most animals, hepatocytes that escaped COX10 ablation were able to proliferate and completely regenerate the liver between days 78 and 155. Conclusions: The results showed that when faced with a severe oxidative phosphorylation defect, hepatocytes in vivo can rely on glycolysis/glycogenolysis for their bioenergetic needs for relatively long periods. Ultimately, defective hepatocytes undergo apoptosis and are replaced by COX-positive cells first observed in the perivascular regions.

Motor protein mutations cause a new form of hereditary spastic paraplegia

Objective: To identify a novel disease gene in 2 families with autosomal recessive hereditary spastic paraplegia (HSP). Methods: We used whole-exome sequencing to identify the underlying genetic disease cause in 2 families with apparently autosomal recessive spastic paraplegia. Endogenous expression as well as subcellular localization of wild-type and mutant protein were studied to support the pathogenicity of the identified mutations. Results: In 2 families, we identified compound heterozygous or homozygous mutations in the kinesin gene KIF1C to cause hereditary spastic paraplegia type 58 (SPG58). SPG58 can be complicated by cervical dystonia and cerebellar ataxia. The same mutations in a heterozygous state result in a mild or subclinical phenotype. KIF1C mutations in SPG58 affect the domains involved in adenosine triphosphate hydrolysis and microtubule binding, key functions for this microtubule-based motor protein. Conclusions: KIF1C is the third kinesin gene involved in the pathogenesis of HSPs and is characterized by a mild dominant and a more severe recessive disease phenotype. The identification of KIF1C as an HSP disease gene further supports the key role of intracellular trafficking processes in the pathogenesis of hereditary axonopathies.

Characterization of the mitofusin 2 R94W mutation in a knock-in mouse model.

Charcot‐Marie‐Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre‐clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock‐in mouse model expressing Mfn2R94W, which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2R94W heterozygous mice show histopathology and age‐dependent open‐field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.