Publications by authors named "Christopher J Carroll"

28 Publications

  • Page 1 of 1

MED27 Variants Cause Developmental Delay, Dystonia, and Cerebellar Hypoplasia.

Ann Neurol 2021 Apr 8;89(4):828-833. Epub 2021 Feb 8.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.

The Mediator multiprotein complex functions as a regulator of RNA polymerase II-catalyzed gene transcription. In this study, exome sequencing detected biallelic putative disease-causing variants in MED27, encoding Mediator complex subunit 27, in 16 patients from 11 families with a novel neurodevelopmental syndrome. Patient phenotypes are highly homogeneous, including global developmental delay, intellectual disability, axial hypotonia with distal spasticity, dystonic movements, and cerebellar hypoplasia. Seizures and cataracts were noted in severely affected individuals. Identification of multiple patients with biallelic MED27 variants supports the critical role of MED27 in normal human neural development, particularly for the cerebellum. ANN NEUROL 2021;89:828-833.
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http://dx.doi.org/10.1002/ana.26019DOI Listing
April 2021

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.

Nat Commun 2020 11 23;11(1):5927. Epub 2020 Nov 23.

Gladstone Institutes and University of California, San Francisco, CA, 94158, USA.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
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http://dx.doi.org/10.1038/s41467-020-19743-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684291PMC
November 2020

Clinical and molecular description of 19 patients with GATAD2B-Associated Neurodevelopmental Disorder (GAND).

Eur J Med Genet 2020 Oct 17;63(10):104004. Epub 2020 Jul 17.

Normandie Univ, UNIROUEN, Inserm U1245, Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, F76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France. Electronic address:

De novo pathogenic variants in the GATAD2B gene have been associated with a syndromic neurodevelopmental disorder (GAND) characterized by severe intellectual disability (ID), impaired speech, childhood hypotonia, and dysmorphic features. Since its first description in 2013, nine patients have been reported in case reports and a series of 50 patients was recently published, which is consistent with the relative frequency of GATAD2B pathogenic variants in public databases. We report the detailed phenotype of 19 patients from various ethnic backgrounds with confirmed pathogenic GATAD2B variants including intragenic deletions. All individuals presented developmental delay with a median age of 2.5 years for independent walking and of 3 years for first spoken words. GATAD2B variant carriers showed very little subsequent speech progress, two patients over 30 years of age remaining non-verbal. ID was mostly moderate to severe, with one profound and one mild case, which shows a wider spectrum of disease severity than previously reported. We confirm macrocephaly as a major feature in GAND (53%). Most common dysmorphic features included broad forehead, deeply set eyes, hypertelorism, wide nasal base, and pointed chin. Conversely, prenatal abnormalities, non-cerebral malformations, epilepsy, and autistic behavior were uncommon. Other features included feeding difficulties, behavioral abnormalities, and unspecific abnormalities on brain MRI. Improving our knowledge of the clinical phenotype is essential for correct interpretation of the molecular results and accurate patient management.
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http://dx.doi.org/10.1016/j.ejmg.2020.104004DOI Listing
October 2020

Genetic background of ataxia in children younger than 5 years in Finland.

Neurol Genet 2020 Aug 5;6(4):e444. Epub 2020 Jun 5.

Department of Child Neurology (E.I., P.I., T.L.), Children's Hospital, University of Helsinki and Helsinki University Hospital; Research Programs Unit, Stem Cells and Metabolism, Faculty of Medicine (E.I., P.I., M.P., S.O., V.B., E.P., A.S.), Institute for Molecular Medicine Finland (FIMM) (P.L.), Neuroscience Center (A.S.), HiLife, University of Helsinki, Finland; and Genetics Research Centre (C.J.C.), Molecular and Clinical Sciences Research Institute, St. George's, University of London, United Kingdom.

Objective: To characterize the genetic background of molecularly undefined childhood-onset ataxias in Finland.

Methods: This study examined a cohort of patients from 50 families with onset of an ataxia syndrome before the age of 5 years collected from a single tertiary center, drawing on the advantages offered by next generation sequencing. A genome-wide genotyping array (Illumina Infinium Global Screening Array MD-24 v.2.0) was used to search for copy number variation undetectable by exome sequencing.

Results: Exome sequencing led to a molecular diagnosis for 20 probands (40%). In the 23 patients examined with a genome-wide genotyping array, 2 additional diagnoses were made. A considerable proportion of probands with a molecular diagnosis had de novo pathogenic variants (45%). In addition, the study identified a de novo variant in a gene not previously linked to ataxia: MED23. Patients in the cohort had medically actionable findings.

Conclusions: There is a high heterogeneity of causative mutations in this cohort despite the defined age at onset, phenotypical overlap between patients, the founder effect, and genetic isolation in the Finnish population. The findings reflect the heterogeneous genetic background of ataxia seen worldwide and the substantial contribution of de novo variants underlying childhood ataxia.
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http://dx.doi.org/10.1212/NXG.0000000000000444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323479PMC
August 2020

Fibroblast Growth Factor 21 Drives Dynamics of Local and Systemic Stress Responses in Mitochondrial Myopathy with mtDNA Deletions.

Cell Metab 2019 12 12;30(6):1040-1054.e7. Epub 2019 Sep 12.

Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland; Department of Neurosciences, Helsinki University Central Hospital, 00290 Helsinki, Finland; Neuroscience Center, University of Helsinki, 00290 Helsinki, Finland. Electronic address:

Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISR) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISR progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISR (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2 metabolic ISR stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3 ISR stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.
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http://dx.doi.org/10.1016/j.cmet.2019.08.019DOI Listing
December 2019

Genetic Basis of Severe Childhood-Onset Cardiomyopathies.

J Am Coll Cardiol 2018 11;72(19):2324-2338

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland; Department of Neurology, Helsinki University Hospital and Clinical Neurosciences, University of Helsinki, Helsinki, Finland; Neuroscience Center, HiLife, University of Helsinki, Helsinki, Finland. Electronic address:

Background: Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized.

Objectives: The purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort.

Methods: The authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used.

Results: The KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP).

Conclusions: Childhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment.
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http://dx.doi.org/10.1016/j.jacc.2018.08.2171DOI Listing
November 2018

Metabolomes of mitochondrial diseases and inclusion body myositis patients: treatment targets and biomarkers.

EMBO Mol Med 2018 12;10(12)

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland

Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect.
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http://dx.doi.org/10.15252/emmm.201809091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284386PMC
December 2018

Reply to 'Letter to Editor by Finsterer J and Zarrouk-Mahjoub S: Phenotypic manifestations of the m.8969G>A variant'.

Neurogenetics 2018 05 26;19(2):133-134. Epub 2018 Feb 26.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland.

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http://dx.doi.org/10.1007/s10048-018-0542-zDOI Listing
May 2018

A urinary biosignature for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS).

Mitochondrion 2019 03 19;45:38-45. Epub 2018 Feb 19.

Mitochondria Research Laboratory, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa. Electronic address:

We used a comprehensive metabolomics approach to study the altered urinary metabolome of two mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS) cohorts carrying the m.3243A>G mutation. The first cohort were used in an exploratory phase, identifying 36 metabolites that were significantly perturbed by the disease. During the second phase, the 36 selected metabolites were able to separate a validation cohort of MELAS patients completely from their respective control group, suggesting usefulness of these 36 markers as a diagnostic set. Many of the 36 perturbed metabolites could be linked to an altered redox state, fatty acid catabolism and one-carbon metabolism. However, our evidence indicates that, of all the metabolic perturbations caused by MELAS, stalled fatty acid oxidation prevailed as being particularly disturbed. The strength of our study was the utilization of five different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data, linking, amongst others, altered one-carbon metabolism to MELAS. The results reported here are important in our understanding of MELAS and might lead to better treatment options for the disease.
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http://dx.doi.org/10.1016/j.mito.2018.02.003DOI Listing
March 2019

Defective mitochondrial ATPase due to rare mtDNA m.8969G>A mutation-causing lactic acidosis, intellectual disability, and poor growth.

Neurogenetics 2018 01 19;19(1):49-53. Epub 2018 Jan 19.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland.

Mutations in mitochondrial ATP synthase 6 (MT-ATP6) are a frequent cause of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndromes, especially a point mutation at nucleotide position 8993. M.8969G>A is a rare MT-ATP6 mutation, previously reported only in three individuals, causing multisystem disorders with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia or IgA nephropathy. We present two siblings with the m.8969G>A mutation and a novel, substantially milder phenotype with lactic acidosis, poor growth, and intellectual disability. Our findings expand the phenotypic spectrum and show that mtDNA mutations should be taken account also with milder, stable phenotypes.
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http://dx.doi.org/10.1007/s10048-018-0537-9DOI Listing
January 2018

Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia.

Am J Hum Genet 2017 Nov;101(5):856-865

Centre Hospitalier Universitaire Sainte Justine Research Center, University of Montreal, Montreal, QC H3T1C5, Canada; Department of Pediatrics, University of Montreal, Montreal, QC H3T 1J4, Canada. Electronic address:

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs102] and c.920delG [p.Gly307Alafs11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673666PMC
November 2017

Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome.

Hum Mol Genet 2017 09;26(17):3352-3361

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki 00290, Finland.

Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain.
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http://dx.doi.org/10.1093/hmg/ddx221DOI Listing
September 2017

Absence of Hikeshi, a nuclear transporter for heat-shock protein HSP70, causes infantile hypomyelinating leukoencephalopathy.

Eur J Hum Genet 2017 02 21;25(3):366-370. Epub 2016 Dec 21.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland.

Genetic leukoencephalopathies are a heterogeneous group of central nervous system disorders with white matter involvement. In a Finnish patient, we identified a novel homozygous disease-causing variant in HIKESHI, c.11G>C, p.(Cys4Ser), leading to hypomyelinating leukoencephalopathy with periventricular cysts and vermian atrophy. A founder Ashkenazi-Jewish disease-causing variant recently linked Hikeshi and its heat-shock protective function to leukoencephalopathy. In our patient, clinical features of lower limb spasticity, optic atrophy, nystagmus, and severe developmental delay were similar to reported patients. Additional features included vermian atrophy, epileptic seizures, and an ovarian tumor. Structural modeling and protein analyses revealed that modified interactions inside Hikeshi's hydrophobic pockets induce protein instability. The patient's cells showed impaired nuclear translocation of HSP70 during heat shock, and decreased ERO1-Lα, an endoplasmic reticulum (ER) oxidoreductase. Overall, we show that: (1) the clinical spectrum associated with Hikeshi deficiency extends to leukoencephalopathy with vermian atrophy and epilepsy; (2) the cellular disease process involves both nuclear chaperone and ER functions.
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http://dx.doi.org/10.1038/ejhg.2016.189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315520PMC
February 2017

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

Am J Hum Genet 2016 09 18;99(3):735-743. Epub 2016 Aug 18.

Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany; DZNE - German Center for Neurodegenerative Diseases, 80336 Munich, Germany; Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University, 80336 Munich, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajhg.2016.06.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010644PMC
September 2016

Mitochondrial DNA Replication Defects Disturb Cellular dNTP Pools and Remodel One-Carbon Metabolism.

Cell Metab 2016 Apr 25;23(4):635-48. Epub 2016 Feb 25.

Research Programs Unit, Molecular Neurology, University of Helsinki, 00290 Helsinki, Finland; Department of Neurology, University of Helsinki, Helsinki University Hospital, 00290 Helsinki, Finland; Neuroscience Center, University of Helsinki, 00790 Helsinki, Finland. Electronic address:

Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy.
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http://dx.doi.org/10.1016/j.cmet.2016.01.019DOI Listing
April 2016

Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3.

EMBO Mol Med 2014 Jun;6(6):721-31

Molecular Neurology, Research Programs Unit, University of Helsinki, Helsinki, Finland Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland Neuroscience Research Centre University of Helsinki, Helsinki, Finland

Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy.
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http://dx.doi.org/10.1002/emmm.201403943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203351PMC
June 2014

Regulation of myocardial interleukin-6 expression by p53 and STAT1.

J Interferon Cytokine Res 2013 Sep 15;33(9):542-8. Epub 2013 May 15.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland.

Cardiovascular diseases are a major cause of morbidity and mortality worldwide. The interferon inducible transcriptional activator signal transducer and activator of transcription-1 (STAT1) and p53 are two critical transcriptional factors that have pivotal roles in cardiac biology and pathology. Here we describe a novel interplay between these two key players that critically regulate the levels of the pleiotropic interleukin 6 (IL6) in the heart. We provide in vivo evidence to demonstrate that, in cardiac tissues, STAT1 is a positive regulator of IL6 expression and it competes with the suppressive effect of p53 to sustain basal IL6 levels. Induction of IL6 expression in response to interferon gamma (IFNγ), a well-characterized activator of STAT1, parallels that of STAT1 phosphorylation and induction of STAT1 target genes, such as the interferon regulatory factor-1 (IRF-1), major histocompatibility complex class II transactivator (C2ta), and β2-microglobulin (B2m). Furthermore, hearts from STAT1 knockout mice fail to induce IL6 expression in response to IFNγ. More importantly, we showed that this regulatory system is not functional in mouse embryonic fibroblasts, suggesting that activation of IL6 expression by STAT1 may be tissue specific. IL6 is a major effector of inflammation and cardiac hypertrophy, two major processes involved in heart failure, and therefore, understanding the molecular mechanisms regulating IL6 expression will enable better therapies and treatments for cardiovascular disease patients.
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http://dx.doi.org/10.1089/jir.2012.0165DOI Listing
September 2013

Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.

J Med Genet 2013 Mar 12;50(3):151-9. Epub 2013 Jan 12.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, r.C523B, Haartmaninkatu 8, Helsinki 00290, Finland.

Background: The genetic complexity of infantile cardiomyopathies is remarkable, and the importance of mitochondrial translation defects as a causative factor is only starting to be recognised. We investigated the genetic basis for infantile onset recessive hypertrophic cardiomyopathy in two siblings.

Methods And Results: Analysis of respiratory chain enzymes revealed a combined deficiency of complexes I and IV in the heart and skeletal muscle. Exome sequencing uncovered a homozygous mutation (L156R) in MRPL44 of both siblings. MRPL44 encodes a protein in the large subunit of the mitochondrial ribosome and is suggested to locate in close proximity to the tunnel exit of the yeast mitochondrial ribosome. We found severely reduced MRPL44 levels in the patient's heart, skeletal muscle and fibroblasts suggesting that the missense mutation affected the protein stability. In patient fibroblasts, decreased MRPL44 affected assembly of the large ribosomal subunit and stability of 16S rRNA leading to complex IV deficiency. Despite this assembly defect, de novo mitochondrial translation was only mildly affected in fibroblasts suggesting that MRPL44 may have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome. Retroviral expression of wild-type MRPL44 in patient fibroblasts rescued the large ribosome assembly defect and COX deficiency.

Conclusions: These findings indicate that mitochondrial ribosomal subunit defects can generate tissue-specific manifestations, such as cardiomyopathy.
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http://dx.doi.org/10.1136/jmedgenet-2012-101375DOI Listing
March 2013

Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy.

Hum Mol Genet 2012 Oct 23;21(20):4521-9. Epub 2012 Jul 23.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, 00290 Helsinki, Finland.

Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.
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http://dx.doi.org/10.1093/hmg/dds294DOI Listing
October 2012

Transgenic overexpression of HSP56 does not result in cardiac hypertrophy nor protect from ischaemia/reperfusion injury.

Int J Biochem Cell Biol 2011 Jan 29;43(1):74-9. Epub 2010 Oct 29.

Medical Molecular Biology Unit, Institute of Child Health, University College London, London WC1N 1EH, UK.

Heat shock proteins are known to be induced during and following different forms of cardiac stress. It has previously been shown that their expression is beneficial for the heart following trauma such as ischaemia-reperfusion (I/R) injury. Heat shock protein 56 (HSP56) belongs to the family of FK506-binding immunophilin proteins and is found in steroid receptor complexes, notably the glucocorticoid receptor. We have previously shown that HSP56 and other HSPs are induced in cardiac myocytes treated with cardiotrophin-1, a cytokine with potent hypertrophic and protective properties on cardiac cells. The hypertrophic action of cardiotrophin-1 on cardiac cells is dependent on HSP56 induction and overexpression of HSP56 is sufficient for inducing hypertrophy in cardiac cells. To investigate this phenomenon in vivo, we have generated transgenic mice overexpressing HSP56 and assessed them for the development cardiac hypertrophy and resistance of their hearts to I/R-injury by Langendorff perfusion. Mice generated demonstrated stable, yet varying expression levels of HSP56. Initial characterisation identified a sex-specific phenotype where male overexpressing mice exhibited a moderate, but significant, reduced body weight compared to wild-type controls. In ex vivo stress analyses we found, unexpectedly, that significant overexpression of HSP56 does not induce myocardial hypertrophy and nor does it protect the intact heart from I/R-injury. These observations now suggest a more intricate HSP56-Sp. Cardiophenotype that requires further studies to determine if HSP56 is necessary in mediating hypertrophy induced by other myocardial stimuli.
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http://dx.doi.org/10.1016/j.biocel.2010.09.020DOI Listing
January 2011

Mitochondrial myopathy induces a starvation-like response.

Hum Mol Genet 2010 Oct 23;19(20):3948-58. Epub 2010 Jul 23.

Research Program of Molecular Neurology, Biomedicum-Helsinki, 00290 Helsinki, Finland.

Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, fibroblast growth factor 21 (Fgf21). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.
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http://dx.doi.org/10.1093/hmg/ddq310DOI Listing
October 2010

Effects of polyclonal IgG derived from patients with different clinical types of the antiphospholipid syndrome on monocyte signaling pathways.

J Immunol 2010 Jun 17;184(12):6622-8. Epub 2010 May 17.

Medical Molecular Biology Unit, Institute of Child Health, Department of Medicine, Centre for Rheumatology Research, University College London, London, UK.

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs, p38 MAPK, and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-kappaB, TLR4, and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+), anti-phospholipid Ab-positive patients without APS, or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK, NF-kappaB, and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.
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http://dx.doi.org/10.4049/jimmunol.0902765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898497PMC
June 2010

Ketogenic diet slows down mitochondrial myopathy progression in mice.

Hum Mol Genet 2010 May 17;19(10):1974-84. Epub 2010 Feb 17.

Research Program of Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki 00290, Finland.

Mitochondrial dysfunction is a major cause of neurodegenerative and neuromuscular diseases of adult age and of multisystem disorders of childhood. However, no effective treatment exists for these progressive disorders. Cell culture studies suggested that ketogenic diet (KD), with low glucose and high fat content, could select against cells or mitochondria with mutant mitochondrial DNA (mtDNA), but proper patient trials are still lacking. We studied here the transgenic Deletor mouse, a disease model for progressive late-onset mitochondrial myopathy, accumulating mtDNA deletions during aging and manifesting subtle progressive respiratory chain (RC) deficiency. We found that these mice have widespread lipidomic and metabolite changes, including abnormal plasma phospholipid and free amino acid levels and ketone body production. We treated these mice with pre-symptomatic long-term and post-symptomatic shorter term KD. The effects of the diet for disease progression were followed by morphological, metabolomic and lipidomic tools. We show here that the diet decreased the amount of cytochrome c oxidase negative muscle fibers, a key feature in mitochondrial RC deficiencies, and prevented completely the formation of the mitochondrial ultrastructural abnormalities in the muscle. Furthermore, most of the metabolic and lipidomic changes were cured by the diet to wild-type levels. The diet did not, however, significantly affect the mtDNA quality or quantity, but rather induced mitochondrial biogenesis and restored liver lipid levels. Our results show that mitochondrial myopathy induces widespread metabolic changes, and that KD can slow down progression of the disease in mice. These results suggest that KD may be useful for mitochondrial late-onset myopathies.
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http://dx.doi.org/10.1093/hmg/ddq076DOI Listing
May 2010

Urocortin prevents mitochondrial permeability transition in response to reperfusion injury indirectly by reducing oxidative stress.

Am J Physiol Heart Circ Physiol 2007 Aug 4;293(2):H928-38. Epub 2007 May 4.

Human Genetics Division, University of Southampton, Southampton, UK.

Urocortin (UCN) protects hearts against ischemia and reperfusion injury whether given before ischemia or at reperfusion. Here we investigate the roles of PKC, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute UCN treatment improved hemodynamic recovery during reperfusion after 30 min of global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of 2-[(3)H]deoxyglucose). UCN pretreatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that UCN decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 h) treatment with UCN reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells that had been pretreated with UCN, suggesting that this is the mechanism by which UCN desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC-epsilon or PKC-alpha translocate to the mitochondria following acute UCN treatment, inhibition of PKC with chelerythrine eliminated the effect of UCN on oxidative stress. Our data suggest that acute UCN treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.
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http://dx.doi.org/10.1152/ajpheart.01135.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950441PMC
August 2007

Urocortin inhibits Beclin1-mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury.

J Mol Cell Cardiol 2006 Jun 12;40(6):846-52. Epub 2006 May 12.

Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK.

Autophagy is known to be a feature of cardiomyopathies and chronic ischaemia. Here we demonstrate that autophagy is also induced by a single cycle of ischaemia/reperfusion (I/R in neonatal and adult rat cardiac myocytes). Consistent with the critical role for Beclin1 in autophagocytosis, reduction of Beclin1 expression in cardiac myocytes by RNAi reduces I/R-induced autophagy and this is associated with enhanced cell survival. Autophagy is also reduced by urocortin, an endogenous cardiac peptide which we have previously shown to reduce other forms of myocyte cell death induced by I/R. The inhibition of autophagy by urocortin is mediated in part by inhibition of Beclin1 expression, an effect which is mediated by activation of the PI3 kinase/Akt pathway but which does not involve activation of p42/p44 MAPK.
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http://dx.doi.org/10.1016/j.yjmcc.2006.03.428DOI Listing
June 2006

The cardioprotective effect of urocortin during ischaemia/reperfusion involves the prevention of mitochondrial damage.

Biochem Biophys Res Commun 2004 Aug;321(2):479-86

Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

We have previously shown, using Affymetrix gene chip technology, that urocortin induces the expression of several diverse genes in cardiac myocytes. An ATP sensitive inwardly rectifying potassium channel, Katp (Kir6.1), the enzyme calcium independent phospholipase A2 (iPLA2), and protein kinase C epsilon (PKCepsilon) and that these genes are involved in the cardioprotective mechanism of action of urocortin. Here we demonstrate that these gene products are localized to cardiac myocyte mitochondria and for the first time show that urocortin protects cardiac myocytes from ischaemia/reperfusion induced cell death by preventing mitochondrial damage. Using pharmacological agents to Katp channels and iPLA2 and synthetic peptide inhibitors of PKCepsilon, we go on to demonstrate that these three gene products are involved in the urocortin induced protection of cardiac myocyte mitochondria. These proteins may interact at the mitochondria to produce the protective effect.
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http://dx.doi.org/10.1016/j.bbrc.2004.06.170DOI Listing
August 2004

BAG-1 proteins protect cardiac myocytes from simulated ischemia/reperfusion-induced apoptosis via an alternate mechanism of cell survival independent of the proteasome.

J Biol Chem 2004 May 20;279(20):20723-8. Epub 2004 Feb 20.

Medical Molecular Biology Unit, Institute of Child Health, University College London, London WC1N 1EH, United Kingdom.

BAG-1 (Bcl-2-associated athanogene-1) proteins interact with the HSC70 and HSP70 heat shock proteins and have been proposed to promote cell survival by coordinating the function of these chaperones with the proteasome to facilitate protein degradation. Consistent with this proposal, previous analyses in cancer cells have demonstrated that BAG-1 requires protein domains important for HSC70/HSP70 and proteasome binding in order to interfere with the growth inhibition induced by heat shock (Townsend, P. A., Cutress, R. I., Sharp, A., Brimmell, M., and Packham, G. (2003) Cancer Res., 63, 4150-4157). Moreover, cellular stress triggered the relocalization of the cytoplasmic BAG-1S (approximately 36 kDa) isoform to the nucleus, and both BAG-1S and the constitutively nuclear localized BAG-1L (approximately 50 kDa) isoform suppressed heat shock-induced apoptosis to the same extent, suggesting a critical role in the nucleus. Because ischemia (I) and reperfusion (R) are important stress signals in acute and chronic heart disease, we have examined the expression and function of BAG-1 proteins in primary cardiac myocytes (CMs) and the Langendorff-perfused intact heart. The expression of both BAG-1 isoforms, BAG-1S and BAG-1L, was rapidly induced following ischemia in rat CM, and this was maintained during subsequent reperfusion. In control hearts, BAG-1S and BAG-1L were readily detectable in both the nucleus and the cytoplasm. However, BAG-1S did not relocate to the nucleus following simulated I/R. BAG-1 interacted with both RAF-1 and HSC70 in CMs and the whole heart, and binding to HSC70 was increased following I/R. Overexpression of the human BAG-1S and BAG-1 M isoforms significantly reduced CM apoptosis following simulated I/R. By contrast, BAG-1L or BAG-1S fused to a heterologous nuclear localization sequence failed to protect CM. Finally, overexpression of BAG-1 deletion and point mutants unable to bind HSC70/HSP70 failed to offer cardioprotection. Surprisingly, a deletion mutant lacking the N-terminal ubiquitin-like domain, which mediates interaction with the proteasome, still promoted cardioprotection. Therefore, BAG-1 has a novel cardioprotective role, mediated via association with HSC70/HSP70, which is critical upon cytoplasmic localization but independent of the BAG-1 ubiquitin-like domain. Our studies demonstrate that BAG-1 can influence cellular response to stress by multiple mechanisms, potentially influenced by the cell type and nature of the stress signal.
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http://dx.doi.org/10.1074/jbc.M400399200DOI Listing
May 2004

YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit.

Hum Mol Genet 2002 May;11(9):1075-94

Cell and Molecular Biology, Leukocyte Biology, Division of Biomedical Sciences, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, UK.

Machado-Joseph disease (MJD; MIM 109150) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the MJD1 gene. We have previously reported the generation of human yeast artificial chromosome (YAC) constructs encompassing the MJD1 locus into which expanded (CAG)(76) and (CAG)(84) repeat motifs have been introduced by homologous recombination. Transgenic mice containing pathological alleles with polyglutamine tract lengths of 64, 67, 72, 76 and 84 repeats, as well as the wild type with 15 repeats, have now been generated using these YAC constructs. The mice with expanded alleles demonstrate a mild and slowly progressive cerebellar deficit, manifesting as early as 4 weeks of age. As the disease progresses, pelvic elevation becomes markedly flattened, accompanied by hypotonia, and motor and sensory loss. Neuronal intranuclear inclusion (NII) formation and cell loss is prominent in the pontine and dentate nuclei, with variable cell loss in other regions of the cerebellum from 4 weeks of age. Interestingly, peripheral nerve demyelination and axonal loss is detected in symptomatic mice from 26 weeks of age. In contrast, transgenic mice carrying the wild-type (CAG)(15) allele of the MJD1 locus appear completely normal at 20 months. Disease severity increases with the level of expression of the expanded protein and the size of the repeat. These mice are representative of MJD and will be a valuable resource for the detailed analysis of the roles of repeat length, tissue specificity and level of expression in the neurodegenerative processes underlying MJD pathogenesis.
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http://dx.doi.org/10.1093/hmg/11.9.1075DOI Listing
May 2002