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1.  L-DOPS corrects neurochemical abnormalities in a Menkes disease mouse model 
Annals of neurology  2012;73(2):259-265.
Objective
Menkes disease is a lethal neurodegenerative disorder of infancy caused by mutations in a copper-transporting ATPase gene, ATP7A. Among its multiple cellular tasks, ATP7A transfers copper to dopamine-beta-hydroxylase (DBH) within the lumen of the Golgi network or secretory granules, catalyzing the conversion of dopamine to norepinephrine. In a well-established mouse model of Menkes disease, mottled-brindled, we tested whether systemic administration of L-threo-dihydroxyphenylserine (L-DOPS), a drug used successfully to treat autosomal recessive norepinephrine deficiency, would improve brain neurochemical abnormalities and neuropathology.
Methods
At 8, 10, and 12 days of age, wild type and mo-br mice received intraperi-toneal injections of 200μg/g body weight of L-DOPS, or mock solution. Five hours after the final injection, the mice were euthanized and brains removed. We measured catecholamine metabolites affected by DBH via high-performance liquid chromatography with electrochemical detection, and assessed brain histopathology.
Results
Compared to mock-treated controls, mo-br mice that received intraperitoneal L-DOPS showed significant increases in brain norepinephrine (P<0.001) and its deaminated metabolite, dihydroxyphenylglycol (DHPG, P<0.05). The ratio of a non-beta-hydroxylated metabolite in the catecholamine biosynthetic pathway, dihydroxyphenylacetic acid, to the beta-hydroxylated metabolite, dihydroxyphenylglycol, improved equivalently to results obtained previously with brain-directed ATP7A gene therapy (P<0.01). However, L-DOPS treatment did not arrest global brain pathology or improve somatic growth, as gene therapy had.
Interpretation
We conclude that 1) L-DOPS crosses the blood-brain barrier in mo-br mice and corrects brain neurochemical abnormalities, 2) norepinephrine deficiency is not the cause of neurodegeneration in mo-br mice, and 3) L-DOPS treatment may ameliorate noradrenergic hypofunction in Menkes disease.
doi:10.1002/ana.23787
PMCID: PMC3597755  PMID: 23224983
2.  Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase 
Human Mutation  2012;33(8):1207-1215.
Copper is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular copper metabolism, an elaborate system of chaperones and transporters has evolved, although no human copper chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited a homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the copper chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163 with tryptophan in domain II of CCS, which interacts directly with SOD1. Biochemical analyses of the patient’s fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal copper homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a copper chaperone.
doi:10.1002/humu.22099
PMCID: PMC3399931  PMID: 22508683
CCS; SOD1; copper; chaperone
3.  Fetal Brain-directed AAV Gene Therapy Results in Rapid, Robust, and Persistent Transduction of Mouse Choroid Plexus Epithelia 
Fetal brain-directed gene addition represents an under-appreciated tool for investigating novel therapeutic approaches in animal models of central nervous system diseases with early prenatal onset. Choroid plexuses (CPs) are specialized neuroectoderm-derived structures that project into the brain's ventricles, produce cerebrospinal fluid (CSF), and regulate CSF biochemical composition. Targeting the CP may be advantageous for adeno-associated viral (AAV) gene therapy for central nervous system disorders due to its immunoprivileged location and slow rate of epithelial turnover. Yet the capacity of AAV vectors to transduce CP has not been delineated precisely. We performed intracerebroventricular injections of recombinant AAV serotype 5-green fluorescent protein (rAAV5-GFP) or rAAV9-GFP in embryonic day 15 (E15) embryos of CD-1 and C57BL/6 pregnant mice and quantified the percentages of GFP expression in CP epithelia (CPE) from lateral and fourth ventricles on E17, postnatal day 2 (P2), and P22. AAV5 was selective for CPE and showed significantly higher transduction efficiency in C57BL/6 mice (P = 0.0128). AAV9 transduced neurons and glial cells in both the mouse strains, in addition to CPE. We documented GFP expression in CPE on E17, within just 48 hours of rAAV administration to the fetal lateral ventricle, and expression by both the serotypes persisted at P130. Our results indicate that prenatal administration of rAAV5 and rAAV9 enables rapid, robust, and sustained transduction of mouse CPE and buttress the rationale for experimental therapeutics targeting the CP.
doi:10.1038/mtna.2013.27
PMCID: PMC3696907  PMID: 23799375
blood-cerebrospinal fluid barrier; choroid plexus; in utero gene therapy; rAAV5; rAAV9; survival surgery
4.  Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy 
Human Molecular Genetics  2011;21(8):1794-1807.
ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.
doi:10.1093/hmg/ddr612
PMCID: PMC3313796  PMID: 22210628
5.  Increased frequency of congenital heart defects in Menkes disease 
Clinical Dysmorphology  2012;21(2):59-63.
ATP7A is a copper-transporting ATPase critical for central and peripheral nervous system function. Mutations in ATP7A cause Menkes disease and occipital horn syndrome (OHS), allelic X-linked recessive conditions that feature vascular abnormalities ascribed to low activity of lysyl oxidase, a copper-dependent enzyme. From a recently created Menkes disease/OHS patient registry, we identified 4 of 95 subjects with major congenital heart defects (4.2%), a proportion exceeding the general population prevalence (≈1%). In conjunction with mouse models of Menkes disease, OHS, and lysyl oxidase deficiency, which feature aortic aneurysms, irregular attachment between vascular endothelium and mesoderm and other defects of embryological development, our observation suggests an important role of copper metabolism in cardiac development. Congenital heart disease may be an under-appreciated abnormality in Menkes disease, and should be considered in a broad differential diagnosis of cardiac defects found prenatally in male fetuses. Conversely, newborn infants with suspected or confirmed Menkes disease should be evaluated for heart disease by careful clinical examination and echocardiography, if indicated.
doi:10.1097/MCD.0b013e32834ea52b
PMCID: PMC3385410  PMID: 22134099
ATP7A; congenital heart disease; lysyl oxidase; Menkes disease; occipital horn syndrome
6.  Neonatal Diagnosis and Treatment of Menkes Disease 
The New England journal of medicine  2008;358(6):605-614.
BACKGROUND
Menkes disease is a fatal neurodegenerative disorder of infancy caused by diverse mutations in a copper-transport gene, ATP7A. Early treatment with copper injections may prevent death and illness, but presymptomatic detection is hindered by the inadequate sensitivity and specificity of diagnostic tests. Exploiting the deficiency of a copper enzyme, dopamine-β-hydroxylase, we prospectively evaluated the diagnostic usefulness of plasma neurochemical levels, assessed the clinical effect of early detection, and investigated the molecular bases for treatment outcomes.
METHODS
Between May 1997 and July 2005, we measured plasma dopamine, norepinephrine, dihydroxyphenylacetic acid, and dihydroxyphenylglycol in 81 infants at risk. In 12 newborns who met the eligibility criteria and began copper-replacement therapy within 22 days after birth, we tracked survival and neurodevelopment longitudinally for 1.5 to 8 years. We characterized ATP7A mutations using yeast complementation, reverse-transcriptase–polymerase-chain-reaction analysis, and immunohistochemical analysis.
RESULTS
Of 81 infants at risk, 46 had abnormal neurochemical findings indicating low dopamine-β-hydroxylase activity. On the basis of longitudinal follow-up, patients were classified as affected or unaffected by Menkes disease, and the neurochemical profiles were shown to have high sensitivity and specificity for detecting disease. Among 12 newborns with positive screening tests who were treated early with copper, survival at a median follow-up of 4.6 years was 92%, as compared with 13% at a median follow-up of 1.8 years for a historical control group of 15 late-diagnosis and late-treatment patients. Two of the 12 patients had normal neurodevelopment and brain myelination; 1 of these patients had a mutation that complemented a Saccharomyces cerevisiae copper-transport mutation, indicating partial ATPase activity, and the other had a mutation that allowed some correct ATP7A splicing.
CONCLUSIONS
Neonatal diagnosis of Menkes disease by plasma neurochemical measurements and early treatment with copper may improve clinical outcomes. Affected newborns who have mutations that do not completely abrogate ATP7A function may be especially responsive to early copper treatment.
doi:10.1056/NEJMoa070613
PMCID: PMC3477514  PMID: 18256395
7.  Molecular correlates of epilepsy in early diagnosed and treated Menkes disease 
Epilepsy is a major feature of Menkes disease, an X-linked recessive infantile neurodegenerative disorder caused by mutations in ATP7A, which produces a copper-transporting ATPase. Three prior surveys indicated clinical seizures and electroencephalographic (EEG) abnormalities in a combined 27 of 29 (93%) symptomatic Menkes disease patients diagnosed at 2 months of age or older. To assess the influence of earlier, presymptomatic diagnosis and treatment on seizure semiology and brain electrical activity, we evaluated 71 EEGs in 24 Menkes disease patients who were diagnosed and treated with copper injections in early infancy (≤6 weeks of age), and whose ATP7A mutations we determined. Clinical seizures were observed in only 12.5% (3/24) of these patients, although 46% (11/24) had at least one abnormal EEG tracing, including 50% of patients with large deletions in ATP7A, 50% of those with small deletions, 60% of those with nonsense mutations, and 57% of those with canonical splice junction mutations. In contrast, five patients with mutations shown to retain partial function, either via some correct RNA splicing or residual copper transport capacity, had neither clinical seizures nor EEG abnormalities. Our findings suggest that early diagnosis and treatment improve brain electrical activity and decrease seizure occurrence in classical Menkes disease irrespective of the precise molecular defect. Subjects with ATP7A mutations that retain some function seem particularly well protected by early intervention against the possibility of epilepsy.
doi:10.1007/s10545-010-9118-2
PMCID: PMC3113468  PMID: 20652413
8.  Somatic Mosaicism in Menkes Disease Suggests Choroid Plexus-mediated Copper Transport to the Developing Brain 
The primary mechanism of copper transport to the brain is unknown, although this process is drastically impaired in Menkes disease, an X-linked neurodevelopmental disorder caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Potential central nervous system entry routes for copper include brain capillary endothelial cells that originate from mesodermal angioblasts and form the blood-brain barrier, and the choroid plexuses, which derive from embryonic ectoderm, and form the blood-cerebrospinal fluid barrier. We exploited a rare (and first reported) example of somatic mosaicism for an ATP7A mutation to shed light on questions about copper transport into the developing brain. In a 20-month-old Menkes disease patient evaluated before copper treatment, blood copper and catecholamine concentrations were normal, whereas levels in cerebrospinal fluid were abnormal and consistent with his neurologically severe phenotype. We documented disparate levels of mosaicism for an ATP7A missense mutation, P1001L, in tissues derived from different embryonic origins; allele quantitation showed P1001L in approximately 27% and 88% of DNA samples from blood cells (mesoderm-derived) and cultured fibroblasts (ectoderm-derived), respectively. These findings imply that the P1001L mutation in the patient preceded formation of the three primary embryonic lineages at gastrulation, with the ectoderm layer ultimately harboring a higher percentage of mutation-bearing cells than mesoderm or endoderm. Since choroid plexus epithelia are derived from neuroectoderm, and brain capillary endothelial cells from mesodermal angioblasts, the clinical and biochemical findings in this infant support a critical role for the blood-CSF barrier (choroid plexus epithelia) in copper entry to the developing brain.
doi:10.1002/ajmg.a.33632
PMCID: PMC3117432  PMID: 20799318
Somatic mosaicism; Menkes disease; ATP7A; copper metabolism; choroid plexus
9.  Translational read-through of a nonsense mutation in ATP7A impacts treatment outcome in Menkes disease 
Annals of neurology  2009;65(1):108-113.
Protein translation ends when a stop codon in a gene’s messenger RNA transcript enters the ribosomal A site. Mutations that create premature stop codons (nonsense mutations) typically cause premature translation termination. An alternative outcome, read-through translation (or nonsense suppression), is well known in prokaryotic, viral, and yeast genes but has not been clearly documented in humans except in the context of pharmacological manipulations. Here, we identify and characterize native read-through of a nonsense mutation (R201X) in the human copper transport gene, ATP7A. Western blotting, in vitro expression analyses, immunohistochemistry, and yeast complementation assays using cultured fibroblasts from a classical Menkes disease patient all indicated small amounts of native ATP7AR201X read-through and were associated with a dramatic clinical response to early copper treatment.
doi:10.1002/ana.21576
PMCID: PMC2917729  PMID: 19194885
10.  Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome 
Journal of Medical Genetics  2007;44(8):492-497.
Background
Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the underlying molecular mechanisms.
Methods
We measured biochemical markers of impaired copper transport in five patients from two unrelated families and used RNase protection, quantitative reverse transcription (RT)‐PCR, Western blot analysis and yeast complementation studies to characterise two ATP7A missense mutations, A1362D and S637L.
Results
In two brothers (family A) with A1362D, RNase protection and Western blot analyses revealed higher amounts of ATP7A transcript and protein in the older, mildly affected patient, who also had a higher plasma copper level and lower cerebrospinal fluid dihydroxyphenylalanine : dihydroxyphenylglycol ratio. These findings indicate greater gastrointestinal absorption of copper and higher activity of dopamine‐β‐hydroxylase, a copper‐dependent enzyme, respectively. In family B, three males with a missense mutation (S637L) in an exon 8 splicing enhancer showed equally reduced amounts of ATP7A transcript and protein by quantitative RT‐PCR and western blot analysis, respectively, despite a more severe phenotype in the youngest. This patient's medical history was notable for cardiac arrest as a neonate, to which we attribute his more severe neurodevelopmental outcome.
Conclusions
These families illustrate that genetic and non‐genetic mechanisms may underlie intrafamilial variability in Menkes disease and its variants.
doi:10.1136/jmg.2007.050013
PMCID: PMC2597922  PMID: 17496194
Menkes disease; intrafamilial variation; gene expression, ATP7A; residual copper transport
11.  Clinical Outcomes in Menkes Disease Patients with a Copper-Responsive ATP7A Mutation, G727R 
Molecular genetics and metabolism  2008;95(3):174-181.
Menkes disease is a fatal neurodegenerative disorder of infancy caused by defects in an X-linked copper transport gene, ATP7A. Evidence from a recent clinical trial indicates that favorable response to early treatment of this disorder with copper injections involves mutations that retain some copper transport capacity. In three unrelated infants, we identified the same mutation, G727R, in the second transmembrane segment of the ATP7A gene product that complemented a S. cerevisiae copper transport mutant, consistent with partial copper transport activity. Quantitative reverse transcription-polymerase chain reaction studies showed approximately normal levels of ATP7AG727R transcript in two patients’ fibroblasts compared to wild type controls, but Western blot analyses showed markedly reduced quantities of ATP7A protein, suggesting post-translational degradation. We confirmed the latter by comparing degradation rates of mutant and wild type ATP7A via cyclohexamide treatment of cultured fibroblasts; half-life of the G727R mutant was 2.9 hr and for the wild-type, 11.4 hr. We also documented a X-box binding protein 1 splice variant in G727R cells - known to be associated with the cellular misfolded protein response. Patient A, diagnosed 6 months of age, began treatment at 228 days (7.6 mos) of age. At his current age (2 years), his overall neurodevelopment remains at a 2 to 4 month level. In contrast, patients B and C were diagnosed in the neonatal period, began treatment within 25 days of age, and show near normal neurodevelopment at their current ages, 3 years (B), and 7 months (C). The poor clinical outcome in patient A with the same missense mutation as patients A and B with near normal oucomes, confirms the importance of early medical intervention in Menkes disease and highlights the critical potential benefit of newborn screening for this disorder.
doi:10.1016/j.ymgme.2008.06.015
PMCID: PMC2654537  PMID: 18752978

Results 1-11 (11)