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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Mechanisms of and Rational Remedies for Inherited Neurometabolic Disorders

Stephen G. Kaler
  • Stephen G. Kaler, MD, Head, Section on Translational Neuroscience
  • Ling Yi, PhD, Staff Scientist
  • Cynthia Abou-Zeid, MD, Postdoctoral Fellow
  • Eun-Young Choi, PhD, Postdoctoral Fellow
  • Marie-Reine Haddad, PhD, Postdoctoral Fellow
  • Diego Martinelli, MD, PhD, Visiting Fellow
  • Kristen Stevens, RN, CPNP, Research Nurse Practitioner
  • Julienne Price, BS, Postbaccalaureate Fellow
  • Sokena Zaidi, BS, Postbaccalaureate Fellow

The Section of Translational Neuroscience strives to dissect and understand mechanisms of human neurometabolic disease and to use the knowledge gained to develop rational remedies, including gene therapy. Core values that guide the Section’s efforts include integrity, humility, hard work with purpose, moving forward rapidly, concern for patients and their families, and mutual support among laboratory members. In addition to molecular genetics, we employ model organisms (mouse, zebrafish, yeast) and cellular, biochemical, and biophysical approaches, and we conduct clinical trials. Preclinical work in the laboratory currently focuses on viral gene therapy in mouse models of Menkes disease and lysosomal storage disease. On the basic neuroscience side, we pursue the molecular mechanisms responsible for certain forms of motor neuron degeneration, utilizing murine and zebrafish models, as well as mouse primary motor neuron cultures.

ATP7A–related copper transport disorders

The P-type ATPase ATP7A is a transmembrane protein with a distinctive intracellular trafficking itinerary essential for proper human copper transport. Mutations in ATP7A lead to Menkes disease, occipital horn syndrome (OHS), or an isolated adult-onset distal motor neuropathy (DMN), phenotypes that encompass variable neurologic disability and lifespan. The past several years have witnessed identification of additional human disorders involving gene products that normally coordinate with ATP7A and which, when mutated, can impair copper metabolism.

Through mutagenesis and immunoprecipitation studies, we recently identified a cryptic ubiquitin regulatory X (UBX) domain in the third luminal loop of ATP7A (between transmembrane segments 5 and 6) that becomes exposed through the conformational effects of a DMN–causing mutation, T994I, and binds to the multi-functional ATPase p97/VCP. Confocal imaging, total internal reflection fluorescence (TIRF) microscopy, and live-cell permeabilization experiments suggest that the abnormal interaction occurs at the plasma membrane of cells. The UBX domains contain an 80–amino acid ubiquitin-like sequence, including a conserved FP (Phe-Pro) dipeptide implicated in p97/VCP binding. The motif in ATP7A (F971/P972) is 22 residues proximal to T994I, and mutation of F971/P972 to alanine residues dramatically diminishes the ATP7A–T994I interaction with p97/VCP. Mutations in p97/VCP itself cause several other inherited motor neuron diseases, confirming a link between this abnormal interaction and motor neuron degeneration.

Mutations in the acetyl-coA transporter gene SLC33A1 cause Huppke-Brendel syndrome, a complex autosomal recessive phenotype that features congenital cataracts, hearing loss, profound neuro-developmental delay, and death during infancy or early childhood. The presence of low serum copper and ceruloplasmin and cerebellar atrophy similar to Menkes disease in affected patients implies possible effects on ATP7A. SLC33A1 normally mediates N-terminal acetylation, a reversible post-translational modification of numerous proteins, including at least six other ATPases. We employed tandem mass spectroscopy to document acetylation of ATP7A, used CRISPR/Cas9 to knock out SCL33A1 in HEK293T cells, and studied ATP7A traffic in response to copper stimulation after over-expression of a Venus-tagged ATP7A construct. In contrast to normal HEK293T cells, ATP7A in SLC33A1 knockout cells fails to traffic from the trans-Golgi compartment to the plasma membrane upon high copper loading. Cultured fibroblasts from three affected patients also show defective endogenous ATP7A trafficking after copper loading. The findings help explain the clinical and biochemical phenotypes of this condition and suggest that acetylation of ATP7A is involved in copper-responsive ATP7A trafficking.

For classic Menkes disease, the impact of existing and developing treatments, including viral gene therapy, would be enhanced by newborn screening (NBS), given that brain degeneration in affected infants typically begins by 6 to 8 weeks of age and leads to death by three years. The distinctively abnormal neurochemical metabolites in Menkes newborns are not amenable to detection in standard high volume NBS platforms owing to the need for an alumina extraction step. Interest in the application of genomic technologies to NBS has rised in recent years based on proof-of-concept studies involving DNA sequence analysis from dried newborn blood spots. In a blinded pilot study of 25 affected Menkes patients, over 95% of ATP7A mutations found by conventional mutation detection methods were detected accurately and efficiently from dried blood spots on NBS cards, auguring well for a future population-based application. These advances in early detection and ATP7A gene replacement have the potential to transform the natural history of Menkes disease by circumventing the largest current barriers to good health in affected patients.

Viral gene therapy for lysosomal storage diseases

Choroid plexus (CP)–targeted gene therapy represents a promising new approach to the treatment of lysosomal storage diseases (LSDs) that impact the central nervous system (CNS). Intrathecal delivery, by injecting enzyme into the cerebrospinal fluid (CSF) during a spinal tap, of recombinant lysosomal enzymes has been successful in ameliorating LSDs in some animal studies and in human clinical trials. However, a major drawback to this approach is the need for repeated (e.g., monthly) intrathecal injections owing to the short half-lives of recombinant enzymes. An alternative strategy is to remodel CP epithelial cells with an adeno-associated virus (AAV) vector containing the cDNA for the enzyme of interest. Given the extremely low turnover rate of CP epithelia, the approach could generate a permanent source of enzyme production for secretion into the CSF and penetration into cerebral and cerebellar structures. For the project supported by our 2014 NIH U01 Award, entitled “Choroid plexus-directed gene therapy for alpha-mannosidosis” and conducted in collaboration with John Wolfe, we are using mouse and cat models to evaluate choroid plexus transduction by several recombinant AAV (rAAV) vectors as well as post-treatment alpha-mannosidase concentration and distribution in brain. Studies in the mouse model will require less virus and the mice will be easier to breed. The cat model (housed at the University of Pennsylvania) features a gyrencephalic brain more similar to the human brain. Thus, the study of these two models are complementary.

In a related clinical natural history study, we evaluated six human subjects with alpha-mannosidosis, ranging in age from 11 to 38, at the NIH Clinical Center. In addition to newly appreciated brain magnetic resonance spectroscopy (MRS) findings, we identified distinctive biochemical and proteomic biomarkers in urine and the CSF, which provide invaluable benchmarks for assessing response to treatment in a planned future first-in-human pilot gene therapy trial.

Maternal and child health issues in survivors of the West Africa Ebola epidemic

In collaboration with NIAID, the Section participated in the Trans-NIH response to the 2014–2016 Ebola epidemic in Liberia, including a vaccine clinical trial (PREVAIL-1) and natural history study of survivors (PREVAIL-3). In contrast to AAV gene therapy, in which the brain's immunoprivileged status is advantageous, the Ebola filovirus poses neuro-cognitive and other health risks in survivors of the acute infection owing to immune sanctuary sites within the CNS.

While pregnancy during acute Ebola virus disease (EVD) was almost invariably associated with fetal loss, little is understood about the antenatal courses and pregnancy outcomes in female EVD survivors who conceived after recovery. In a retrospective cohort of 70 EVD survivors who conceived after recovery, the rate of adverse pregnancy outcomes suggested that EVD also engenders reproductive health risks after clinical disease has been resolved, especially when pregnancy occurs within two months of recovery (Reference 1). However, a recently completed prospective evaluation of 94 additional pregnancy outcomes in EVD survivors who conceived 14–24 months after discharge from an Ebola Treatment Unit suggests no maternal-infant transmission of Ebola when pregnancy occurs this long after recovery. The findings have important public health implications for EVD survivors of childbearing age and their health care providers.

Clinical research protocols

  1. Principal Investigator, 90-CH-0149: Early copper histidine treatment in Menkes disease: relationship of molecular defects to neurodevelopmental outcomes
  2. Principal Investigator, 09-CH-0059: Molecular bases of response to copper treatment in Menkes disease, related phenotypes, and unexplained copper deficiency
  3. Principal Investigator, 14-CH-0106: Clinical biomarkers in alpha-mannosidosis
  4. Associate Investigator, Partnership for Research on Ebola Virus in Liberia PREVAIL III (15-I-N122); Monrovia, Liberia
  5. Sub-Investigator, Partnership for Research on Ebola Virus in Liberia PREVAIL I (15-I-N071); Monrovia, Liberia
  6. Associate Investigator; Phase II Study of AAV9-GAA gene transfer in Pompe disease (NHLBI U01 Award, Co-PIs: B. Byrne/A. Arai)

Patents filed

Patent 4239-81164-01: Identification of subjects likely to benefit from copper treatment. International Filing Date: 06 October, 2008

PCT/US2016/058124: Codon-optimized reduced-size ATP7A cDNA and uses for treatment of copper transport disorders. Filing date: October 21, 2016.

Additional Funding

  • 2015 NIH Bench-to-Bedside Award (Kaler/Petris/Feldman). Mechanisms and treatment of motor neuron disease associated with copper metabolism defects
  • U01-CH-079066-01. Choroid plexus-directed gene therapy for alpha-mannosidosis
  • U01-HL121842-01A1. Phase II study of AAV9-GAA gene transfer in Pompe disease
  • 2016 NIH Bench-to-Bedside Award (Kaler/Dickson). Phenotypic effects of gene therapy to the choroid plexus epithelium for Sanfilippo B
  • CRADA with Cyprium Therapeutics, Inc. New York, NY


  1. Fallah MP, Skrip LA, Dahn BT, Nyenswah TG, Flumo H, Glayweon M, Lorseh TL, Kaler SG, Higgs ES, Galvani AP. Pregnancy outcomes in Liberian women who conceived after recovery from Ebola virus disease. Lancet Glob Health 2016 4:678-679.
  2. Kaler SG. Microbial peptide de-coppers mitochondria: implications for Wilson disease. J Clin Invest 2016 126:2412-2414.
  3. Bhattacharjee A, Yang H, Duffy M, Robinson E, Conrad-Antoville A, Lu YW, Capps T, Braiterman L, Wolfgang M, Murphy MP, Yi L, Kaler SG, Lutsenko S, Ralle M. The activity of Menkes disease protein ATP7A is essential for redox balance in mitochondria. J Biol Chem 2016 291:16644-16658.
  4. Hu Frisk JM, Kjellén L, Kaler SG, Pejler G, Öhrvik H. Copper regulates maturation and expression of an MITF:tryptase axis in mast cells. J Immunol 2017 99(12):4132-4141.
  5. Kennedy SB, Bolay F, Kieh M, Grandits G, Badio M, Ballou R, Eckes R, Feinberg M, Follmann D, Grund B, Gupta S, Hensley L, Higgs E, Janosko K, Johnson M, Kateh F, Logue J, Marchand J, Monath T, Nason M, Nyenswah T, Roman F, Stavale E, Wolfson J, Neaton JD, Lane HC; PREVAIL I Study Group. Phase 2 placebo-controlled trial of two vaccines to prevent Ebola in Liberia. N Engl J Med 2017 377:1438-1447.


  • Eva Baker, MD, PhD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
  • Andy Bhattacharjee, PhD, Parabase Genomics, Boston, MA
  • Lauren Brinster, VMD, Division of Veterinary Resources, Office of Research Services, NIH, Bethesda, MD
  • Sara Cathey, MD, Greenwood Genetics Center, Greenwood, SC
  • Jose Centeno, PhD, Walter Reed Army Medical Center, Silver Spring, MD
  • John Chiorini, PhD, Molecular Physiology and Therapeutics Branch, NIDCR, Bethesda, MD
  • Patricia Dickson, MD, Harbor-UCLA Medical Center, Los Angeles, CA
  • Mosoka P. Fallah, PhD, Ministry of Health, Monrovia, Liberia; NIAID Prevail-III Study
  • David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Courtney Holmes, CMT, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Peter Huppke, MD, Georg August Universität, Göttingen, Germany
  • Robert Kotin, PhD, University of Massachusetts Medical Center, Worcester, MA
  • Avindra Nath, MD, Section of Infections of the Nervous System, NINDS, Bethesda, MD
  • Richard Parad, MD, MPH, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
  • Nicholas Patronas, MD, Diagnostic Radiology Department, Clinical Center, NIH, Bethesda, MD
  • Michael Petris, PhD, University of Missouri-Columbia, Columbia, MO
  • Martina Ralle, PhD, Oregon Health Sciences University, Portland, OR
  • Evan Sadler, MD, PhD, Washington University, St. Louis, MO
  • Alan N. Schechter, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
  • Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
  • Wen-Hann Tan, MD, Boston Children's Hospital, Boston, MA
  • John Wolfe, VMD, PhD, University of Pennsylvania, Philadelphia, PA


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