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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2019 Annual Report of the Division of Intramural Research

Translational Research in Inherited Neurodegenerative and Motor Neuron Diseases

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
  • Kristen Stevens, RN, CPNP, Research Nurse Practitioner
  • Julienne Price, BS, Postbaccalaureate Fellow
  • Robert M. Kotin, PhD, Special Volunteer/Research Collaborator

* As of December 1, 2019, Dr. Kaler is now at Nationwide Children’s Hospital.

The laboratory is committed to dissecting the mechanisms and pathophysiologies of inherited neurodegenerative and motor neuron diseases and using this knowledge to improve health through rational remedies, including gene therapy. Patients and families affected by these disorders provide the impetus for scientific inquiry. In addition to molecular genetics, the laboratory employs model organisms (yeast, mouse, zebrafish) and cellular and biochemical approaches, and conducts clinical protocols. Choroid plexus–targeted adeno-associated virus (AAV) gene therapy for alpha-mannosidosis, a prototypical lysosomal storage disease, and the study of motor neuron degeneration mediated by the p97/valosin-containing protein (p97/VCP) represent our current main directions. Copper histidinate (CuHis), a new molecular entity originally developed at the NIH Clinical Center for the Section's long-term clinical work on Menkes' disease, received fast-track designation from the Food & Drug Administration (FDA) in 2018. New drug approval will pave the way for a trial of subcutaneous CuHis in combination with cerebrospinal spinal fluid (CSF)–directed ATP7A gene therapy for this illness.

Choroid plexus–targeted viral gene therapy for alpha-mannosidosis, a prototypical lysosomal storage disease

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 recombinant lysosomal enzymes into the cerebrospinal fluid [CSF] during a spinal tap) 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. We devised an alternative strategy 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, which are postmitotic neuroectoderm-derived cells, the approach can provide a permanent source of enzyme production for secretion into the CSF and penetration into cerebral and cerebellar structures. We are using a mouse model to evaluate choroid plexus transduction by several recombinant AAV (rAAV) vector serotypes and to determine the posttreatment lysosomal alpha-mannosidase (LAMAN) activity and distribution in the brain globally. These preclinical studies open the door to IND (investigational new drug)–generating studies for a future first-in-human clinical trial.

In a natural history study of alpha-mannosidosis, we evaluate human subjects with this condition 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 are potentially useful benchmarks for assessing response to investigational treatments.

ATP7A–related disorders, including motor neuron degeneration

ATP7A is an evolutionarily conserved copper-transporting P-type ATPase associated with X-linked recessive Menkes disease and occipital horn syndrome, a milder version of this phenotype. Based on clinical trials conducted by the Section using subcutaneous injections of a lyophilized form of copper histidinate (CuHis), this new molecular entity was granted Fast Track status by the FDA and is anticipated to be the first federally approved treatment for this difficult neurometabolic disorder. Recent companion preclinical studies in a mouse model of Menkes' disease illustrate the synergistic effect of AAV serotype 9 gene therapy with subcutaneous CuHis. The Section's therapeutic advances in Menkes' disease highlight the importance of future newborn screening for this disorder, for which targeted next-generation sequencing appears to be a reliable and cost-effective approach.

Recently, two unique ATP7A missense mutations, T994I and P1386S, were shown to cause adult-onset isolated distal motor neuropathy without the clinical or biochemical features of other ATP7A disorders. The mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma-membrane localization compared with wild-type ATP7A. We identified an abnormal interaction between the vesicular trafficking protein p97/VCP and ATP7A-T994I. Mutations in p97/VCP cause at least three other motor neuron diseases: amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, and inclusion body myopathy with early-onset Paget's disease and frontotemporal dementia (IBMPFD). We identified a putative UBX domain (found in ubiquitin-regulatory proteins) in the luminal loop of ATP7A immediately adjacent to the T994I mutation, at which p97/VCP binds. TIRF (total internal reflection fluorescence microscopy) imaging, cell fractionation, and immunoprecipitation analyses determined the intracellular localization of the interaction at or near the cell plasma membrane. The findings have implications for similar phenomena involving other proteins, and we are pursuing further studies to better understand the pathophysiological mechanism(s) implicated in motor neuron degeneration associated with p97/VCP.

Mutations in the acetyl-CoA transporter SLC33A1 cause a complex autosomal recessive phenotype known as Huppke-Brendel syndrome. Low serum copper and ceruloplasmin and cerebellar atrophy similar to Menkes' disease in affected patients, as well as hepatic copper overload similar to Wilson's disease, imply possible effects on ATP7A and ATP7B, the copper-transporting ATPases implicated in those respective disorders. SLC33A1 normally mediates transport of acetyl-CoA, required for lysine acetylation, which is a reversible post-translational modification of some transmembrane proteins. To explore the possible connection between these diverse gene products, we used CRISPR/Cas9 to knock out SCL33A1 in Hek293T culture cells and studied ATP7A trafficking in response to copper stimulation. For these experiments, we overexpressed fluorescently tagged ATP7A or ATP7B constructs in Hek293T cells. In contrast to normal Hek293T cells, both copper ATPases failed to traffic from the trans-Golgi network to the plasma membrane in SLC33A1 knock-out cells. We also studied fibroblasts available from four affected patients; all showed partial defects in endogenous ATP7A trafficking in response to copper. We then employed tandem mass spectroscopy to document acetylation of numerous lysine residues in ATP7A and ATP7B, which we now are systematically mutating to identify the critical site(s) implicated. This previously unappreciated post-translational modification appears to mediate normal Cu–ATPase trafficking and in part explains the Huppke-Brendel phenotype.

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

In collaboration with Elizabeth Higgs and Mosoka Fallah, the Section remains involved in a natural history study of Ebola survivors (PREVAIL-3), specifically children and adolescent survivors, as well as a Birth Cohort substudy that evaluates pregnancy outcomes in female Ebola survivors of child-bearing age. In contrast to AAV gene therapy, in which the brain's immunoprivileged status is advantageous, the Ebola filovirus poses neurocognitive and other health risks in survivors of the acute infection owing to immunological niches or sanctuary sites within the CNS.

Clinical research protocols

  1. Principal Investigator (PI), 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

  • U.S. Patent Application No. 62/620,811; filed January 23, 2018: Methods and Compositions for Treating Lysosomal Storage Diseases
  • U.S. Patent Application No. 15/769,294; filed April 18, 2018: Codon-Optimized Reduced-Size ATP7A cDNA and Uses for Treatment of Copper Transport Disorders

Additional Funding

  • U01-CH-079066-04. Choroid plexus-directed gene therapy for alpha-mannosidosis
  • U01-HL121842-01A1. Phase II study of AAV9-GAA gene transfer in Pompe disease
  • Cooperative Research and Development Agreement (CRADA) with Cyprium Therapeutics, Inc. New York, NY


  1. Sharma P, Reichert M, Lu Y, Markello TC, Adams DR, Steinbach PJ, Fuqua BK, Parisi X, Kaler SG, Vulpe CD, Anderson GJ, Gahl WA, Malicdan MCV. Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype. PLoS Genet 2019;15(5):e1008143.
  2. Yi L, Kaler SG. Interaction between the AAA ATPase p97/VCP and a concealed UBX domain in the copper transporter ATP7A is associated with motor neuron degeneration. J Biol Chem 2018;293:7606-7617.
  3. Haddad MR, Choi EY, Zerfas PM, Yi L, Martinelli D, Sullivan P, Goldstein DS, Centeno JA, Brinster LR, Ralle M, Kaler SG. Cerebrospinal fluid-directed rAAV9-rsATP7A plus subcutaneous copper histidinate advance survival and outcomes in a Menkes disease mouse model. Mol Ther Methods Clin Dev 2018;10:165-178.
  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;199:4132-4141.
  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.


  • Andrew Arai, MD, Cardiovascular Branch, NHLBI, Bethesda, MD
  • Eva Baker, MD, PhD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
  • Juan Bonifacino, PhD, Section on Intracellular Protein Trafficking, NICHD, Bethesda, MD
  • Carsten Bönnemann, MD, Neurogenetics Branch, NINDS, Bethesda, MD
  • Lauren Brinster, VMD, Division of Veterinary Resources, Office of Research Services, NIH, Bethesda, MD
  • Barry Byrne, MD, PhD, Powell Gene Therapy Center, University of Florida, Gainesville, FL
  • 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
  • William Gahl, MD, PhD, Medical Genetics Branch, NHGRI, Bethesda, MD
  • David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Elizabeth S. Higgs, MD, DTM&H, Division of Clinical Research, NIAID, Rockville, MD
  • Peter Huppke, MD, Georg August Universität, Göttingen, Germany
  • Jamie Marko, MD, Radiology and Imaging Sciences Department, Clinical Center, NIH, Bethesda, MD
  • 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
  • Michael Petris, PhD, University of Missouri-Columbia, Columbia, MO
  • Martina Ralle, PhD, Oregon Health Sciences University, Portland, OR
  • Alan N. Schechter, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
  • Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
  • Patricia Sullivan, MT, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Wen-Hann Tan, MD, Boston Children's Hospital, Boston, MA
  • Cynthia Tifft, MD, PhD, Office of the Clinical Director, NHGRI, Bethesda, MD
  • Charles Venditti, MD, PhD, Medical Genomics and Metabolic Genetics Branch, NHGRI, Bethesda, MD
  • John Wolfe, VMD, PhD, University of Pennsylvania, Philadelphia, PA

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