Disrupted Membrane Homeostasis and Accumulation of Ubiquitinated Proteins in a Mouse Model of Infantile Neuroaxonal Dystrophy Caused by PLA2G6 Mutations

Ibrahim Malik^*, John Turk, David J. Mancuso, Laura Montier^*, Mary Wohltmann, David F. Wozniak^¶, Robert E. Schmidt^¶||, Richard W. Gross^** and Paul T. Kotzbauer^*¶**

From the Department of Neurology,^*the Divisions of Endocrinology, Metabolism, and Lipid Researchand Bioorganic Chemistry and Molecular Pharmacologyin the Department of Medicine, the Departments of Psychiatryand Pathology and Immunology,||Hope Center for Neurological Disorders,^¶and the Department of Molecular Biology and Pharmacology,^*^*Washington University School of Medicine, St. Louis; and the Department of Chemistry,Washington University, St. Louis, Missouri

Mutations in the PLA2G6 gene, which encodes group VIA calcium-independentphospholipase A2 (iPLA₂β), were recently identified inpatients with infantile neuroaxonal dystrophy (INAD) and neurodegenerationwith brain iron accumulation. A pathological hallmark of thesechildhood neurodegenerative diseases is the presence of distinctivespheroids in distal axons that contain accumulated membranes.We used iPLA₂β-KO mice generated by homologous recombinationto investigate neurodegenerative consequences of PLA2G6 mutations.iPLA₂β-KO mice developed age-dependent neurological impairmentthat was evident in rotarod, balance, and climbing tests by13 months of age. The primary abnormality underlying this neurologicalimpairment was the formation of spheroids containing tubulovesicularmembranes remarkably similar to human INAD. Spheroids were stronglylabeled with anti-ubiquitin antibodies. Accumulation of ubiquitinatedprotein in spheroids was evident in some brain regions as earlyas 4 months of age, and the onset of motor impairment correlatedwith a dramatic increase in ubiquitin-positive spheroids throughoutthe neuropil in nearly all brain regions. Furthermore accumulatingubiquitinated proteins were observed primarily in insolublefractions of brain tissue, implicating protein aggregation inthis pathogenic process. These results indicate that loss ofiPLA₂β causes age-dependent impairment of axonal membranehomeostasis and protein degradation pathways, leading to age-dependentneurological impairment. iPLA₂β-KO mice will be usefulfor further studies of pathogenesis and experimental interventionsin INAD and neurodegeneration with brain iron accumulation.