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

Abstract

Mutations in the PLA2G6 gene, which encodes group VIA calcium-independent phospholipase A2 (iPLA 2 ␤), were recently identified in patients with infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation. A pathological hallmark of these childhood neurodegenerative diseases is the presence of distinctive spheroids in distal axons that contain accumulated membranes. We used iPLA 2 ␤-KO mice generated by homologous recombination to investigate neurodegenerative consequences of PLA2G6 mutations. iPLA 2 ␤-KO mice developed age-dependent neurological impairment that was evident in rotarod, balance, and climbing tests by 13 months of age. The primary abnormality underlying this neurological impairment was the formation of spheroids containing tubulovesicular membranes remarkably similar to human INAD. Spheroids were strongly labeled with anti-ubiquitin antibodies. Accumulation of ubiquitinated protein in spheroids was evident in some brain regions as early as 4 months of age, and the onset of motor impairment correlated with a dramatic increase in ubiquitin-positive spheroids throughout the neuropil in nearly all brain regions. Furthermore accumulating ubiquitinated proteins were observed primarily in insoluble fractions of brain tissue, implicating protein aggregation in this pathogenic process. These results indicate that loss of iPLA 2 ␤ causes age-dependent impairment of axonal membrane homeostasis and protein degradation pathways, leading to age-dependent neurological impairment. iPLA 2 ␤-KO mice will be useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration with brain iron accumulation.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

“…Numerous enlarged structures had an onion-like appearance with concentric whorls of membranous material. C, electron micrograph of a dystrophic axon in iPLA 2 ␥ Ϫ/Ϫ mice showing the presence of tubulovesicular elements with a "typical cleft" similar to lesions previously seen in the iPLA 2 ␤ Ϫ/Ϫ mouse (16). These clefts typically are admixed with sheets of membrane that are sometimes found in stacked or whorled arrangements.…”

“…Primary antibodies used for immunoblotting were anti-porin (Calbiochem, 1:200) and anti-COX IV (Invitrogen, 1:500). Tissue processing and immunohistochemistry were performed as previously described (16). In brief, mice were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture, and tissues were fixed by intracardiac perfusion with phosphate-buffered saline followed by 4% paraformaldehyde.…”

“…Transmission Electron Microscopy-Electron microscopy was performed as previously described (16). In brief, dissected tissue or mitochondrial fractions were fixed overnight in chilled 3% glutaraldehyde in cacodylate.…”

“…Conversely, phospholipases also participate in the pathologic sequelae of neuronal ischemia, axonal dystrophy (e.g. infantile neuroaxonal dystrophy (INAD)), and Alzheimer disease (16,17). However, the types of phospholipases and the biochemical mechanisms that mediate these responses in neuronal tissues are at their earliest stages of understanding.…”

Les protéines à domaine patatine

“…L'invalidation du gène codant cette enzyme chez la souris conduit à une diminution de la fertilité chez les mâles [15], mais surtout à une diminution de la tolérance au glucose due à un défaut de sécrétion de l'insuline par les îlots β du pancréas [16]. À l'inverse, la surexpression de l'iPLA 2 β chez la souris induit une augmentation de la sécrétion d'insuline, mais elle a aussi d'autres conséquences : une diminution de la masse osseuse due à une adipogenèse accrue dans la moelle osseuse au détriment de l'ostéogenèse [17] et des dysfonctionnements neurologiques, conséquence d'une perturbation de l'homéostasie des membranes lipidiques axonales [18]. Très récemment, des mutations dans le gène codant l'iPLA 2 β ont été identifiées dans deux pathologies neurodégénératives infantiles illustrant son rôle fondamental dans le développement neuronal chez l'homme 2 [19].…”