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)beta), 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)beta-KO mice generated by homologous recombination to investigate neurodegenerative consequences of PLA2G6 mutations. iPLA(2)beta-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)beta causes age-dependent impairment of axonal membrane homeostasis and protein degradation pathways, leading to age-dependent neurological impairment. iPLA(2)beta-KO mice will be useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration with brain iron accumulation.

Figure 1

Age-dependent progression of neurological impairment in iPLA₂β-KO mice. Motor coordination and balance were assessed in groups of iPLA₂β-KO and WT mice, 4 and 13 months of age, with the rotarod test. A–C: The performance of 4-month-old KO mice was similar to WT mice on the stationary rod (A), the constant speed rotarod (B), and the accelerating rotarod (C) conditions. Although KO mice performed slightly worse during initial trials, no significant differences between the genotypes were observed at age 4 months. In addition, KO mice demonstrated improved performance across trials and test days suggesting motor learning capabilities, and their performance was equivalent to that of WT controls by the end of testing. D–F: In contrast to the 4-month-old KO mice, the performance of 13-month-old KO animals was profoundly impaired in all three rotarod conditions. Although the KO mice exhibited large and significant performance deficits on the stationary rod (D) and constant speed rotarod (E) conditions, they still showed improvement across trials and test days suggesting some motor learning capabilities. The performance of the KO mice was worst on the more challenging accelerating rotarod condition (F) where they showed no significant improvement during training. Asterisks in D–F indicate P values for pairwise comparisons between WT and KO genotypes.

Figure 2

Thirteen-month-old iPLA₂β-KO mice are impaired in other sensorimotor tests involving balance, motor co-ordination, and strength, and they exhibited increased general locomotor activity. A and B: KO mice were able to remain on a narrow Plexiglas ledge (A) or a small circular platform (B) for significantly less time than WT littermate controls. C and D: KO mice also took significantly longer to turn and climb to the top of a 90° inclined screen (C) compared to WT littermates and were able to stay upside down on an inverted screen for a significantly shorter period of time compared to WT littermate controls (D). E: Although differences were not statistically significant, KO mice appeared to have greater difficulty in climbing down a vertical pole although their performance was highly variable. F: KO mice displayed significantly increased levels of general locomotor activity when total ambulations (whole body movements) were measured for 1 hour. Bars represent mean values and error bars represent SEM. P values are indicated in parentheses above error bars.

Figure 3

Spheroid formation in the striatum of iPLA₂β-KO mice. A: H&E staining of a striatum section from an iPLA₂β-KO mouse shows a round eosinophilic inclusion in the neuropil (arrowhead). B and C: Many more spheroids are visualized by immunohistochemistry using an antibody to ubiquitin compared to H&E staining. Comparison of ubiquitin staining in 8-month-old WT (B) and KO (C) mouse brain demonstrates numerous ubiquitin-positive spheroids in the neuropil of KO mice. Spheroids present in KO mouse brain, indicated by arrowheads, exhibit heterogeneous ubiquitin staining and range in size from 1 to 10 μm. Scale bar = 10 μm.

Figure 4

Transmission EM reveals tubulovesicular membranes and other material accumulating within spheroids. A–F: Images from transmission EM, performed on sections from striatum of 8-month-old KO and WT mice, illustrate ultrastructural features of spheroids. B, D, and F are higher magnification images of A, C, and E, respectively. Spheroids (asterisks) were typically surrounded by an intact membrane. Common to all spheroids was the presence of accumulating membranes. Tubulovesicular membranes were abundant and interrupted or bordered by clefts containing sheets of membranes, often in stacked or whorled structures. These structural features of spheroids strongly resemble those reported in human INAD. Transmission EM in the cerebellar granule layer (G) and molecular layer (H) also indicates accumulation of tubulovesicular membranes in spheroids. Magnifications: ×6400 (A, C, F), ×8400 (B), ×12,800 (D), ×3200 (E), ×4200 (G), ×1280 (H).

Figure 5

Age-dependent increase in spheroid density among multiple brain regions of iPLA₂β-KO mice as assessed by ubiquitin immunohistochemistry. Representative photographs from striatum (A–C), cerebellum (D–F), and cortex (G–I) are shown at ages 4, 8, and 13 months from left to right. A progressive increase in the density of ubiquitin-positive spheroids (labeled with brown diaminobenzidine tetrahydrochloride chromagen) in the neuropil was observed with increasing age in these regions and in nearly all other regions of the brain. Scale bar = 10 μm.

Figure 6

Quantitative assessment of progressive increases in spheroid density in striatum (A), cerebellum (B), and cortex (C) of iPLA₂β-KO mice. Brain sections from iPLA₂β-KO mice at 4, 8, and 13 months were stained with a rabbit polyclonal ubiquitin antibody, and average spheroid density in each region was determined by image analysis of multiple random fields in each region. Graphs show mean spheroid density at each age. Error bars indicate SD.

Figure 7

Ubiquitinated proteins accumulate in insoluble fractions obtained by sequential extraction of iPLA₂β-KO mouse brain. A: Anti-ubiquitin Western blot analysis of fractions obtained by sequential biochemical extraction of KO and WT mouse brain tissue samples containing frontal cortex plus striatum. The profiles of ubiquitinated proteins extracted by high salt (HS), high salt with Triton (HS/T), and RIPA buffers are similar, with perhaps a small increase in ubiquitinated proteins extracted from KO brain by HS/T. However there is a clear increase in a complex pattern of high molecular weight ubiquitinated protein species extracted by SDS buffer from KO brain tissue. The pattern of accumulating high molecular weight protein species was complex, indicating the presence of multiple polyubiquitinated proteins. B: Accumulation of ubiquitinated proteins in insoluble fractions was observed in multiple independent fractionation experiments using brain tissue samples containing frontal cortex plus striatum (FC/S) or striatum alone (S) from 13-month-old WT and KO mice.

Figure 8

Neuroaxonal dystrophy affects the sympathetic nervous system and sensory axons in the gracile nucleus in iPLA₂β-KO mice. A and B: Images from transmission EM illustrate prominent spheroids with accumulating tubulovesicular membranes in axon terminals within the sympathetic superior mesenteric-celiac ganglia. H&E staining revealed large spheroids in the gracile nucleus at 4 months. C: Spheroids increased in size and number at 13 months (D). Scale bars: 6 μm (A); 2 μm (B); 10 μm (C, D).

Figure 9

Accumulation of α-synuclein protein in spheroids in the striatum of iPLA₂β-KO mice. Immunohistochemistry using monoclonal synuclein antibody syn303 demonstrates staining of spheroids with morphology similar to those labeled with ubiquitin antibodies. Representative photographs (A, B) are shown from the striatum of a 13-month-old iPLA₂β-KO mouse. Syn303 staining was primarily observed in striatum of mice aged 13 months or older, with only rare staining of spheroids in other brain regions. Scale bar = 10 μm.