Autophagy-lysosome pathway associated neuropathology and axonal degeneration in the brains of alpha-galactosidase A-deficient mice

Abstract

Background: Mutations in the gene for alpha-galactosidase A result in Fabry disease, a rare, X-linked lysosomal storage disorder characterized by a loss of alpha-galactosidase A enzymatic activity. The resultant accumulation of glycosphingolipids throughout the body leads to widespread vasculopathy with particular detriment to the kidneys, heart and nervous system. Disruption in the autophagy-lysosome pathway has been documented previously in Fabry disease but its relative contribution to nervous system pathology in Fabry disease is unknown. Using an experimental mouse model of Fabry disease, alpha-galactosidase A deficiency, we examined brain pathology in 20-24 month old mice with particular emphasis on the autophagy-lysosome pathway.

Results: Alpha-galactosidase A-deficient mouse brains exhibited enhanced punctate perinuclear immunoreactivity for the autophagy marker microtubule-associated protein light-chain 3 (LC3) in the parenchyma of several brain regions, as well as enhanced parenchymal and vascular immunoreactivity for lysosome-associated membrane protein-1 (LAMP-1). Ultrastructural analysis revealed endothelial cell inclusions with electron densities and a pronounced accumulation of electron-dense lipopigment. The pons of alpha-galactosidase A-deficient mice in particular exhibited a striking neuropathological phenotype, including the presence of large, swollen axonal spheroids indicating axonal degeneration, in addition to large interstitial aggregates positive for phosphorylated alpha-synuclein that co-localized with the axonal spheroids. Double-label immunofluorescence revealed co-localization of phosphorylated alpha-synuclein aggregates with ubiquitin and LC3.

Conclusion: Together these findings indicate widespread neuropathology and focused axonal neurodegeneration in alpha-galactosidase A-deficient mouse brain in association with disruption of the autophagy-lysosome pathway, and provide the basis for future mechanistic assessment of the contribution of the autophagy-lysosome pathway to this histologic phenotype.

Figure 1

LC3 levels are enhanced throughout α-Gal A-deficient mouse brain. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a-c, h-j, o-q, u-w) or -/0 (d-f, k-m, r-t, x-z) mice were immunolabeled with an antibody against LC3 (a, d, h, k, o, r, u, x) and counterstained with bis-benzimide (b, e, i, l, p, s, v, y). Inset in panel f indicates LC3-positive puncta. The images shown were taken from the cerebellum (CB), pons (PONS), hippocampus (HIP), and cortex (CTX). Scale bars = 50 microns. Graphs in (g and n) show relative mean fluorescence intensity of LC3 signal normalized to averaged α-Gal A +/0 levels (n = 3) (*p < 0.05, Student’s t-test).

Figure 2

Evidence of enhanced neuritic and perinuclear LAMP-1 levels. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a-d) or -/0 (e-h) mice were immunolabeled with an antibody against LAMP-1 and nuclei were counterstained with hematoxylin. Higher magnification areas of interest from α-Gal A -/0 sections are included for detail (i-l). Arrowheads indicate areas of increased perinuclear (f, j) and neuritic (e, g, h, I, k, l) LAMP-1 immunoreactivity, while arrows indicate vascular LAMP-1 staining. The images shown were taken from the cerebellum, pons, hippocampus, and cortex as indicated. Scale bars = 50 microns.

Figure 3

LAMP-1 levels are enhanced throughout α-Gal A-deficient brain and exhibit vascular localization. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a-d, i-l, q-t, y-bb) or -/0 (e-h, m-p, u-x, cc-ff) mice were immunolabeled with an antibody against LAMP-1 (a, e, i, m, q, u, y, cc), and fluorescent potato lectin (b, f, j, n, r, v, z, dd) and counterstained with bisbenzimide (c, g, k, o, s, w, aa, ee). Red arrowheads (a, e) indicate areas of intense vascular LAMP-1 immuoreactivity and green arrowheads (b, f) indicate vascular staining in similar locations. White arrows (d, h, p, x, ff) indicate the preferential localization of LAMP-1-positive staining to the vascular endothelium. The images shown were taken from the cerebellum (CB), pons (PONS), hippocampus (HIP), and cortex (CTX) as indicated. Scale bars = 50 microns.

Figure 4

Representative ultrastructural analysis of α-Gal A-deficient and wild-type mouse brain. Ultra-thin sections of the cerebellum (CB; panels a, b, d, e, g, h) and cortex (CTX; panels c, f, i) of 21 month-old α-Gal A hemizygous (-/0; panels d-i) and α-Gal A wild-type (+/0; panels a-c) mice were prepared for electron microscopic analysis. Brain ultrastructure from the α-Gal A +/0 mouse exhibited normal appearing blood vessels (panel a) and perikarya (panel b) and neuronal cells with normal appearing nuclei and cytoplasm (panel c). The endothelial cell lining a blood vessel in α-Gal A -/0 cerebellum contains a prominent inclusion (panel d; panel g represents inset box) exhibiting electron densities (arrowheads). In both cerebellum (panel e; panel h represents inset box) and cortex (panel f; panel i represents inset box) there appeared numerous lipopigment aggregates consisting of lipid droplets associated with electron-dense osmiophilic material. Scale bars = 500 nm.

Figure 5

Aggregates immunopositive for phosphorylated α-synuclein in the pons of α-Gal A-deficient brain. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a, b, f) or -/0 (c, d, g) mice were immunolabeled with an antibody against phosphorylated α-synuclein. Panels a-d show similar regions within the pons, and (b and d) are 8x magnifications of the boxes in (a and c). Black arrowheads indicate areas of intense phosphorylated α-synuclein immunoreactivity (d). Phosphorylated-α-synuclein aggregates above 10 microns in diameter were counted per field and graphed in panel e, which shows that none were present in α-Gal A +/0 pons while α-Gal A -/0 pons demonstrated an average of approximately 5 aggregates per field. Panels (f and g) show similar perinuclear phosphorylated α-synuclein staining patterns in the hippocampus between α-gal A +/0 (f) and -/0 (g) brains. Scale bars = 100 microns (a-d), 50 microns (f, g).

Figure 6

Phosphorylated α-synuclein aggregates localized to axonal spheroids specific to the α-Gal A-deficient brain. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a) or -/0 (b) mice were stained with hematoxylin and eosin and serial sections from α-Gal A -/0 mice in the same group were stained with either hematoxylin and eosin (d) or an antibody against phosphorylated α-synuclein (e). H&E staining indicates large eosinophilic axonal spheroids in α-gal A -/0 brains (white arrows, b) that are not evident in α-gal A +/0 (a). This observation is quantified in (c), comparing the number of axonal spheroids counted per field between α-Gal A +/0 and α-Gal A -/0 mice (n = 3). Panels d and e are serial sections demonstrating axonal spheroids (white arrows, d) that are immunopositive for phosphorylated-α-synuclein (white arrows, e) while panel (f) demonstrates the percentage of axonal spheroids that were positive for α-synuclein in serial sections (n = 3). Scale bars = 50 microns.

Figure 7

Phosphorylated α-synuclein lesions in the α-Gal A-deficient pons co-localize with ubiquitin and LC3. Sagittal brain sections from male 20- to 24-month-old α-Gal A +/0 (a-d) or -/0 (b-l) mice were immunolabeled with antibodies against phosphorylated α-synuclein (a, e, i) and ubiquitin (b, f) or LC3 (j). Colored arrowheads indicate large lesions containing phosphorylated α-synuclein, ubiquitin, or LC3, and arrows indicate co-localization of large phosphorylated α-synuclein-containing lesions with either ubiquitin (h) or LC3 (l). Co-localization analysis was performed using the thresholded Manders test. Manders co-localization between ubiquitin (M1) and phosphorylated-α-synuclein (M2) was calculated to be (tM1 = 0.506 and tM2 = 0.856, Costes p-value = 1, n = 3), indicating strong co-localization of phosphorylated-α-synuclein signal with ubiquitin in the pons of α-Gal A -/0 mice. Manders co-localization between phosphorylated-α-synuclein (M1) and LC3 (M2) was calculated to be (tM1 = 0.499 and tM2 = 0.765, Costes p-value = 0.958, n = 3), indicating strong co-localization of LC3 signal with phosphorylated-α-synuclein in the pons of α-Gal A -/0 mice. Scale bars = 50 microns.