Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III)

Abstract

Deficiency of arylsulfatase G (ARSG) leads to a lysosomal storage disease in mice resembling biochemical and pathological features of the mucopolysaccharidoses and particularly features of mucopolysaccharidosis type III (Sanfilippo syndrome). Here we show that Arsg KO mice share common neuropathological findings with other Sanfilippo syndrome models and patients, but they can be clearly distinguished by the limitation of most phenotypic alterations to the cerebellum, presenting with ataxia as the major neurological finding. We determined in detail the expression of ARSG in the central nervous system and observed highest expression in perivascular macrophages (which are characterized by abundant vacuolization in Arsg KO mice) and oligodendrocytes. To gain insight into possible mechanisms leading to ataxia, the pathology in older adult mice (>12 months) was investigated in detail. This study revealed massive loss of Purkinje cells and gliosis in the cerebellum, and secondary accumulation of glycolipids like GM2 and GM3 gangliosides and unesterified cholesterol in surviving Purkinje cells, as well as neurons of some other brain regions. The abundant presence of ubiquitin and p62-positive aggregates in degenerating Purkinje cells coupled with the absence of significant defects in macroautophagy is consistent with lysosomal membrane permeabilization playing a role in the pathogenesis of Arsg-deficient mice and presumably Sanfilippo disease in general. Our data delineating the phenotype of mucopolysaccharidosis IIIE in a mouse KO model should help in the identification of possible human cases of this disease.

Figure 1.

Different types of storage vacuoles in CNS cells. (A) Electron micrograph of a massively enlarged perivascular macrophage with several distended vacuoles, whose major content is washed out during preparation and an electron-dense core. (B) Similar macrophage as depicted in (A) with additional lipid-rich lipofuscin (light grey). (C) PC perikaryon with a large vacuole containing heterogeneous material. (D) Phagocytosing microglia/macrophage from the molecular layer of the cerebellum filled with debris and different types of phagolysosomes. (E) Storage body from the soma of a PC with partially lamellar and partially granular appearance. (F) Typical large storage body in the PC soma with irregular, lamellar electron-dense structure with similarity to ceroid lipofuscin with structured areas. (G) PC dendrite abundantly vacuolated with heterogeneous, partially water-soluble and partially electron-dense material ranging from structured to unstructured areas. Scale bars: a = 2.5 µm; b, c, d and g = 5 µm; e = 250 µm; f = 1 µm.

Figure 2.

Expression of ARSG in the murine central nervous system. (A) Immunoblot analysis of different brain areas (Cb = cerebellum, Hc = hippocampus, Cx = cortex, Ob = olfactory bulb, Bs = brain stem, Th = thalamus, Sc = spinal cord) shows highest expression of ARSG in the spinal cord and the brain stem and lowest expression in the olfactory bulb. Homogenates from Arsg KO mice (−/−) served as controls for specificity of the antibody. ARSG-specific bands are labelled with arrows. A non-specific band also present in KO mice is labelled with an asterisk. (B) ARSG was specifically detected with a polyclonal antibody in wild-type sections with particularly high expression in the anterior horn of the spinal cord and the inferior colliculus. Absence of specific DAB staining reveals specificity of the antibody. Scale bars = 200 µm (C) Double immunofluorescence shows co-localization of ARSG and LAMP1 with additional cytoplasmic staining for ARSG in cells of the spinal cord. ARSG staining is absent in the spinal cord of KO mice. Scale bars = 15 µm. (D) Double immunofluorescence staining of ARSG with markers for different CNS cell types reveals modest expression in microglia (Iba1⁺), highest expression in perivascular macrophages (CD68⁺) but no expression in astrocytes (GFAP⁺). Oligodendrocytes (Olig-2⁺ and CNPase⁺) reveal extensive expression of ARSG, while neurons (NeuN⁺) reveal only subtle staining. Endothelial (Pecam-1⁺) cells lack ARSG staining. All pictures from the inferior colliculus, except CNPase (granular layer of the cerebellum). Scale bars = 15 µm. (E) Semi-quantitative grading of the ARSG expression in wild-type cells and comparison with lysosomal storage phenotype in Arsg KO mouse cells reveal that highest expression in perivascular cells is paralleled with the highest vacuolization. However, oligodendrocytes with high expression do not reveal signs of lysosomal storage. ⁽¹⁾not determined due to non-specific antibody reactivity in PCs; ⁽²⁾lysosomes containing phagocytosed debris were considered to be unrelated to the primary enzyme defect.

Figure 3.

Myelination of the cerebellum of Arsg KO mice. (A) Myelination in general is not severely impaired in 20-month-old mice as determined by immunoblot for myelin basic protein (MBP) of cerebellar extracts and immunofluorescence staining as shown in (B) (white matter tracts of the cerebellum). Scale bars = 80 µm. (C) White matter tracts contain activated microglia in contrast to resting microglia in WT mice. Scale bars = 40 µm. (D) Some activated, phagocytic microglia contain myelin figures, indicating phagocytosis of lipids. Scale bars = 1 µm.

Figure 4.

PC death, axonal spheroids and astrogliosis of the cerebellum of Arsg KO mice. (A) In 22-month-old animals, an apparent atrophy of the cerebellum is evident. (B) Double immunofluorescence for calbindin as a marker for PCs and GFAP, an astrocytic marker, reveals dramatic loss and depletion of PCs by hypertrophic astrocytes in the molecular layer of the cerebellum (as shown for layer VI). Scale bar = 75 µm. (C) PC axons from knockout animals regularly show axonal swellings (lower left, fiber tracts of the cerebellum) as determined by calbindin staining. Numerous PC have several swellings in single axons (right, PC layer/granular layer). Dendrites are abnormally enlarged (lower panel). Scale bars = 25 µm. (D) Electron micrographs of axonal spheroids range from small swellings with a diameter of 2 µm to larger spheroids with a diameter of up to 11 µm. Axonal spheroids are filled with heterogeneous material ranging from dense lysosomal structures and multivesicular bodies to mitochondria. Neurofilaments are abundantly present in spheroids. Scale bars: left = 5 µm, right = 1 µm. (E) Immunoblot analysis of cerebellar homogenates of 12- and 24-month-old animals reveals a significant decrease of calbindin which is almost complete at 24 months. GFAP in contrast is upregulated. *P < 0.05 and ***P < 0.001.

Figure 5.

Accumulation of gangliosides and cholesterol in the cerebellum of Arsg KO mice. (A) While GM2 ganglioside staining is undetectable in WT cerebellum, small vesicular GM2 labeling is observed in the ARSG KO cerebellum. (B) GM3 staining is dramatically increased in the molecular layer of the cerebellum in granular distribution in amoeboid cells, presumably phagocytic macrophages (age for GM2 and GM2: 22 months). Scale bar = 20 µm. (C) A clear increase in the amounts of free cholesterol (as determined by Filipin fluorescence staining) is evident already in young mice (6 months) with intense staining of PC somata and dendrites in a vesicular pattern. Filipin-positive perivascular macrophages are also evident at the sulci of the cerebellum (arrow heads). Scale bar = 200 µm.

Figure 6.

Accumulation of aggregated proteins in the cerebellum. (A) Western blots of detergent-soluble fractions of the cerebellum show no increase in LC3-II of 12-month-old and 24-month-old Arsg KO mice, but slight increase of p62 and phospho-p62 in detergent-insoluble extracts. (B) Electron micrograph of a PC axon reveals increased amounts of autophagosomes that are, however, rarely seen, whereas in the molecular layer of the cerebellum large filamentous structures (C) of up to 10 µm can be regularly found. Scale bars: b = 1 µm; c = 5 µm; inset = 1 µm. (D) Immunohistochemical staining of p62 and ubiquitin of the cerebellum reveals large aggregates with a diameter of up to 6 µm in the molecular layer of the cerebellum, which are absent in wild-type mice (age 22 months). Scale bar = 25 µm. (E) Co-immunofluorescence of p62 (red) and ubiquitin (green) reveals extensive co-localization. Scale bar = 10 µm. (F) In contrast, p62 aggregates do not co-localize at all or only weakly with Lamp1 (upper panel). They are often found in very close proximity to Lamp1-positive lysosomes (lower panels) and higher magnification reveals p62 decorating lysosomal membranes (arrow heads). Scale bars = 5 µm. (G) Co-localization with autofluorescent lipofuscin indicates similarly p62 decorating lipofuscin aggregates. Scale bar = 5 µm. (H) Western blot of detergent-insoluble cerebellar extracts from 12-month-old and 24-month-old animals for ubiquitin reveals increased levels of several ubiquitinated protein species of ∼25–35 kDa (labeled with a bracket).

Figure 7.

Secondary storage of SCMAS in the cerebral cortex but not the cerebellum. (A) Immunofluorescence staining of SCMAS in 22-month-old wild-type and Arsg KO mice reveals clearly increased amounts of SCMAS in layer V of the cerebral cortex, but comparable levels of SCMAS in the cerebellum of wild-type and Arsg KO mice. Scale bars = 100 µm (upper panel); 20 µm (lower panel). (B) SCMAS partially, but not completely, co-localizes with LAMP1 in cortical neurons of Arsg KO mouse as depicted by higher magnification. Scale bar = 10 µm.

Figure 8.

Comparison of microgliosis and astrogliosis of Sgsh KO mice (MPS IIIA) and Arsg KO mice (MPS IIIE). (A) Immunofluorescence staining of CD68, a marker for macrophages, and activated microglia reveals generalized microgliosis in the cerebral cortex, hippocampus and cerebellum of 10-month-old MPS IIIA mice, whereas microgliosis is restricted to the cerebellum of 24-month-old MPS IIIE mice. Note enlarged perivascular macrophages in MPS IIIE mice in the cerebral cortex and the hippocampus. (B) Microgliosis is accompanied by severe astrogliosis as revealed by GFAP staining in all regions of the CNS in MPS IIIA mice, but restricted to the cerebellum in MPS IIIE mice with intense Bergmann gliosis in the molecular layer and subtle white matter astrogliosis. Scale bars = 300 µm (70 µm in magnified panels).