Article Figures & Data
Figures
- Figure 1.
β-Glucuronidase distribution at 4 weeks after intraventricular injection. β-glucuronidase activity was revealed by in situ assay. Enzyme expression is observed throughout the brain, bilaterally, with highest staining density along the ventricular system. A, Low-power photomicrograph showing β-glucuronidase activity throughout the brain of treated MPS VII mice. Scale bar, 400 μm. B-G, Higher magnification of selected regions including the olfactory bulb (B; arrow, positive cell in granule cell layer), the RMS (C), the ependyma (EP) and corpus callosum (D), the CA3 region of the hippocampus (E), the thalamus (F), and the cerebellum (G). Some positive cells are associated with small vessels (arrowheads). Scale bar, 50 μm. LV, Lateral ventricle; 3V, the third ventricle; Hi, hippocampus; CC, corpus callosum; Th, thalamus; 4V, the fourth ventricle; CB, cerebellum, Co, cortex.
- Figure 2.
β-Glucuronidase-positive cells are in close association with the brain vasculature. A-C, Immunohistochemistry for β-glucuronidase (A; red) and PCAM CD31 (B; green), an endothelial cell marker, reveals close association but not overlap (C; merge). Arrowheads denote β-glucuronidase-positive cells.
- Figure 3.
β-Glucuronidase activity levels in the brain. Data are shown as percentage of heterozygous levels and are mean ± SEM. Het, Heterozygotes; Hi, hippocampus; Co, cortex; CB, cerebellum; BS, brainstem. Error bars represent SEM.
- Figure 4.
The effects of AAV4βgluc delivery on substrate levels in MPS VII mouse brain. A-H, Substrate levels were assessed using anti-chondroitin sulfate (A-D) and anti-heparan sulfate (E-H) antibodies. A, C, E, G, In untreated MPS VII mice, substrate accumulation was notable in the extracellular spaces and cytoplasm. B, D, F, H, At 4 weeks after injection, chondroitin and heparan sulfate levels in AAV4βgluc-treated MPS VII mice decreased and were indistinguishable from sections harvested from normal littermates. Scale bar, 50 μm.
- Figure 5.
CNS pathology is improved after AAV4βgluc delivery to the lateral ventricles of MPS VII mice. A-M, Lysosomal storage is observed throughout the brain in untreated (A, C, E, G, I, K, M) but reduced in treated (B, D, F, H, J, L, N) mutant mice. G-J, The hippocampus (G, H) and the striatum (J) are nearly devoid of remaining storage 4 weeks after gene transfer. A-N, Evaluation of the ependyma (A, B), meninges (C, D), cerebral cortex (E, F), cerebellum (K, L), and olfactory bulb (M, N) shows extensive improvements in lysosomal distention after AAV4βgluc delivery. Scale bar, 20 μm.
- Figure 6.
AAV4βgluc reduces lysosomal storage vacuoles in MPS VII mice brain. Cells in the hippocampus, cerebral cortex, and striatum were assessed for the presence or absence of storage vacuoles. Data are presented as percentage of cells ± SEM. Error bars represent SEM.
- Figure 7.
AAV4βgluc reverses behavioral deficits in MPS VII mice in context fear-conditioning assays. One-way ANOVA revealed group (n = 3, heterozygous, treated, and untreated MPS VII mice) as a significant factor (F(2,220) = 10.754; p < 0.0001). A, Baseline measurements in context 1, day 1. Both treated and untreated MPS VII mice have significant elevated baseline levels of freezing compared with heterozygous controls (*p < 0.0001; Fisher's PLSD). B, The increase in freezing time from baseline after placement in context 1 on day 2. Results from treated MPS VII mice and heterozygotes are comparable (p = 0.63; Fisher's PLSD). Increases in freezing time for untreated MPS VII mice were significantly less than the other groups (*p < 0.0001; Fisher's PLSD). C, Difference in freezing time between context 1 and 2, day 2. Compared with untreated MPS VII mice, treated MPS VII mice and heterozygous controls freeze significantly less in context 2 compared with context 1 (*p < 0.0001; Fisher's PLSD). Error bars represent SEM.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplementary Figure. Brain tissues harvested from normal mice have no detectable chondroitin sulfate- and minimal levels of heparan sulfate-immunoreactivity. (A, B), chondroitin sulfate immunoreactivity in the cerebral cortex and ependyma, respectively. (C, D), heparan sulfate immunoreactivity in the hippocampus and striatum, respectively. IHC with secondary antibody only were also negative (data not shown).
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