Fig. 2.

Quantification and visualization of glycosphingolipids stored in mouse brain. Glycosphinglolipids (GSLs) in untransduced and transduced Sandhoff mouse brain were analyzed by high-performance thin-layer chromatography (A and B) and electron microscopy (C). (A) GSLs were extracted from a wild type aged 21 weeks (lanes 1, 2, and 13), an untransduced Sandhoff mouse aged 16 weeks (lanes 3, 4 and 14), or the brains of Sandhoff mice transduced with rAAVα+β at a single site in the right striatum (lanes 5–12 and 14–18). Vector was injected at 4 weeks of age; the animals were killed at 16 (lanes 5, 6, and15), 20 (lanes 7, 8, and 16), 24 (lanes 9, 10, and 17), and 30 weeks of age (lanes 11, 12, and 18). Right (lanes 1, 3, 5, 7, 9, and 11) and left cerebrum (lanes 2, 4, 6, 8, 10, and 12) and cerebella (lanes 13–18) were dissected and individually analyzed. Pure GM1, GM2, and GA2 gangliosides and the myelin component, galactocerebroside (Galc), were used as standards (STD). (B) GA2 and GM2 content was quantified densitometrically and is represented as the percentage of the content in the untreated Sandhoff mouse, after correcting for loading differences, by using the internal Galc standard. Storage was diminished in all treated Sandhoff brains but increased progressively with age. (C) Neuronal ultrastructure in brain sections from wild-type (d), untransduced (c), and singly rAAVα+β-transduced Sandhoff mice (a and b). A single striatal injection of viral vector was given at 4 weeks, and the tissue harvested at 16 weeks of age. Neurons in the transduced ipsilateral cerebral cortex had no membranous cytoplasmic cell bodies (b), whereas those in the contralateral cortex (a) were distended by the storage vesicles (arrowheads in a) with distortion of the nuclei, as in untreated Sandhoff animals (c). N, nucleus. (Scale bar: 2 μm.)