Comparative analysis of brain lipids in mice, cats, and humans with Sandhoff disease

Abstract

Sandhoff disease (SD) is a glycosphingolipid (GSL) storage disease that arises from an autosomal recessive mutation in the gene for the beta-subunit of beta-Hexosaminidase A (Hexb gene), which catabolizes ganglioside GM2 within lysosomes. Accumulation of GM2 and asialo-GM2 (GA2) occurs primarily in the CNS, leading to neurodegeneration and brain dysfunction. We analyzed the total lipids in the brains of SD mice, cats, and humans. GM2 and GA2 were mostly undetectable in the normal mouse, cat, and human brain. The lipid abnormalities in the SD cat brain were generally intermediate to those observed in the SD mouse and the SD human brains. GM2 comprised 38, 67, and 87% of the total brain ganglioside distribution in the SD mice, cats, and humans, respectively. The ratio of GA2-GM2 was 0.93, 0.13, and 0.27 in the SD mice, cats, and humans, respectively, suggesting that the relative storage of GA2 is greater in the SD mouse than in the SD cat or human. Finally, the myelin-enriched lipids, cerebrosides and sulfatides, were significantly lower in the SD brains than in the control brains. This study is the first comparative analysis of brain lipids in mice, cats, and humans with SD and will be important for designing therapies for Sandhoff disease patients.

Figure 1

HPTLC analysis of cortical gangliosides in normal and Sandhoff disease mice (M), cats (C), and humans (H). The amount of ganglioside spotted per lane was equivalent to 1.5 μg sialic acid. The plate was developed in one ascending run with chloroform: methanol: 0.02% aqueous calcium chloride (55:45:10 by vol). Gangliosides were visualized with resorcinol-HCl spray.

Figure 2

Ratio of GA2 to GM2 in Sandhoff disease mice (N = 3), cats (N = 4), and humans (N = 2). Values represent mean + SEM, except for the human Sandhoff samples, where the value represents mean + interquartile range.

Figure 3

HPTLC of cortical neutral lipids in normal and Sandhoff disease mice (M), cats (C), and humans (H). The amount of neutral lipids spotted per lane was equivalent to approximately 70 μg brain dry weight. The plate was developed to a height of 4.5 cm with chloroform: methanol: acetic acid: formic acid: water (35:15:6:2:1 by volume), then developed to the top with hexanes: diisopropyl ether: acetic acid (65:35:2 by volume). The bands were visualized by charring with 3% cupric acetate in 8% phosphoric acid solution. CE, cholesterol esters; TG, triglycerides; IS, internal standard; Chol, cholesterol; CM, ceramide; CB, cerebroside (doublet, indicated by arrow); PE, phosphatidylethanolamine; PC, phosphatidlycholine; SM, sphingomyelin; GA2, asialo GM2; LPC, lyso-phosphatidylcholine.

Figure 4

HPTLC of cortical acidic lipids in normal and Sandhoff disease mice (M), cats (C), and humans (H). The amount of acidic lipids spotted per lane was equivalent to approximately 200 μg brain dry weight. The plate was developed to a height of 6 cm with chloroform: methanol: acetic acid: formic acid: water (35:15:6:2:1 by volume), then developed to the top with hexanes: diisopropyl ether: acetic acid (65:35:2 by volume). The bands were visualized by charring with 3% cupric acetate in 8% phosphoric acid solution. FA, fatty acids; IS, internal standard; CL, cardiolipin; PA, phosphatidic acid; Sulf, sulfatides (doublet, indicated by arrow); PS, phosphatidylserine; PI, phosphatidyinositol.

Figure 5

Concentration of cerebrosides and sulfatides as a percentage of normal in Sandhoff disease mice (N = 3), cats (N = 4), and humans (N = 2). Values represent mean + SEM, except for the human samples, where the value represents mean + interquartile range. Trace amounts of cerebrosides and sulfatides were detected in the SD human samples.