Figure 3: APP is a natural substrate of Neu1 and is abnormally processed in Neu1^−/− lysosomes.

(a,b) APP levels are increased in Neu1^−/− total hippocampal (HIP) lysates as demonstrated by immunoblots (a) and by densitometric analysis (b). (c) APP was immunoprecipitated from WT and Neu1^−/− HIP and probed on blots with Sanbucus nigra lectin (SNA). (d) Equal amounts of HIP lysates were treated with N-glycanase, sialidase and probed on blots with anti-APP N-terminal antibody. (e) Immunoblots of WT and Neu1^−/− HIP-enriched LF shows APP accumulation in the Neu1^−/− samples. (f) Quantification of e. (g,i) β-CTF levels are increased in Neu1^−/− HIP lysates and Neu1^−/− HIP-enriched LF, respectively. (h,j) Quantification of g and i. (k) Representative Aβ peptide staining in WT and Neu1^−/− brain sections shows immunoreactivity only in Neu1^−/− specimens (mouse-specific pan-Aβ-antibody used on 5-month-old brain sections). Scale bar, 20 μm. (l,m) Levels of mouse Aβ42 assayed in the cerebrospinal fluid (CSF) and in the culture medium of neurospheres, respectively. Normalized APP and CTFs refer to the ratio of WT versus Neu1^−/− bands. These were calculated by densitometric analysis of single bands on immunoblots probed with anti-APP or β-CTF antibodies, and amido black bands and anti-tubulin in WT and Neu1^−/− samples. Red asterisks in c denote oversialylated bands. Asterisks on graphic bars indicate statistically significant results, as determined by the Student’s t-test. Data are represented as the mean±s.d. (error bars); n=3.