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Figure 6 | Molecular Neurodegeneration

Figure 6

From: LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer’s disease

Figure 6

LRP10 affect the trafficking of APP in neuronal SH-SY5Y cells. (A-F) LRP10 colocalized and altered the distribution of endogenous APP. SH-SY5Y cells were stably transfected with empty pcDNA3 vector (A, D) or high levels of HA-tagged-LRP10wt (B, E) or -LRP102DXXAA (C, F). Cells were fixed, permeabilized, and immunostained with anti-APP (green), anti-HA (red), and anti-TGN46 or anti-EEA1 (blue) antibodies. The labeled cells were examined by confocal fluorescence microscopy. (A, D) In control cells (pcDNA3), endogenous APP was detected mainly in the Golgi region where it partially overlapped with TGN46 (A, inset arrowheads) and surrounding vesicles where it partially overlapped with EEA1 (D, inset, arrowheads). (B, E) LRP10wt-HA was detected in the TGN (B, inset) and surrounding EEA1-labeled endosomes (E, inset). Endogenous APP was mainly detected in the Golgi region, where it partially overlapped with LRP10wt-HA (B, inset). The merged image shows a clear overlap between LRP10wt-HA and endogenous APP in the TGN (B, arrowheads, insets). (C, F) LRP102DXXAA-HA was redistributed mainly to peripheral EEA1-labeled early endosomes (F, inset). Endogenous APP was also detected in peripheral EEA1-labeled endosomes (F, inset), where it colocalized with HA-tagged LRP102DXXAA (F, arrowheads, inset). Scale bar, 10 μm. (G) The level of cell surface APP was altered by the expression of LRP10. SH-SY5Y cells stably expressing pcDNA3 vector or high levels of HA-tagged-LRP10wt or -LRP102DXXAA were surface-biotinylated with sulfo-NHS-SS-biotin. Biotinylated proteins were precipitated with neutravidin, and samples (Surface proteins) were analyzed by immunoblotting with antisera directed against HA or APP. The total cell lysate (Total proteins) was also analyzed to assess APP and LRP10 expression levels. The three APP variants (APP695, APP751, and APP770) were detected by the anti-APP antibodies. The asterisk indicates an accumulation of mature APP (glycosylated APP751/770) in the presence of LRP10wt. Actin was used as a loading control. (H) The maturation of APP was altered in SH-SY5Y cells expressing LRP10wt. SH-SY5Y control cells or cells stably expressing LRP10wt or LRP102DXXAA were pulse-labeled with [35 S]methionine for 5 min and chased at 37 °C for the indicated time. Radiolabeled APP was immunoprecipitated from the cell extracts and was analyzed by SDS-PAGE and autoradiography. The three APP variants (APP695, APP751, and APP770) are indicated by arrowheadss. Accumulations of mature APP (*, indicates mature APP751/770; +, indicates mature APP695) were observed in cells expressing LRP10wt. Graph of the ratio of mature (m) APP versus immature (im) APP in the indicated SH-SY5Y stable clones as determined by densitometric scanning of pulse-chase experiments exemplified in the autoradiogram above. The results indicated that there is a delayed turnover of the mature, fully glycosylated APP variants when they are co-expressed with LRP10wt. Results are expressed as means ± SD (n = 3). *, p < 0.05; **, p < 0.005 (compared to control cells).

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