Isoform specificity in the processing of APP and generation of Aβ by the Numb proteins. A, effect of Numb proteins on the amounts of APP and TfR. Left, representative immunoblot showing total levels of APP and TfR protein in the indicated PC12 clones. Each lane was loaded with 50 μg of protein. The relative amounts of APP and TfR were normalized to those of actin. Densitometric analyses were performed on the immunoblots (right). The values in the histogram represent the mean ± S.D. n = 3; ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to empty vector-transfected cells. B, effect of Numb proteins on the cleavage of APP. APP derivatives in the conditioned medium and in lysates of the indicated PC12 clones were quantified by Western blot followed by densitometric analyses. APP derivatives in conditioned culture medium were immunoprecipitated with an antibody to the amino-terminal domain of the APP protein (22C11), which precipitates both sAPPα and sAPPβ, and an antibody to Aβ (6E10; Signet), which recognizes sAPPα. Steady-state levels of membrane-associated APP derivatives, C99 and C83, were immunodetected with antibodies to the carboxyl-terminal domain of the APP protein. The β-actin signal represents the internal loading control. Densitometric analyses were performed on the immunoblots (left). The values in the histogram represent the mean ± S.D.; n = 3; ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to vector-transfected control. Essentially similar results were obtained using two additional stable clones. C, effect of Numb proteins on the generation of Aβ peptides. SY5Y cells stably expressing the Swedish mutation of APP were transfected with plasmids encoding the FLAG-Numb proteins. Conditioned media were harvested and analyzed by a sandwich ELISA for specific quantitation of Aβ40 and Aβ42. Expression of FLAG-Numb proteins was detected by immunoblotting using an antibody to FLAG (left). The histogram shows the amounts of Aβ40 and Aβ42 in the indicated clones (right). The values represent the mean ± S.D.; n = 3; ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to vector-transfected cells.

Isoform specificity of Numb effects on APP metabolism is dependent on the interaction of the Numb proteins with APP and its subsequent internalization. A, expression of a Numb protein lacking the PTB (ΔPTB-Numb) did not alter APP metabolism. Left, representative immunoblot showing total levels of APP holoprotein in the indicated PC12 clones. Each lane was loaded with 50 μg of protein. APP was quantified by Western blot with an antibody to the amino-terminal domain of the APP protein, followed by densitometric analyses (right). The relative amounts of APP were normalized to those of actin. B, inhibition of endocytosis by overexpression of mutant dyn 1K44E abolished the effects of the Numb proteins on APP metabolism. Twenty-four hours after transduction, cell lysates were analyzed by immunoblotting, followed by densitometry (right). A representative immunoblot shows total levels of APP in the indicated PC12 clones. Overexpression of the dyn 1K44E was confirmed using antibodies raised to dyn 1 and to the hemagglutinin tag. The β-actin signal represents the internal loading control. The values in the histogram represent the mean ± S.D. n = 3; ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to vector-transfected cells. C, localization of APP and Numb in PC12 cells by confocal immunofluorescence microscopy. Following fixation and permeabilization, rabbit anti-Numb- and goat anti-rabbit-conjugated fluorescein isothiocyanate, mouse anti-APP (22C11), and goat anti-mouse-conjugated CY3 secondary antibody were used to detect Numb and APP protein, respectively. The right panel shows the merged image of APP and Numb. D, intracellular accumulation of APP and membrane-associated APP derivatives in a SPTB-Numb (left) and LPTB-Numb clone (right). Following fixation and permeabilization, APP was detected with antibodies to the amino- and carboxyl-terminal domains of the APP protein. E, localization of APP to endocytic compartments by confocal immunofluorescence microscopy. Following fixation and permeabilization, rabbit anti-Rab5A and goat anti-rabbit conjugated fluorescein isothiocyanate were used to detect the early endosomes (green signal), and mouse anti-APP (22C11) and goat anti-mouse-conjugated CY3 secondary antibody were used to detect APP protein (red signal). The right side of each image shows the merged image of APP and Rab5, depicting early endosomes. The images are representative of those obtained from at least three stably transfected clones.

Effect of the Numb proteins on the sorting of APP to the degradative pathway. A, time course of the accumulation of APP and APP-CTFs in LPTB-Numb but not SPTB-Numb clones after treatment with a lysosomal inhibitor. The indicated stable PC12 clones were treated with choroquine (100 μm) for 0, 2, and 6 h. Equal amounts of cellular lysates were separated by 16.5% Tris-Tricine SDS-PAGE and immunoblotted with an antibody to the carboxyl terminus of APP. β-Actin was used as the internal loading control. Densitometric analyses were performed on the immunoblots (bottom). The values in the histogram are the mean ± S.D. of three independent measurements. ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to vector-transfected cells. Similar results were obtained using two additional stably transfected Numb clones. B, time course of the accumulation of APP in LPTB-Numb but not SPTB-Numb clones upon treatment with NH₄Cl. The indicated clones were treated with NH₄Cl (50 μm) for 0, 2, 6, and 8 h and processed for immunoblotting using an antibody to the carboxyl-terminal of APP. C, time course of the accumulation of APP and APP-CTFs in SH-SY5Y cells overexpressing the empty vector (Vector) or a vector encoding the Swedish mutant APP (APP) upon chloroquine treatment. The indicated SY-SY5Y clones were treated with choroquine (100 μm) for 0, 2, and 6 h. Each lane was loaded with 50 μg of protein. β-Actin was used as the internal loading control. D, time course of the accumulation of APP in the SPTB-Numb but not LPTB-Numb clones after treatment with the γ-secretase inhibitor DAPT. The indicated clones were treated for 0, 2, 6, and 12 h with 10 μm DAPT. Each lane was loaded with 50 μg of protein. β-Actin was used as the internal loading control. Densitometric analyses were performed on the immunoblots (bottom). Values are the means ± S.D. of three independent measurements. ^*, p < 0.01 (ANOVA with Scheffe post hoc tests) relative to empty vector-transfected cells. E, inhibition of the proteasomes failed to affect the regulation of APP metabolism by the Numb proteins. Time course of the levels of APP and APP-CTFs in the indicated clones after treatment with 10 μm lactacystin. Equal amounts of protein from each sample were immunoblotted to detect APP and APP-CTFs. Each lane was loaded with 50 μg of protein and verified with an antibody to actin. Essentially similar results were obtained using two additional stably transfected Numb clones.

Detection of alternatively spliced Numb transcripts under normal and stressed conditions. A, domain structure of the Numb protein. The 11-amino acid insertion in the PTB is indicated in black. The forward (F) and reverse (R) primers were designed to amplify a region flanking the PTB insertion. The specificity of the primer set was confirmed by performing PCR using an empty plasmid (pCDNA3.1) or plasmids containing each of the four Numb isoforms. The expected sizes of PCR fragments were 114 and 147 base pairs for the SPTB- and LPTB-Numb transcripts, respectively. B, time course of SPTB-Numb and LPTB-Numb mRNA levels in PC12 cells subjected to TFW. C, densitometric analyses were performed on the data shown B. The values in the histogram are the mean ± S.D. of three independent measurements. ^*, p < 0.01 (Student's t test) relative to untreated control PC12 cells. D, effect of stress-induced Numb isoform change on APP processing. Top, semiquantitative real time PCR analysis of APP mRNA levels in PC12 before and after TFW for the indicated times. The glyceraldehyde-3-phosphate dehydrogenase signal represents the internal loading control. Bottom, representative immunoblots showing total levels of APP in PC12 cells left untreated (Con) or subjected to TFW for 24 h. Each lane was loaded with 50 μg of protein. β-Actin was used as the internal loading control. Densitometric analyses were performed on the immunoblots. The values in the histogram represent the mean ± S.D. of three independent measurements. ^*, p < 0.05 (Student's t test) relative to nontreated cells. E, effect of stress-induced Numb isoform change on the generation of Aβ. PC12 cells were left untreated (Con) or subjected to TFW for 24 h. Conditioned media were harvested and analyzed by a sandwich ELISA for specific quantitation of Aβ40. The histogram shows the relative amounts of Aβ40. The values represent the mean ± S.D.; n = 3; ^*, p < 0.01 (Student's t test) relative to nontreated control cells.

Lack of isoform specificity on the expression of APP and the activities of the APP secretases. A, expression of the Numb proteins did not alter the expression of APP. Left, semiquantitative real time PCR analysis of APP mRNA in the indicated PC12 clones. The glyceraldehyde-3-phosphate dehydrogenase signal represents the internal loading control. The values in the histogram are the mean ± S.D. of three independent measurements (right). Essentially similar results were obtained using two additional stably transfected clones. B, expression of the Numb proteins did not alter the activities of APP-processing secretases. The lysates were tested for secretase activities by the addition of secretase-specific peptide substrates conjugated to the reporter molecules EDANS and DABCYL (R&D Systems). Fluorescently labeled peptides were detected only upon cleavage by the respective secretases. After incubation at 37 °C for 2 h, reactions were transferred to a 96-well plate, and fluorescence was measured as described under “Experimental Procedures.” The values expressed as arbitrary units of fluorescence (AUF) are the mean ± S.D. of at least three independent experiments. C, Numb proteins did not alter the protein level of β-secretase (BACE1) and of the components of the γ-secretase complex. Representative immunoblots show total levels of β-secretase, presenilin-1, nicastrin, Aph-1, and Pen-2 in the indicated clones. Each lane was loaded with 50 μg of protein. Blots were reprobed with an antibody against actin to confirm equal levels of protein loading among samples.

Effect of the Numb proteins on the sorting of APP to the recycling pathway. A, the Numb proteins differentially alter the amount of cell surface APP. Immunostaining of cell surface APP with an antibody to the amino-terminal end of APP is shown. In nonpermeabilized cells (without Triton X-100), significantly more APP is detected on the surface of a SPTB-Numb clone compared with a LPTB-Numb clone or a clone transfected with empty vector. B, detection of cell surface APP by biotinylation. Cells were surface-biotinylated, lysed, and incubated with streptavidin-agarose beads. Biotinylated proteins were immunoblotted with antibodies to APP and TfR. C, quantitative data showing the ratio of cell surface APP and TfR in the indicated clones. Values are the mean ± S.D. of at least three independent experiments. ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests) relative to vector-transfected cells. D, recycling blockade depletes cell surface APP protein level. The indicated clones were pretreated with monensin for 60 min at 37 °C in Dulbecco's modified Eagle's medium/bovine serum albumin and incubated in Dulbecco's modified Eagle's medium/bovine serum albumin for 60 min afterward. Cells were then surface-biotinylated using a membrane-impermeable cross-linker and immunoprecipitated with streptavidin beads. Precipitates were subjected to SDS-PAGE, and blots were probed with antibodies to APP and TfR. E, quantitative data showing the changes in the ratio of cell surface APP and TfR in the vector-transfected and a SPTB-Numb clone after monesin treatment. Values are plotted as percentage of untreated control (vehicle) and are the mean ± S.D. of at least three independent experiments. ^*, p < 0.05 (Student's t test) relative to vector-transfected cells.

Effect of the Numb proteins on the levels of small Rab GTPases. A, representative immunoblots showing total amounts of Rab4A, Rab5A, Rab7, Rab11, and LAMP1 in the indicated clones. Each lane was loaded with 50 μg of protein and verified with an antibody to actin. B, densitometric analyses were performed on the immunoblots shown in A. The values in the histogram are the mean ± S.D. of three independent measurements. ^*, p < 0.01; #, p < 0.05 (ANOVA with Scheffe post hoc tests). C, semiquantitative real time PCR analysis of Rab5A mRNA in the indicated PC12 clones. The glyceraldehyde-3-phosphate dehydrogenase signal represents the internal loading control.

Summary diagram showing the effects of the alternatively spliced Numb isoforms on the trafficking and processing of APP.