Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation*

Background: Sterols can alter APP metabolism. Results: Cyclopamine, a phytosterol, alters APP-CTF degradation rate, decreases APP-CTF bioavailability for γ-secretase cleavage, and reduces Aβ and AICD generation. Conclusion: Cyclopamine decreases Aβ and AICD production by altering APP-CTF retrograde trafficking. Significance: Cyclopamine is a novel modulator of APP metabolism and trafficking, which can illuminate new avenues for Alzheimer disease treatment. Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-β (Aβ), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aβ is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aβ generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aβ generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.

Cell Culture and Transfection-HeLa cells were maintained at 37°C, 5% CO 2 in complete DMEM (Corning Glass) supplemented with 10% FBS (Atlanta Biologicals), 100 units/ml penicillin, and 100 g/ml streptomycin (Corning Glass), 2 mM L-glutamine (Corning Glass). Cells were grown to 80% confluence and serum-starved (0.5% FBS/DMEM) for 24 h prior to pharmacological or genetic manipulation. For pharmacological manipulation, drugs were diluted in 0.5% FBS/DMEM. For genetic overexpression experiments, cells were grown to 80% confluence and then transfected using with TurboFect transfection reagent (Thermo Scientific) according to the manufacturer's protocol. Culture media were removed 24 h post-transfection, and cells were collected or further treated with pharmacological agents (0.5% FBS/DMEM) for an additional 24 h.
Primary Neuron Culture-Primary cortical neuron cultures were isolated from postnatal day 1 (P1) Sprague-Dawley rat pups. Briefly, cortices were dissected out, minced, and treated with papain (100 units; Worthington) for 15 min at 37°C. Next, tissue was treated with type II-O trypsin inhibitor from chicken egg white (Sigma) for 15 min at 37°C. Tissue was washed with fresh Neurobasal medium (Invitrogen) supplemented with B-27 (Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Tissue was triturated, centrifuged at 1000 rpm for 10 min, and then resuspended in the fresh and complete Neurobasal medium. 2 ϫ 10 6 cells per 35-mm well were plated onto poly-DL-lysine (50 g/ml; Sigma)-coated tissue culture plates. Cortical neurons were treated with pharmacological agents on 6 days in vitro for 24 h, and lysates were collected for further biochemical analysis. All animals were used in accordance with animal protocols approved by the Institutional Animal Care and Use Committee (IACUC Protocol number 19787). Animals were delivered to and maintained at the Calhoun Animal Facility (Drexel University, PA). Animal procedures were performed strictly in accordance with the National Institutes of Health Guide for the care and use of Laboratory Animals approved by the Drexel University Animal Care and Use Committee.
Drosophila Stocks and Genetics-Drosophila husbandry was performed as described previously (37). For experiments utilizing the ␥-secretase reporter GMR-APP-Gal4; UAS-Grim/Cyo model (38), flies were crossed on standard cornmeal agar food supplemented with cyclopamine (100 nM) or DMSO vehicle control (0.1%), and flies were collected 24 h post-eclosion, and their compound eye was inspected. Assessment of penetrance and severity of the rough-eye phenotype was accomplished by photographing the compound eye using a Canon PowerShot S70 digital camera mounted to a Leica Mz 125 stereomicroscope. Severity of rough-eye phenotype was scored ϩ (mild) to ϩϩϩ (severe). One "ϩ" refers to where less than 1 ⁄ 2 of the eye was apoptotic and therefore appears "rough". A score of "ϩϩ" (moderate) defined increased penetrance, where apoptosis affected approximately 1 ⁄ 2 of the eye. Severe "ϩϩϩ" rough-eye phenotype described when more than 1 ⁄ 2 of the eye appeared rough, and eye size was significantly reduced. For objective quantification, five blinded laboratory personnel analyzed all experiments.
Immunoblotting-Lysates were collected in complete RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40) supplemented with Halt protease and phosphatase inhibitor and EDTA (Thermo-Fisher). Lysates were briefly cleared at 20,000 ϫ g at 4°C and stored at Ϫ20°C. Protein concentrations were determined using the BCA assay kit according to the manufacturer's protocol (Pierce). 40 g of lysate was supplemented with NuPAGE LDS sample buffer (Invitrogen) and heated to 75°C for 10 min. Protein was separated on 4 -12% NuPAGE BisTris gels (Invitrogen) using MES running buffer (Invitrogen) and then transferred onto Immobilon PVDF membrane (Millipore). Odyssey blocking buffer (Li-Cor Biosciences) was used for blocking and resuspending primary and secondary antibodies. Membranes were scanned using Li-Cor Odyssey infrared scanning instrument.
A␤ ELISA-HeLa cells and primary rat cortical neurons were treated with pharmacological agents for 24 h, and conditioned supernatants were collected and cleared at 20,000 ϫ g for 20 min at 4°C. Fresh cleared supernatants were used for A␤ 40 ELISA kit (Wako, Japan) according to the manufacturer's pro-tocol. Briefly, samples were diluted 1:1 using kit diluent and incubated overnight at 4°C. Samples were compared with the ELISA kit positive control and negative control (diluent alone). Samples were incubated and analyzed using a luminescence plate reader.
In Vitro ␥-Secretase Assay-We utilized a well established cell-free ␥-secretase activity assay that utilizes a fluorogenic peptide substrate corresponding to the APP ␥-secretase cleavage site (39,40). HeLa cells grown to 100% confluence in 150-mm culture dishes were collected in ice-cold PBS and pelleted at 5000 rpm for 5 min. The pellet was homogenized in 500 l of Buffer B (20 mM HEPES, pH 7.5, 150 mM KCl, 2 mM EGTA, protease and phosphatase inhibitors) using a 27-gauge needle. The resulting homogenate was cleared at 45,000 rpm at 4°C for 1 h. Supernatant was stored at Ϫ80°C, and pellet was washed with 500 l Buffer B and passed through 27 gauge needle on ice. The suspension was cleared again at 45,000 rpm for 1 h at 4°C. Supernatant was discarded and pellet resuspended in 75 l Buffer B ϩ 1% CHAPSO and passed through a 27-gauge needle on ice. The resulting membrane samples were solubilized on a rotator at 4°C for 2 h. Solubilized samples were cleared at 45,000 rpm for 1 h at 4°Cl; supernatant (total cell membrane) was collected and pellet discarded. Total protein was determined using BCA assay (Pierce) and 200 g of protein were used for in vitro ␥-secretase activity assay. Briefly, membranes were resuspended in ␥-secretase assay buffer (100 mM Tris-HCl, pH 6.8, 4 mM EDTA, 0.5% CHAPSO), and pretreated with vehicle control, L-685,458, or cyclopamine. Because the membrane preparation enriches total ␥-secretase in the sample, the amount of pharmacological agent was increased accordingly. Therefore, 20 M drug in a total vehicle volume of 1 l per was used. 150 l of total volume per well of a 96-well plate was used. Membranes were pretreated for 3 h at 37°C and 5% CO 2 , and then fluorogenic ␥-secretase substrate (Calbiochem, EMD Millipore) was added to membranes and further incubated at 37°C and 5% CO 2 for the indicated time points at which time membranes were removed and fluorescence was measured using a plate reader (Promega). BSA was used as negative control in place of membranes.
Subcellular Fractionation-HeLa cells grown to 80% confluence in 100-mm culture dishes were treated with 5 M cyclopamine or DMSO for 24 h, rinsed and collected in PBS, and then cleared at 1000 rpm for 7 min. The cell pellet was resuspended in homogenization buffer (250 mM sucrose, 150 mM NaCl, 25 mM Tris, 1 mM EDTA, protease and phosphatase inhibitor mixture) and homogenized using ball-bearing 12-m clearance cell buster. Homogenates were cleared at 1000 ϫ g for 15 min at 4°C, and post-nuclear supernatant was loaded into discontinuous density gradient (50, 30, and 10%) medium (Optiprep, Sigma) in Opti-Seal centrifuge tubes (Beckman). Homogenates were spun at 30,000 rpm for 19 h at 4°C and 300 l fractions collected.
Immunofluorescence-Cells were fixed using 4% paraformaldehyde, 0.1% Triton X-100, blocked in 2% BSA for 30 min, and incubated with primary antibodies overnight at 4°C. Cells were rinsed with PBS and stained with secondary antibodies at room temperature for 1 h, washed with PBS, and mounted. Cells were imaged using Olympus Fluoview 1000 inverted confocal microscope. Quantification of three-dimensional confocal image stacks was accomplished using SlideBook 5.0 or Volocity Image analysis software (PerkinElmer Life Sciences).
Surface Biotinylation-HeLa cells were treated with 5 M cyclopamine or DMSO for 24 h. Cells were placed on ice to halt membrane dynamics, rinsed with ice-cold PBS, and incubated with sulfo-NHS-SS-biotin (1 mg/ml in PBS; Thermo Scientific) for 40 min on ice with gentle rocking. Biotin was quenched with 100 mM glycine in PBS for 15 min. Cells were collected in PBS and pelleted at 500 ϫ g for 5 min at 4°C. The pellet was lysed in 200 l of standard RIPA lysis buffer containing protease and phosphatase inhibitors. Lysate was sheared using a 27-gauge needle on ice and solubilized for 2 h at 4°C on a rotator. Lysate was cleared by centrifugation at 10,000 ϫ g for 5 min, and 50 l from each sample was set aside for "total" protein analysis. The rest of the supernatant was loaded into a capped spin column (Pierce; 69725) with NeutrAvidin-coated agarose resin (Thermo Scientific) at a 1:1 ratio and incubated overnight at 4°C on a rotator. Columns were centrifuged at 10,000 rpm for 1 min, and flowthrough ("unbound" control) was collected and saved. Resin was washed several times with complete RIPA. Then 50 l of NuPAGE LDS Sample Buffer (Invitrogen) with 5% ␤-mercaptoethanol was loaded into each column and incubated for 30 min at room temperature on a shaker. To collect the surface-biotinylated protein, columns were centrifuged at maximum speed for 2 min. Biotinylated protein was separated on 4 -12% NuPAGE BisTris gels (Invitrogen) and then transferred, and the membrane was probed for surface APP. Nonbiotinylated lysates were collected as control samples. Biotinylated "surface" samples were compared with total lysate samples.
Statistical Analysis-All graphs and diagrams represent mean values Ϯ S.E. of all triplicates from at least three independent experiments. Either two-tailed or one-tailed Student's t test was used to compare two treatment groups and calculate significance from at least three independent experiments (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.005). For in vivo Drosophila melanogaster experiments, G test (goodness of fit) was used to determine significance of phenotypic penetrance in experimental populations. Degree of significance and corresponding p value criteria for G test were identical to previously mentioned Student's t test.

RESULTS
Cyclopamine Treatment Results in APP C-terminal Fragment Accumulation-To test whether cyclopamine modulates APP metabolism, we treated primary rat cortical neurons with cyclopamine (41, 42). We did not observe an appreciable change in the full-length APP (FL-APP) holoprotein after 24 h of 5 M cyclopamine treatment (Fig. 1, A and B). However, the 8 -12-kDa APP products of ␣and ␤-secretase (␣-and ␤-CTFs; collectively known as APP-CTFs) significantly increased when compared with vehicle control-treated neurons (p ϭ 0.0190)  -1744) antibody. E, NICD levels normalized to ⌬ENotch levels. F, APP-CTF-Gal4 and ⌬ENotch levels normalized to ␤-actin. G-J, representative images of rough-eye phenotype from GMR-APP-Gal4; UAS-Grim (␥-secretase reporter) flies raised on normal food, 100 nM cyclopamine, or vehicle control (DMSO). Flies were scored 1 day after eclosion as follows: mild (ϩ), moderate (ϩϩ), and severe (ϩϩϩ). K, relative changes in penetrance of rough-eye phenotype in flies raised on normal food (n ϭ 168), vehicle-containing food (n ϭ 72), or cyclopamine-containing food (n ϭ 112). Population of flies with mild, moderate, or severe rough-eye phenotype is illustrated as percent of total population per experimental group. Statistical analysis in vivo experiments are as follows: G test, ***, p Ͻ 0.005; **, p Ͻ 0.01, was performed. Student's t test was used for statistical analysis of cell based in vitro studies. Values denote means Ϯ S.E. n.s., not significant. (Fig. 1, A and C). To determine whether these effects can be observed in other models, we utilized HeLa cells because they are easily manipulated and have been previously utilized to study APP processing and trafficking (19,43). Using naive HeLa cells, we performed cyclopamine time and dose dependence experiments. Cells were treated for 24 h with increasing concentrations of cyclopamine from 0.5 to 10 M (Fig. 1D). Compared with vehicle control (Fig. 1E, dashed line), we observed a significant increase in APP-CTF levels with as little as 0.5 M cyclopamine (p ϭ 0.000615) (Fig. 1E). No change in FL-APP was observed in cells exposed to 0.5, 1, and 5 M of drug (Fig.  1E). A small, yet significant increase in FL-APP was observed upon 10 M cyclopamine treatment. To address time dependence of cyclopamine's effects on APP-CTF accumulation, we performed a time course experiment. Because we observed robust increases in APP-CTFs after 24 h with as little as 0.5 M drug, we hypothesized that using 5 M cyclopamine would significantly increase APP-CTF levels within a shorter exposure time. Accumulation of APP-CTFs was evident by 3 h of exposure (p ϭ 0.000488), and further accumulation continued for the remainder of the time course (by 24 h p ϭ 9.50 ϫ 10 Ϫ6 ) (Fig. 1, F and G). The lack of significant changes in FL-APP levels upon 5 M cyclopamine exposure suggests APP gene transcription is not altered. In fact, using quantitative PCR, we analyzed APP mRNA in naive HeLa cells upon cyclopamine treatment and did not observe changes in APP transcript levels as compared with vehicle control (data not shown).
APP proteolysis is initiated by ␣or ␤-secretase. This cleavage liberates soluble N-terminal APP ectodomains (sAPP). Treatment of naive HeLa cells with cyclopamine did not alter sAPP levels (Fig. 1, H and I). This suggests the increase in APP-CTFs is not due to modulation of ␣or ␤-secretase cleavage of APP by cyclopamine. The observed increase in APP-CTFs and the lack of change in FL-APP levels resemble the effects of ␥-secretase inhibitors but to a diminished degree (Fig. 1, J-L) (44,45).
Cyclopamine Decreases ␥-Secretase-mediated Cleavage of APP in Vitro and in Vivo-Because cyclopamine treatment increased levels of APP-CTFs, analogously to ␥-secretase inhibitor treatment, we hypothesized that cyclopamine would decrease levels of ␥-secretase cleavage products, namely A␤ and the AICD. Both A␤ and AICD are produced upon ␥-secretase cleavage of APP-CTFs. To test this hypothesis, we exposed naive HeLa and primary rat cortical neuron cells to cyclopamine for 24 h. Cyclopamine-treated cells secreted significantly less A␤ compared with vehicle control in primary cortical neurons and HeLa cells (p ϭ 0.00567) (Fig. 2A). The other product of ␥-secretase cleavage, AICD, is difficult to detect. Therefore, we used a previously described APP-Gal4 construct to aid in detection (36,46). We exposed HeLa cells transiently overexpressing APP-Gal4 to cyclopamine for 24 h and detected AICD-Gal4 levels using Western blot analysis. The ␥-secretase inhibitor L-685,458 served as a positive control because it prevents AICD generation. As predicted, in comparison with vehicle control, cyclopamine significantly decreased AICD-Gal4 levels (p ϭ 0.000228) (Fig. 2, B and C). However, these effects were much more modest than those observed upon ␥-secretase inhibition with L-685,458.
To determine whether the observed effects were specific to APP, we monitored ␥-secretase cleavage of Notch in response to cyclopamine treatment. Similar to AICD, endogenous NICD is also difficult to detect. To overcome this difficulty, we transiently overexpressed Myc-⌬ENotch in HeLa cells and treated with cyclopamine for 24 h (47,48). Cyclopamine significantly decreased NICD levels (p ϭ 8.87 ϫ 10 Ϫ5 ) to a similar extent as observed in A␤ and AICD levels (Fig. 2, D and E). These effects on AICD and NICD were much more modest than those observed upon ␥-secretase inhibition. Similar to endogenous APP-CTFs, cyclopamine increased APP-CTF-Gal4 levels (Fig. 2F). Interestingly, no change in ⌬ENotch levels was observed suggesting that the effects could be specific (Fig. 2F).
Given these results and the availability of an in vivo ␥-secretase reporter, we tested the ability of cyclopamine to modulate ␥-secretase cleavage of APP in vivo. Briefly, in 2003, Guo et al. (38) developed and characterized a D. melanogaster ␥-secretase reporter. These transgenic flies express the APP ␥-secretase substrate, APP-C99-Gal4, specifically in the fly eye ommatidia. These flies also carry a UAS element upstream of GRIM, a cell death activator. Upon ␥-secretase cleavage of APP-C99-Gal4, the resulting AICD-Gal4 can bind to the UAS element and induce GRIM expression. GRIM expression leads to death of ommatidia and results in a rough-eye phenotype (38,49,50). To test whether cyclopamine decreases ␥-secretase-mediated cleavage of APP-C99-Gal4, we raised APP-C99-Gal4;UAS-GRIM flies on normal, vehicle, or cyclopamine supplemented food. Flies were collected 1 day post-eclosion, and their eyes were scored for rough-eye phenotype. Flies raised on cyclopamine displayed decreased severity of the rough-eye phenotype (p ϭ 2.40 ϫ 10 Ϫ34 ) (Fig. 2, G-K). More specifically, 10% of the flies raised on cyclopamine displayed severe rough-eye phenotype compared with the 47% raised on vehicle food. Although only 10% of the vehicle-treated flies displayed "mild" rough-eye phenotypes, 53% of cyclopamine-treated flies displayed this phenotype (Fig. 2K). Together, these data demonstrate that cyclopamine treatment decreases ␥-secretase-mediated cleavage of APP-CTFs in vitro and in vivo.
Cyclopamine Does Not Alter ␥-Secretase Activity-Because in vivo and in vitro cyclopamine treatment leads to decreased ␥-secretase cleavage of APP-CTFs, we investigated whether cyclopamine inhibits ␥-secretase activity. One major step in ␥-secretase complex maturation is the autoproteolysis of preseni-lin1 (PSEN1) to form the active N-and C-terminal fragments. Therefore, detection of the PSEN1-CTF is an indicator of an active ␥-secretase complex (11). To this end, we exposed naive HeLa cells to cyclopamine for 24 h and observed an increase in APP-CTFs levels; however, PSEN1-CTF levels did not change in response to cyclopamine treatment (Fig. 3, A and B).
To assess overall ␥-secretase activity, we utilized an in vitro, fluorescence-based activity assay (40). We isolated total cellular membranes from naive HeLa cells and treated these membranes with vehicle, L-685,458, or cyclopamine (51-54). As expected, treatment with L-685,458 decreased cleavage of the fluorogenic ␥-secretase peptide substrate resulting in decreased fluorescence intensity over time. Surprisingly, treatment with cyclopamine did not alter ␥-secretase activity (Fig. 3C). These results suggest that cyclopamine decreases ␥-secretase-mediated cleavage of APP without directly affecting ␥-secretase activity. One mechanism that could explain these results is that cyclopamine mediates a change in the subcellular localization of APP and/or ␥-secretase.
Cyclopamine Alters APP-CTF Subcellular Localization-Proteolytic processing of APP is dependent on its subcellular localization. To investigate whether cyclopamine alters APP subcellular localization, naive HeLa cells were exposed to cyclopamine for 0, 6, or 24 h, and APP subcellular distribution was visualized using immunofluorescence. Analogous to the time course experiment in which we observed increased APP-CTFs by Western blot (Fig. 1G), here cyclopamine treatment induced significant accumulation of APP-positive puncta detected with an antibody raised to the APP C terminus (p ϭ 0.00120) (Fig. 4, A-C). Visualization of APP distribution using an antibody specific to the N-terminal portion of APP did not reveal similar cyclopamine-induced APP puncta. In fact, a lack of colocalization was observed between the Nand C-terminal APP antibodies in the cyclopamine-induced APP puncta (Fig. 4, D and E). This suggests that the cyclopamine-induced puncta are APP-CTFs and not FL-APP nor sAPP. FL-APP is not a suitable substrate for ␥-secretase cleavage. The increase in APP-CTF subcellular puncta and the lack of change in FL-APP and sAPP protein levels suggests that cyclopamine does not alter APP biosynthetic path- way. Furthermore, it also suggests that cyclopamine may induce alterations in APP-CTF endocytosis.
To further investigate this latter possibility, we measured surface APP levels using cell surface biotinylation and Western blot analysis. We treated naive HeLa cells with cyclopamine for 24 h, and we observed a significant decrease in surface FL-APP (p ϭ 0.00446) (Fig. 4, F and G). Therefore, the observed accumulations of APP-CTF-positive puncta coupled with decreased surface FL-APP suggests that cyclopamine alters internalization and possibly retrograde trafficking that is required for ␥-secretase-mediated cleavage of APP-CTFs.
Cyclopamine Alters Retrograde Trafficking and Promotes APP-CTFs Localization to Lysosomes-Previous reports indicate that upon endocytosis, FL-APP and APP-CTFs are localized to early endosomes and then sorted to either one of three possible trafficking pathways. One route is for FL-APP to be recycled back to the plasma membrane. For ␣or ␤-secretasecleaved APP fragments, APP-CTFs, a second route is available that allows these fragments to be retrogradely trafficked to the trans-Golgi network (TGN) for ␥-secretase cleavage (19). Finally, APP-CTFs can be trafficked to the lysosome for degradation. To gain insight into these possibilities, we investigated the subcellular localization of APP-CTFs using immunofluorescence and subcellular fractionation.
To determine to which subcellular compartment APP-CTFs localizes, we quantified colocalization of APP-CTFs with these markers. Although the overall colocalization is low, we noticed that upon cyclopamine treatment there was a significant increase in APP colocalization with EEA1-MP6R-, and LAMP1-positive puncta (p ϭ 1.17 ϫ 10 Ϫ6 , p ϭ 1.12 ϫ 10 Ϫ14 , and p ϭ 4.39 ϫ 10 Ϫ33 ; respectively) ( Fig. 7, F-H). There was a significant reduction in colocalization of APP-CTFs with TGN46 (p ϭ 3.21 ϫ 10 Ϫ5 ) (Fig. 7I). No change in colocalization of APP-CTFs with LC3 was observed (Fig. 7J). In addition, we detected APP-CTF-positive puncta in close association with the ESCRT multivesicular body (MVB) markers, Tsg101 and Chmp2a (Fig. 8, A and B). These data suggest that cyclopamine decreases retrograde trafficking of APP-CTFs to the TGN while increasing trafficking to lysosomes.
To independently verify that APP-CTF localization is altered upon cyclopamine treatment, we utilized subcellular fractionation of vehicle-and cyclopamine-treated HeLa cells (Fig. 9, A  and B). Very modest changes in the distribution of subcellular markers such as EEA1 and LAMP1 were observed upon cyclopamine treatment. With respect to APP-CTF distribution, in vehicle-treated cells 69% of APP-CTFs are found in fractions 5-7, which partially overlap with the TGN marker (TGN46) (Fig. 9, C and H). However, in cyclopamine-treated cells we observed a shift in APP-CTF distribution; APP-CTFs in fractions 5-7 decreased to 37%, and an increase was observed in fractions 9 and 10. These latter fractions are enriched for the lysosomal marker LAMP1 (Fig. 9, C and G). In contrast to APP-CTFs, we did not detect an observable change in FL-APP distribution upon cyclopamine treatment (Fig. 9D). Thus, cyclopamine decreases trafficking of APP-CTFs to the TGN where ␥-secretase-mediated A␤ generation occurs, and it increases APP-CTF transport to the lysosome. Because we did not observe a change in the distribution of PSEN1-CTF and FL-APP upon cyclopamine treatment, this suggests the effects are specific to APP-CTFs.
Increased localization to lysosomes would suggest increased degradation of APP-CTFs. Surprisingly, we observed cyclopamine treatment increased APP-CTF levels. One way to rationalize our observations is that cyclopamine may attenuate lysosomal degradation of APP-CTFs.
Cyclopamine Decreases APP-CTF Lysosomal Degradation-To investigate whether cyclopamine affects APP-CTF degradation, we pretreated HeLa cells with cyclopamine for 24 h and then added cycloheximide to inhibit protein synthesis for an additional 0 -4 h. At the end of this additional 4 h, APP-CTFs decreased by 76% in vehicle-treated cells, although only a 38% decrease was observed in cyclopamine-treated cells (p ϭ 0.00218) (Fig. 10, A and B). Cyclopamine treatment nearly doubled the APP-CTF half-life from 2 to 3.6 h (Fig. 10B). Similar to our previous findings, cyclopamine did not alter the FL-APP rate of degradation (p ϭ 0.0645) (Fig. 10C).
To test whether these APP-CTF degradation changes are due to decreased lysosomal maturation, we monitored cathepsin D levels, a reliable marker of lysosomal maturation (56,57). We detected a modest but significant (p ϭ 0.030) decrease in the ratio of mature (31 kDa) to immature (53 kDa) cathepsin D levels (Fig. 10, D and E). Together, our results implicate that cyclopamine leads to increased preferential retrograde trafficking of APP-CTFs to lysosomes and decreased lysosomal degradation of these APP-CTFs.

DISCUSSION
Here, we have discovered novel effects on APP trafficking and lysosomal maturation induced by treatment with cyclopamine. Specifically we demonstrate an accumulation of APP-CTFs in lysosomes and a decrease in A␤ and AICD generation. After translation, APP is trafficked to the plasma membrane via the secretory pathway. APP can then be cleaved by ␣or ␤-secretase at the plasma membrane or early endosome after endocytosis, respectively. These APP-CTFs are then retrogradely trafficked to the TGN for ␥-secretase cleavage and A␤ generation (19). APP retrograde trafficking is highly regulated because APP proteolysis is dynamic and can lead to rapid changes in A␤ production (58). A consequence of decreased APP-CTF trafficking to the TGN is the decrease in ␥-secretasemediated cleavage of APP-CTFs and the concomitant decrease in A␤ and AICD generation. Hence, modulating APP retrograde trafficking independent of secretase activity can have novel implications for therapeutic avenues to treat AD.
Cyclopamine is a naturally occurring phytosterol isolated from the corn lily plant. The animal sterol, cholesterol, promotes amyloidogenic processing and increases A␤ generation, whereas cyclopamine exhibits the opposite effects on A␤ generation (29,30). Phytosterols were recently shown to modify APP metabolism. Some phytosterols increased amyloidogenic processing and A␤ levels, and others decreased A␤ levels (32). In fact, stigmasterol demonstrated anti-amyloidogenic properties, decreased ␤-secretase cleavage of APP, and decreased A␤ generation. Moreover, mice fed stigmasterol-enriched diets showed decreased ␥-secretase complex protein expression but lacked direct inhibition of ␥-secretase activity in isolated mouse brain tissue (32). We also observed that cyclopamine treatment decreases ␥-secretase-mediated proteolysis of APP without inhibiting ␥-secretase activity directly. However, we observed cyclopamine treatment leads to the accumulation of APP-CTFs derived from ␣and ␤-secretase equally. This is because the observed cyclopamine effects were downstream of APP-CTF generation at the plasma membrane (␣-secretase) or early endosome (␤-secretase).
Cyclopamine decreased APP-CTF retrograde trafficking to the TGN in HeLa cells. Choy et al. (19) demonstrated that ␥-secretase cleavage of endocytic APP-CTFs and A␤ generation occurs at the TGN. Because cyclopamine does not inhibit ␥-secretase activity, it is clear that the changes in APP-CTF levels are not due to changes in activity. Instead, these changes could be due to APP-CTF retrograde trafficking, thereby preventing colocalization with ␥-secretase. Because we observed decrease localization of APP-CTFs at the TGN, we rationalize that altered trafficking is the mechanism by which cyclopamine decreases A␤ generation (Fig. 11). Alternatively, after APP-CTF endocytosis, these fragments can be trafficked to the late endosome/MVB and then to the lysosome for degradation. Here, we demonstrate cyclopamine increases APP-CTF trafficking to lysosomes. This increased trafficking to lysosomes could result in increased protein degradation and decreased APP-CTF levels. Surprisingly however, we observed increased FIGURE 10. Cyclopamine leads to moderate decrease in lysosomal maturation and significantly attenuates APP-CTF rate of lysosomal degradation. A, Western immunoblot analysis of FL-APP and APP-CTFs using a C-terminal APP antibody (c1/6.1). For FL-APP and APP-CTF analysis, naive HeLa cells were pretreated with 5 M cyclopamine (Cyc) or vehicle (DMSO) for 24 h and then exposed to 50 g/ml cycloheximide (CHX) for the indicated times. B and C, FL-APP and APP-CTF protein levels were normalized to ␤-actin first. The graphs represent protein levels as percent remaining of total protein at time 0 h. The lines represent the linear least squares fit where the slope of the line is the rate of protein degradation. D, naive HeLa cells treated with vehicle (DMSO) or 5 M cyclopamine for 24 h followed by mature and immature cathepsin D detection in cell lysates via Western immunoblotting. E, bar diagram represents ratio of mature to immature cathepsin D protein quantification normalized to ␤-actin. Values denote mean Ϯ S.E. Student's t test was used for statistical analysis as follows: **, p Ͻ 0.01; *, p Ͻ 0.05. APP-CTF levels and attenuated lysosomal degradation of APP-CTFs upon cyclopamine treatment. Interestingly, the accumulation of APP-CTFs was completely reversible upon washout of cyclopamine (data not shown). Therefore, it will be interesting to determine whether the APP-CTF accumulations are completely degraded after removal of the drug, and whether this acute drug exposure will still result in decreased A␤ levels. We observed similar changes in APP processing in HeLa cells and neurons. Our future studies will determine whether the changes in trafficking and lysosomal maturation observed in HeLa cells are also observed in neuronal cells.
Upon reaching the plasma membrane, FL-APP sheds the sAPP ectodomain. Lack of change in FL-APP and sAPP levels indicates cyclopamine does not alter the APP biosynthetic pathway. Decreased surface FL-APP suggests enhanced endocytosis after shedding, which can explain the increase in early endosome immunofluorescence intensity. The changes in A␤, AICD, and APP-CTF levels in the absence of changes in FL-APP and sAPP levels imply that the effects of cyclopamine on APP metabolism are specific. The lack of change in FL-APP and sAPP levels upon cyclopamine treatment may also suggest it is a good candidate for possible AD therapy as it induces APP-CTF sequestration in lysosomes resulting in modest decreases in A␤ levels while not affecting FL-APP levels and sAPP generation. However, the chronic accumulation of APP-CTFs in the lysosome may be detrimental to protein homeostasis.
Both APP and Notch require primary cleavage at the plasma membrane for downstream endocytosis and retrograde trafficking to the TGN for ␥-secretase and AICD/NICD generation (48, 59 -61). Cyclopamine's effects may be specific to APP-CTFs because we did not see the exact same changes in exogenous ⌬ENotch processing. We did observe decreased NICD levels similar to the decrease in AICD levels. However, we did not observe increased ⌬ENotch levels as we did for exogenous APP-CTF-Gal4 and endogenous APP-CTFs. Because ⌬ENotch overexpression may result in nonphysiological ⌬ENotch trafficking and processing, we are reticent to conclude that cyclopamine's effects are specific to APP.
Cyclopamine is known to bind to and inhibit Smoothened (Smo) (62). Smo is a G protein-coupled receptor that is a component of the Sonic hedgehog (Shh) signaling pathway (63)(64)(65). It will be interesting to determine what role, if any, Smo and Shh play in APP trafficking and proteolysis. Given the ability of cyclopamine to decrease A␤ levels, it will be intriguing to determine whether cyclopamine is effective in treating AD transgenic animal models. One possible concern in utilizing cyclopamine is that it is a potent teratogen. The vast majority of AD patients are well beyond their reproductive age, which makes the teratogenicity less of a concern.
A balance between retrograde and lysosomal degradation trafficking pathways ensures proper distribution of APP and Notch holoproteins and their metabolites. Because these metabolites have been shown to be involved in regulating cell FIGURE 11. Model representation of APP-CTF retrograde trafficking and lysosomal localization upon cyclopamine exposure. Trafficking and cleavage of FL-APP and APP-CTFs in normal conditions (A) compared with cyclopamine treatment (B); FL-APP proteolysis and production of APP-CTFs occur at the plasma membrane (␣-secretase) and early endosomes (␤-secretase). APP-CTFs are then trafficked, via the retrograde pathway, to TGN for subsequent ␥-secretase cleavage and A␤ generation (19). Alternatively, APP-CTFs are trafficked to late endosomes/multivesicular bodies thus destined for lysosomal degradation. B, cyclopamine treatment favors the lysosomal degradation trafficking pathway (bold arrows) of APP-CTFs thereby preventing ␥-secretase proteolysis of APP-CTFs and A␤ generation. death/survival and synaptic plasticity, completely ablating the production of these metabolites would be detrimental (66 -74). The modest but significant decrease observed in A␤, AICD, and NICD levels suggests cyclopamine may not have the negative consequences that ␥-secretase inhibition displays in some AD patients (75,76).