Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A

Primary neuronal cultures were generated from B6.Cg-Tg (APPswe, PSEN1dE9)85Dbo/Mmjax mice (APP/PS1) AD transgenic (tg) and wild-type (wt) mouse embryos. The APP sequence in APP/PS1 encodes a chimeric mouse/human APP (Mo/HuAPP695swe) that was humanized by modifying three amino acids, and introducing the Swedish AD mutation. The PS1 sequence encodes human presenilin 1 lacking exon 9 (dE9) that models AD-associated mutations in PS1. Both APPswe and PS1 are independently controlled by the prion protein promoter. Primary neuronal cultures were prepared from cortices and hippocampi of embryonic day 15 embryos as previously described [9]. In brief, E15 brain tissue was dissociated by trypsinization and trituration in DMEM with 10% fetal bovine serum (Gibco). Dissociated neurons were cultured on poly-D-lysine (Sigma) coated plates or glass coverslips (Bellco Glass Inc.) and were maintained until 12 and 19 DIV in neurobasal medium (Gibco), B27 supplement (Gibco), glutamine (Invitrogen) and antibiotics (ThermoScientific).

Wild type mouse N2a neuroblastoma cells (N2a) or N2a cells stably transfected with the 670/671 Swedish mutation human APP (Swe) [26] or wild-type α-synuclein with HA-Tag (α-syn) were grown on 10 cm dishes or coverslips.

Cells were grown on Thermanox coverslips (Nalgene, Nunc) and fixed with 2% PFA, 2.5% glutaraldehyde in 0.1 M cacodylate. Cells were then secondarily fixed with 1% osmium tetroxide followed by incubation with 1% tannic acid to enhance contrast. Cells were dehydrated using increasing percentages of ethanol before being embedded onto Epoxy resin (Agar scientific, UK) stubs. Coverslips were cured overnight at 65 °C. Ultrathin sections were cut using a diamond knife mounted to a Reichert ultracut S ultramicrotome and sections were collected onto copper grids. Grids were post-stained with drops of lead citrate. Sections were viewed on a FEI Tecnai transmission electron microscope (Eindhoven, The Netherlands) at a working voltage of 80 kV. BSA-gold was prepared as previously described [27]. For quantification of MVB diameter, MVBs were defined as organelles containing intraluminal vesicles and monomeric rather than flocculated BSA-gold.

Aβ1-42 peptides (Sigma) were incubated at 37 °C for 1 h to induce fibril formation in vitro. Grids were inverted onto the drops of Aβ1-42, negatively stained with 2% uranyl acetate, washed with water and dried on filter paper before being viewed by EM.

Cells were transfected using Lipofectamine 2000 (Invitrogen) for N2a cells or Lipofectamine 3000 (Invitrogen) for primary neurons. N2a cells were transfected in Opti-MEM while primary neurons were transfected directly in their growth medium. The plasmids p3xFLAG-CMV-10-hVPS4A-wt and p3xFLAG-CMV-10-hVPS4A-dn E228Q were generated as described [28]. The control plasmids p3xFLAG-CMV-7-BAP Control Plasmid was purchased from Sigma-Aldrich and pcDNA3-CMV-GFP from Addgene. pcDNA3-synapsin-FLAG-wtVPS4 and pcDNA3-synapsin-FLAG-dnVPS4 were constructed from pcDNA3-synapsin-FLAG and PCR products from the p3xFLAG-CMV-10-hVPS4A-wt and p3xFLAG-CMV-10-hVPS4A-dn respectively. Control plasmid pAAV-synapsin-GFP was purchased from Addgene.

The following antibodies were used (see also Additional file 1: Table S1): 369 [29] (Buxbaum et al., 1990) for Western blot (WB) 1:1000; 6E10 (BioLegend, previously Covance SIG-39320) IF: 1:500, WB 1:1000; 12F4 (BioLegend, previously Covance SIG-39142) for immunofluorescence (IF) 1:250; Amyloidβ (1-42) (IBL, 18,582); Amyloidβ (1-42) (Invitrogen, 700,254) IF 1:1000; beta-actin (Sigma, A 5316) WB 1:2000; CD63 (ThermoFisher Scientific, MAI-19281) WB 1:1000; CHMP2B (Abcam, ab33174) IF 1:250, WB 1:1000; Clavestin-1 + 2 (Bioss, bs-6569R-A647) IF 1:250; DAPI (Sigma, D9542) IF 1:2000; drebrin (Abcam, ab11068) IF 1:1000; FLAG (Biolegend, 637,302) IF 1:1000, (Sigma, F1804) WB 1:1000; Flotillin-1 (BD Biosciences, 610,821) IF 1:400; GM130 (BD Biosciences, 610,822) IF: 1:500; GSK3β (Cell Signaling Technology, 12,456) IF 1:400; pGSKα/β (Cell Signaling Technology, 9331S) WB 1:1000; HA-Tag (Cell Signaling Technology, 3724) WB 1:1000; Hrs and Hrs-2 (Enzo, ALX-804-382-C050) IF 1:100; LAMP1 (Abcam, ab24170) IF 1:1000; LAMP1 (Abcam, ab25245) IF: 1:1500; LC3β (Cell Signaling Technology, 2775) WB 1:1000; Amyloid fibrils OC (Merck Millipore, AB2286) IF 1:1000; P2:1 (ThermoFisher Scientific, OMA1-03132) IF 1:500; Phospho-tau pSer396 (ThermoFisher, 44-752G); Rab7 (Abcam, ab50533) IF 1:500; Synaptophysin (Merck Millipore, MAB5258) IF 1:1000; Tsg101 (Genetex, GTX70255) IF 1:250, WB 1:1000; VPS4 (SantaCruz, sc-133,122) IF 1:100, WB 1:1000; secondary antibodies conjugated to Alexa Fluor-488, −546, −647 (IF 1:500; Invitrogen) or to HRP (WB 1:2000; R&D Systems, Minneapolis, MN).

Bafilomycin A (Sigma), torin 1 (Tocris) or rapamycin (Fisher BioReagents) were added to pre-warmed culture media at appropriate concentrations. Starvation media for induction of autophagy was 33% Opti-MEM in Hank’s Balance Salt solution (HBSS). Aβ1-40 or Aβ1-42 peptides (Tocris) were reconstituted in DMSO to 250 μM, sonicated for 10 min and followed by 15 min of centrifugation at 12 k rpm before adding the supernatant to the culture media for the depicted times. All experiments used 0.5 μM of Aβ1-40 or Aβ1-42, except for EM and LAMP-1 positive vesicle size experiments that used 5 μM and 1 μM respectively.

Mice were anesthetized with isoflurane and perfused transcardially with saline followed by 4% PFA in 0.1 M PBS (pH 7.4) at RT. After dissection, brains were postfixed by immersion in 4% PFA in 0.1 M PBS (pH 7.4) at 4 °C for 2 h or overnight. After fixation, brains were cut in 40 μm thick sections with a sliding microtome. Sections were kept in storage buffer composed of 30% sucrose and 30% ethylene glycol in PBS at −20 °C. Free-floating sections were blocked for 1 h in RT with serum and triton-X and then incubated in primary antibodies overnight at 4 °C, followed by appropriate fluorescent Alexa secondary antibodies for 1 h at RT.

Medium was collected and centrifuged and cells were washed twice, harvested in ice cold PBS, and centrifuged. Cell pellets were lysed with 6% sodium dodecyl sulfate (SDS) containing 10 μl/ml β-mercaptoethanol, sonicated, and then heated at 95 °C for 6 min. After centrifugation, supernatants and medium were mixed with loading buffer, heated at 95 °C for 5 min and loaded into 10–20% Tricine gels (Invitrogen). Samples were subjected to electrophoresis and transferred to polyvinylidine difluoride membranes (Millipore). Membranes were blocked in PBS containing 0.1% Tween-20 (PBST) and 5% milk, and incubated in primary antibodies overnight and then with HRP-conjugated secondary antibodies for 1 h diluted in PBS containing 0.1% Tween-20 (PBST) and 5% milk. The immunoreaction was visualized by a chemiluminescence system (Pierce or BioRad). Bands were quantified using Image Lab (Bio-Rad Laboratories). For visualization of Aβ, membranes were boiled in PBS for 5 min prior to blocking. For analysis of exosomes, WB was performed as above but without β-mercaptoethanol in the 6% SDS lysis buffer.

For analysis of LC3β cells were lysed in RIPA buffer (Thermo Fisher Scientific) with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific). Lysates with NuPAGE LDS sample buffer and NuPAGE reducing agent were loaded on NuPAGE 4-12% BisTris gels and run with NuPAGE MES SDS buffer (Invitrogen).

For analysis of α-synuclein in medium, total protein was extracted using a trichloroacetic acid (TCA)/acetone precipitation protocol. Briefly, freshly collected samples were cleared by centrifugation at 10000 rpm for 10 min to pellet debris and intact cells. The supernatant was transferred to a new tube and added with ¼ volume of ice-cold 20% TCA followed by incubation on ice for 3 h. The proteins were pelleted by centrifugation at 14000 rpm and washed twice with cold acetone.

For native conditions, cell pellets were lysed on ice in NativePAGE sample buffer (1X, Life Technologies) containing 1% digitonin (Life Technologies) and Halt proteinase inhibitor cocktail (1X, Thermo Scientific) by pipetting up and down and incubating on ice for 15 min. Lysates were centrifuged at 20000 x g for 30 min at 4 °C and protein concentrations of the supernatants were determined with BCA assay. Equal amounts of protein were loaded on a 3-12% NativePAGE Novex Bis-Tris gel (Life Technologies).

Exosomes were purified from cell culture medium by differential ultracentrifugation as described previously [30]. Briefly, Swe N2a cells were cultured and transfected for 48 h in exosome-free medium. Collected medium was depleted of cells and cellular debris by sequential low speed centrifugation. Exosomes were then isolated by centrifugation of the collected supernatant at 100,000×g at 4 °C for 70 min. The resultant pellet was washed in PBS and centrifuged for 70 min at 100,000×g at 4 °C.

Statistical analysis was performed with PRISM 6 software (Graph-Pad Software, San Diego, CA, USA) by using unpaired t-test or ANOVA with Tukey’s multiple comparisons test or ANOVA with Dunnett’s multiple comparisons test. All data are expressed as the mean ± SD. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Endogenous Aβ42 is present in both dendrites and axons of cultured primary APP transgenic neurons and localizes especially with markers of late endosomes/MVBs [21]. In order to examine whether increased levels of Aβ can lead to the enlarged endosomal phenotype seen in AD, MVB size was compared between APP/PS1 transgenic and wt primary mouse neurons. Cells were incubated with BSA-gold 2 h (h) before fixation to confirm identity of the endocytic compartments. Remarkably, electron micrographs showed a significantly greater MVB diameter (52% increase; p < 0.001) in APP/PS1 transgenic compared to wt neurons at 12 days in vitro (DIV) (Fig. 1a and b) consistent with the larger diameter of endosomes described in human AD. Since full length APP, β-CTFs, Aβ and the presenilin mutation in the APP/PS1 transgenic neurons potentially all could be the cause of this effect, wt neurons at 12 DIV were treated with freshly prepared human synthetic Aβ1-40 or Aβ1-42 for 48 h, to test if Aβ was sufficient to induce the endosomal enlargements. The neurons demonstrated a 69% and 114% increase in the diameter of MVBs with Aβ1-40 or Aβ1-42 treatment, respectively (p < 0.001; Fig. 1c and d) as measured on EM images compared to controls. Further, an increased size by 165% of LAMP1-positive late endosomes/lysosomes was already evident by immunofluorescent labelling at 3 h in pyramidal neurons treated with Aβ1-42 (p < 0.0001, Fig. 1e and f). LAMP1-changes at different time points of Aβ1-42 treatment, ranging from 30 s to 48 h, are shown in Additional file 2: Figure S1B. Taken together, our results indicate that enlarged MVBs, one of the earliest pathological features in AD, can be caused by Aβ, which however does not rule out important contributions also of other APP components such as β-CTFs.

Interestingly, fibril-like structures were apparent by EM in endocytic organelles of wt primary neurons incubated with synthetic human Aβ1-42 for 48 h that were not seen in untreated neurons (Fig. 2a), and were more prominent in neurons treated with Aβ1-42 than Aβ1-40. For comparison, Aβ1-42 fibrilized in vitro showed similar size and morphology of fibrils on EM as in the endocytic vesicles (Fig. 2b). Loss of endolysosomal impermeability was also seen with Aβ1-42 treatment of neurons with BSA-gold leaking out into the cytoplasm (Fig. 2c). To further investigate the fibril-like structures, wt murine neurons were treated with elevated levels of human Aβ1-42 at different time points and immunolabelled with the conformational dependent Aβ antibody OC to label amyloid fibrils and fibrillar oligomers [31] and antibody 6E10 to specifically label the added human Aβ. Using the same parameters as in the EM experiments, neurons treated with Aβ1-40 only led to weak OC antibody labelling compared to vehicle treated cells, while Aβ1-42 treated neurons showed robust OC labelling (Additional file 3: Figure S2).

Already at 45 min of treatment with 0.5 μM Aβ1-42 (Fig. 2d) and up to 24 h of incubation, antibodies OC and 6E10 revealed an almost completely overlapping vesicular pattern of labelling, indicating that the amyloid fibrils and/or fibrillar oligomers consisted of Aβ1-42, which was particularly prominent along neuronal processes. Of note, OC labelling increased in intensity with time of treatment with Aβ1-42. Interestingly, at 48 h the OC positive structures were enlarged and elongated, displaying a part that was human Aβ antibody 6E10 positive and an extension that was 6E10 negative (Fig. 2e). Surface labelling of non-permeabilized cells revealed that the elongated OC labelling at 48 h was now to a certain extent extracellular, whereas the punctate OC and 6E10 co-labelling at 24 h was generally intracellular (Fig. 2f). This suggests that with time, fibrils extend out of neurites into the extracellular space and/or that organelles containing the fibrils fuse with the plasma membrane. To better define the subcellular site of Aβ aggregation, cells were double labelled with OC antibody and the late endosomal/lysosomal marker LAMP1. At 45 min colocalization with OC was evident in small LAMP1-positive vesicles in the processes but not in the larger LAMP1-positive vesicles in the cell soma (Additional file 4: Figure S3A). Since lysosomes normally do not localize to axons and dendrites, other than their very proximal part, this suggests that Aβ42 starts to aggregate in late endosomes/MVBs of neurites. At 48 h, OC and LAMP1 still colocalized mainly in neuronal processes, but in LAMP1-positive structures that now appeared somewhat enlarged and irregular in their shape. Elongated OC-positive structures could be seen extending out from the more punctate LAMP1 labelling (Fig. 2g and Additional file 4: Figure S3A). The late endosomal marker Rab7 indicated that at least a subset of the vesicular OC labelling at 24 h colocalized with Rab7 positive late endocytic compartments in neurites (Additional file 4: Figure S3C). Taken together these data indicate that Aβ1-42 can be taken up by neurons in culture and forms amyloidogenic fibrils and/or fibrillar oligomers in late endocytic compartments particularly within neuronal processes that eventually appear to extend extracellularly.

Given prior evidence supporting that sorting via the MVB pathway was impaired by Aβ accumulation in cultured AD transgenic neurons and that Aβ dependent translocation into MVBs seemed affected [21], we next investigated possible dysfunction of the ESCRT pathway in models of AD. In APP/PS1 transgenic primary neurons Aβ42 was found associated with CHMP2B-positive vesicles (Fig. 3a). In young 3-month-old Tg19959 mice, a different transgenic mouse model of β-amyloidosis harbouring the Swedish and Indiana APP mutations, CHMP2B immunolabelling was most prominent in the area of hippocampus and entorhinal cortex that also expressed increased levels of Aβ/APP (Additional file 5: Figure S4A-B). Although levels of total CHMP2B and VPS4 were not significantly changed in wt compared to APP/PS1 primary neurons lysed in 6% SDS (Additional file 5: Figure S4C), levels of high molecular weight complexes of CHMP2B on blue native polyacrylamide gel electrophoresis were increased in APP/PS1 neurons treated with Aβ1-42 for 3 h (Fig. 3b). Other ESCRT proteins were not resolved on native gels, likely due to masking of antibody epitopes under native conditions.

Since AD transgenic neurons showed an increased diameter of MVBs and treatment with Aβ1-42 led to prominent fibril-like structures in late endocytic organelles, and as prior immuno-EM work has indicated early Aβ accumulation in dystrophic neurites [10], we next examined whether aggregating Aβ42 in endosomes might lead to the presence of ESCRT components in amyloid plaques. Indeed, APP/PS1 transgenic mice with plaque pathology demonstrated that the ESCRT-III associated protein VPS4 was markedly increased in plaques (Fig. 3c and Additional file 5: Figure S4D), where it partially colocalized with Aβ42. ESCRT–I component Tsg101, a marker of MVBs, also showed increased labelling in plaques (Fig. 3d). In contrast, labelling of CHMP2B and the earlier ESCRT-0 component Hrs appeared decreased in plaques compared to surrounding brain parenchyma (Additional file 5: Figure S4E-F). Brain sections were also co-labelled with an antibody against clavestin-1 and 2 (Fig. 3d), which are neuron-specific proteins in the endo-lysosomal pathway [32]. The strong colocalization between Tsg101 and clavesin-1 and 2 in amyloid plaques supports the neuronal origin of ESCRT components in plaques. Moreover, much of the strong Aβ42 labelling outside of plaques in AD transgenic brain occurred in vesicle-like structures of dystrophic neurites that also contained clavesin-1 and 2 and Tsg101, consistent with accumulation of Aβ42 within endosomal compartments of neurons. To confirm these results in a different AD transgenic mouse model of β-amyloidosis, Tg19959 mice were examined with the onset of plaque pathology. In early plaques of 3-month-old Tg19959 mice Tsg101 (Fig. 3e) and VPS4 (not shown) also colocalized strongly with Aβ42.

In order to model the effect of dysfunctional ESCRT-dependent MVBs on Aβ accumulation, a dominant negative, E228Q ATPase-deficient form of VPS4A (dnVPS4A) was expressed for 24 h in N2a neuroblastoma cells harbouring stably transfected human Swedish mutant APP. N2a cells were used here because of their high transfection efficiency compared to primary neurons. To assess that N2a cells react to Aβ1-42 in a similar manner as primary neurons, N2a cells were treated with exogenously added Aβ1-42 for different time points. N2a cells treated with Aβ1-42 also exhibited increased size of LAMP1-positive vesicles (Additional file 6: Figure S5). The ATPase VPS4 is a key component of the ESCRT machinery as it is the only energy-consuming enzyme, promotes disassembly and recycling of ESCRT-III oligomers, and is recruited to the ESCRT-III complex by direct interaction with CHMP2B. We found that the expression of dnVPS4A markedly increased the intracellular pool of Aβ by 424% (p < 0.001, Fig. 4a and d) while concurrently decreasing the amounts of Aβ secreted into the medium by 87% (p < 0.0001, Fig. 4c and d). DnVPS4A also increased higher molecular weight bands between 17 kDa and 34 kDa that might represent Aβ oligomers within cells (Fig. 4a) as they were not seen with C-terminal specific APP antibody 369 (Fig. 4b). In addition, dnVPS4A increased the levels of APP within cells by 149% (p < 0.05) and secreted APPα in conditioned media by 149% (p < 0.05). Overexpression of wild type VPS4A (wtVPS4A) also significantly reduced secretion of Aβ by 72% (p < 0.001), although not to the extent of the dominant negative construct. Such a partially dominant negative effect of over-expressing wtVPS4 has been described [28].

Confocal immunofluorescence microscopy of Swe N2a cells transfected with dnVPS4A confirmed the increase of Aβ and full-length APP (Fig. 4e). Increased labelling by immunofluorescent microscopy with the conformational antibody OC in dnVPS4A-transfected cells confirmed the increase in Aβ oligomers and showed that the fibrillar oligomers and/or fibrils labelled by antibody OC colocalized with enlarged vesicles labelled positive for dnVPS4A protein and flotillin-1 (Fig. 4f and g). Triple labelling revealed that Aβ42 accumulated in enlarged vesicles positive for the MVB marker Tsg101 in dnVPS4A expressing cells labelled by the FLAG-tag (Fig. 4h). Thus, dysfunctional MVBs accumulate Aβ42 that, at least in part, is in an oligomeric and/or fibrillar form. In contrast to effects on Aβ and in agreement with a prior report [28], dnVPS4A increased secretion of α-synuclein in α-syn N2a cells without altering the total pool of intracellular α-synuclein (Additional file 7: Figure S6). Since the CMV-FLAG-dnVPS4A induced toxicity in primary neurons, we constructed a plasmid under the weaker synapsin promoter. Expressing dnVPS4A under this synapsin promoter in wt or APP/PS1 primary neurons showed increased labelling of Aβ42 (Fig. 4i), supporting that dysfunctional ESCRT-dependent MVB formation leads to increased levels of Aβ within neurons.

The increased intracellular levels of Aβ with dnVPS4A could be due to changes in the production of Aβ, reduced secretion and/or reduced fusion of MVBs with the lysosome for degradation. In order to investigate the turnover of APP and Aβ, Swe N2a were treated with the protein synthesis inhibitor cycloheximide (Additional file 8: Figure S7A-B and Additional file 9: Figure S8D). This revealed a rapid turnover of APP in cell lysates with a half-life of about 45 min, while cellular Aβ had a half-life of about 4 h. In contrast, the degradation of APP and Aβ in conditioned media was slower, not reaching 50% of control levels within 6 h of cycloheximide treatment. To investigate the role of lysosomal degradation, Swe N2a cells were treated for 24 h with 5 nM bafilomycin A1 (BafA1), which inhibits the vacuolar H+ ATPase. BafA1 treatment had the same effects on Aβ as dnVPS4A, markedly increasing intracellular levels of Aβ and reducing the secretion of Aβ (Fig. 5a-b and Additional file 9: Figure S8A-D). These results suggest that dnVPS4A blocks MVBs with Aβ on route to lysosomes for degradation. Consistently, when the endo-lysosomal pH gradient was blocked with BafA1, dnVPS4A transfected cells no longer showed higher cellular levels of Aβ than the wtVPS4A or GFP transfected cells (Fig. 5c). Hence, when the ability of lysosomes to degrade Aβ is abolished, blocking Aβ on route to the lysosomes via dnVPS4 does not lead to further Aβ accumulation.

To investigate the secretion of Aβ via fusion of MVBs with the plasma membrane and concomitant release of exosomes, exosomes from dnVPS4A-transfected Swe N2a cells were isolated and analysed (Fig. 5d). Secretion of exosomes was not prevented but instead increased by dnVPS4A in Swe N2a cells, possibly due to upregulation of ESCRT independent (CD63 dependent) MVB formation [33‐35].

A major question in AD is how Aβ links to tau pathology. MVBs are necessary for the sequestration of GSK3 [36] which phosphorylates tau at serine 396 (S396). Both elevated tau phosphorylation at S396 [37] and hyperactive GSK3 is implicated in AD [38, 39]. Therefore, we examined the effects of dnVPS4A on tau phosphorylation. Tau phosphorylation at residue S396 was significantly increased by 56% with dnVPS4A (p < 0.05; Fig. 6a and b), suggesting increased activation of GSK3β and increased GSK3β dependent phosphorylation of tau caused by dysfunctional ESCRT dependent MVBs. Immunofluorescence microscopy revealed increased GSK3β labelling of cells upon dnVPS4A transfection (Additional file 10: Figure S9A), although changes in total GSK3β or phosphorylated GSK3α/β (S21/9) levels were not detected by Western blot in cell lysates of Swe N2a cells transfected with dnVPS4A (Additional file 10: Figure S9B). One possible explanation for this apparent discrepancy is that the active GSK3β in the cytosol and early endosomes (facing the cytosol) is easier to visualize with immunofluorescence than the sequestered GSK3β inside the ILVs of MVBs.

Since autophagic organelles are markedly increased in AD and autophagy is thought to be impaired in the disease [14], we next examined LC3β-II and p62, markers of autophagy, in dnVPS4A transfected cells accumulating intracellular Aβ. Expression of dnVPS4A in Swe N2a cells showed increased levels of LC3β-II, which correlate with increased numbers of autophagosomes in the cell, by 35% (p < 0.05, Fig. 7a) and increased levels of p62 by 52% (p < 0.05, Fig. 7b). Inducing autophagy by 1 μM Rapamycin (p < 0.01, Fig. 7c), 250 nM Torin1 or starvation (Fig. 7d) reduced the dnVPS4A-induced increase in intracellular Aβ. Thus, in the setting of dysfunctional MVBs with Aβ that is inefficiently trafficked to lysosomes for degradation and/or inefficiently secreted, stimulation of autophagy is associated with decreased cellular Aβ.