Impaired Autophagy in APOE4 Astrocytes

Abstract

Alzheimer’s disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aβ plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aβ plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aβ plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aβ plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aβ plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

Keywords

Alzheimer’s disease amyloid-β apolipoprotein E4 autophagy

INTRODUCTION

Alzheimer’s disease (AD), the most prevalent form of dementia in the elderly, is characterized by cognitive decline, occurrence of brain senile plaques, neurofibrillary tangles (NFT), and synapse and neuronal loss in the brain [1 –3]. The senile plaques contain a 40-42-amino acid-long amyloid-β (Aβ) peptide derived from amyloid-β protein precursor (AβPP) [3, 4], whereas the NFT contain hyperphosphorylated aggregates of the microtubule-associated protein tau [5]. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in families with a higher risk of late onset AD and sporadic AD. There are three major alleles of ApoE; E2 (ApoE2), E3 (ApoE3), and E4 (ApoE4), of which ApoE4 strongly increases AD risk relative to E2 and E3 in a dose-dependent fashion. The allele frequency of ApoE4 in sporadic AD is >50%, and it increases the risk for AD by lowering the disease onset by 7 to 9 years per allele copy [6].

The finding that Aβ deposition is specifically elevated in ApoE4-positive AD patients, together with corresponding animal and cellular models studies which revealed that ApoE4 and the amyloid cascade interact synergistically, led to the hypothesis that key pathological effects of ApoE4 are mediated by cross-talk interactions with the amyloid cascade [7]. We have previously shown that activation of the amyloid cascade triggers specific accumulation of Aβ in hippocampal neurons of ApoE4 mice, but not of ApoE3 mice [8]. This accumulation of Aβ induces synaptic degeneration and, subsequently, neuronal loss in the ApoE4 mice [9]. Additional studies have shown that the early neuropathological and cognitive effects of ApoE4 in young naïve mice are associated with the accumulation of Aβ in the affected hippocampal neurons [10].

Several cellular mechanisms are believed to mediate the disposal of Aβ plaques and NFT, one of which is autophagy [11, 12]. Autophagy serves as an intracellular degradation pathway involved in the turnover of long-lived proteins, dysfunctional organelles, and aggregated proteins [13]. There are three forms of autophagy, of which macroautophagy is the most studied (hereafter called autophagy). Central to autophagy is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to the lysosome. Studies with AβPP transgenic mice have shown that autophagosomes appear prior to the deposition of Aβ and that the elimination of Aβ depends on functional autophagy [14]. However, other studies revealed that autophagy could be either induced or impaired in AD [15, 16]. The current view suggests that autophagy is activated as a protective mechanism to remove Aβ aggregates; however, as the disease progresses, the autophagic apparatus becomes dysfunctional. Therefore, at later stages of AD, impaired autophagy might become harmful [17, 18]. Indeed, the maturation of autophagosomes is disrupted in AD and is associated with pathological autophagosome accumulation [15, 16].

Although autophagy and ApoE4 are both strongly associated with AD, the possibility that the pathological effects of ApoE4 in AD and on the clearance of Aβ are related to autophagy has not been investigated. Astrocytes and microglia play an important role in defense mechanisms against pathological abnormalities that occur in neurodegenerative diseases [19 –21] and in which autophagy plays an important role [14]. We presently examined the possibility that autophagy is affected isoform specifically by ApoE4 in astrocytes. This was pursued by investigating the effects of ApoE3 and ApoE4 on autophagy in astrocytes under resting and stimulating conditions. The extent to which this is related to the isoform specific effects of ApoE4 on the senile plaques clearance and digestion by astrocytes was also examined. The results reveal that autophagy is inhibited isoform specifically by ApoE4. Also, ApoE4 impairs the clearance of Aβ, which may be related to the decreased autophagy.

MATERIALS AND METHODS

Materials

Antibodies were obtained from the following sources: monoclonal mouse anti-actin (MP Biomedicals, Santa Ana, CA; 691001), polyclonal rabbit anti-LC3B (Sigma-Aldrich, St. Louis, MO; L7543), polyclonal rabbit anti p62 (MBL International, Woburn, MA; PM045), polyclonal rabbit anti phospho-Thr389-S6 kinase (Sigma, S6311), polyclonal rabbit anti S6 kinase (Sigma-Aldrich, S4047) and monoclonal mouse anti-Beta Amyloid (6E10) (Covance, Emeryville, CA; SIG-39300). Reagents are as follows: Earle’s Balanced Salt Solution (EBSS; Sigma-Aldrich, E3024) chloroquine (CQ; Sigma-Aldrich, C6628), Rapamycin (Rapa; Cayman Chemical, Ann Arbor, MI; 13346) and Beta Amyloid _1 - 42, HiLyte Fluor^TM 488-label (Anaspec, Campus Drive Fremont, CA; 60479).

Cell lines

The human ApoE3 and ApoE4 expressing astrocytes were previously described [22]. Cells were grown in Dulbeco’s Modified Eagle’s Medium/ Nutrient Mixture F-12 HAM (Sigma-Aldrich, D6421) supplemented with antibiotics and 10% heat-inactivated fetal bovine serum (FBS, Hyclone, CH30160.03). Cells were incubated at 37°C in 5% CO₂ in air, and the medium was changed every 3-4 days. Cells were passaged when 70% confluent using trypsin/Di-sodium ethylenediaminetetra-acetic acid (Biological Industries, 03-045-1). Cells were cultured at 30% confluence in growth medium, 1-2 days before each experiment.

Primary astrocytes cultures

The study was conducted according to the NIH Guidelines for Use and Care of Laboratory Animals and following the approval by Animal Care Committee of the Tel Aviv University, #L-12-020. Mouse primary astrocytes were prepared as previously described [23 –25], with minor modifications. Cerebral cortices from 1- to 3-day-old neonatal ApoE3 and ApoE4 target replacement mice were dissected in ice-cold Hanks’ Balanced Salt Solution (HBSS), carefully stripped of their meninges, and digested with 0.25% trypsin for 10 min at room temperature. Trypsinization was stopped by addition of an equal volume of Dulbeco’s Modified Eagle’s Medium (Sigma-Aldrich, D5796) to which we added 0.02% deoxyribonuclease I. The cells were dispersed into a single-cell level by repeated pipetting, then pelleted in centrifuge for 7 min at 700 g, 4°C and resuspended in culture medium supplemented with 10% FCS and antibiotics. Cells were seeded at a density of 300,000 cells/ml (equivalent to 62,500 cells/cm²) in flasks and cultured at 37°C in humidified 5% CO₂–95% air. A day after the preparation, all flasks were washed twice with 5 ml PBS, to allow washing of preparation remnants. Medium was replaced every 4 days. These cultures (“mixed glia”) reached confluence after 7–10 days and were used between 15 and 20 days after preparation. Astrocytes were isolated from the mixed glia cultures by mild trypsinization (0.06% trypsin) resulting in detachment of an intact layer of cells containing almost all the astrocytes. After reaching confluence (∼7–10 days), the astrocytes were treated with 10μM Ara-C for 4 days to eliminate proliferating cells. At this stage the culture contains 95% astrocytes [25].

Transfections

Transfection was performed using the Lipofectamine^® 2000 reagent (Invitrogen, Carlsbad, CA) as follows: the cells were incubated with 1.4μg plasmid DNA and 2.1μl transfection reagent in 6-well plates containing 1.5 ml medium supplemented with 10% Opti-MEM^® for 5 h, after which another 1.5 ml of medium was added. Medium was replaced after 24 h.

Lysate preparation and immunoblotting

Cells were grown in medium with or without the indicated stimuli. After treatment, cells were solubilized in lysis buffer (50 mM HEPES pH = 7.5, 150 mM NaCl, 10% glycerol, 1% triton X, 1 mM EDTA pH = 8, 1 mM EGTA pH = 8, 1.5 mM MgCl₂, 200μM Na₃VO₄, 150 nM aprotinin, 1μM leupeptin, 500μM AEBSF). Lysates were cleared by boiling in gel sample buffer followed by centrifugation as described [26, 27]. Lysates were resolved by SDS-polyacrylamide gel electrophoresis through 10–12.5% gels and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked for 1 h in TBST buffer (0.05 M Tris HCl pH 7.5, 0.15 M NaCl and 0.1% Tween 20) containing 6% milk, blotted with primary antibodies for 2 h, followed by secondary antibody linked to horseradish peroxidase for 1 h. Immunoreactive bands were detected with the enhanced chemiluminescence reagent. Densitometric analysis of the results was performed using the ImageJ program.

Electron microscopy

Cells were grown in Matek dishes with coverslip in the bottom. Cells were first fixed in 4% paraformaldehyde followed by 2% paraformaldehyde and 1% glutaraldehyde, then fixed in osmium tetroxide and embedded in epon araldite, sectioned at 90 nm thickness and analyzed with an EM10 Zeiss electron microscope as previously described [28]. Electron micrographs were obtained at a magnification of ×5000 and ×25,000.

In situ Aβ plaque degradation in the presence of ApoE3 and ApoE4 astrocytes

This assay is based on measurement of the ability of the astrocytes to clear Aβ plaques following their co-incubation with Aβ plaques containing brain sections from 5XFAD mice [29, 30]. Accordingly, 20μm coronal frozen brain sections were prepared from 9-13-month-old 5XFAD mice using cryostat and placed on glass slides, 3-4 consecutive sections in each slide. The slides were kept at –80°C until being used.

At the beginning of the experiment, the slices were incubated for 48 h at 37°C with ApoE3 and ApoE4 astrocytes (10⁵ cells in 100μl medium per slice). Medium without astrocytes was used as control. The treated slides were gently placed in round petri dishes with thick wet paper, on the placement of the dishes. PapPen was used to draw liquid repellent borders of the sections to avoid spillage of coating solution out of the slides. In the pharmacological experiments, the cells were treated with either rapamycin (100 nM) or with chloroquine (10μM). After 48 h the slides were gently washed of all containing remains by placing them in PBS for 10 min. Then, the slides were fixed with 4% PFA for 10 min, followed by 0.1% Triton solution for 10 min after which they were incubated with blocking solution (8% Horse Serum, 0.3% Triton, 1 gr/ml BSA, 0.02% Sodium Azide in PBS) for 30 min, followed by incubation with primary anti-Beta Amyloid (6E10) antibody at a dilution of 1:750 in blocking solution for 1 h at room temperature. The slides were then washed 5 times with PBST. Secondary antibody (Alexa Flour^© 488 donkey α-mouse IgG, A-21202) was added at dilution of 1:250 and incubated at room temperature for 45 min, followed by 5 washes with PBST. The slides were then stained with DAPI (Vektashield, Burlingame, CA; H-1200). Analysis of the Aβ plaque burden was performed as percentage of the area covered in plaques using the ImageJ software.

Quantification of fluorescent Aβ uptake by flow cytometry

Cells were incubated with HiLyte Fluor^TM 488-labeled Aβ_1 - 42 at the indicated concentration in serum-free medium at 37°C for 2 h. The cells were then washed with PBS three times, trypsinized, centrifuged at 1,500 g for 5 min and resuspended in PBS. Cell fluorescence was analyzed in a fluorescence-activated cell sorter (FACScan; Becton Dickinson, Franklin Lakes, NJ) within 30 min.

Statistical analysis

All experiments were performed at least three times. Results are presented as mean±SE. One-tailed Student’s t-test was used to assess the differences between means. Results were considered statistically significant when p < 0.05. In the in situ plaques elimination assays and the electron microscopy results were analyzed using One Way ANOVA for group analysis followed by the indicated post hoc analysis.

RESULTS

Autophagy and autophagic flux is inhibited in ApoE4 expressing astrocytes

To explore the role of ApoE in autophagy, we examined the extent to which autophagy is differentially regulated by ApoE alleles, utilizing immortalized mouse astrocytes cell lines derived from ApoE knock-in mice expressing either human ApoE4 or ApoE3 [22]. Accordingly, we first used a known trigger of autophagy, namely, amino acid deprivation (incubation in EBSS), for the indicated time periods to determine its effects on the expression of the autophagic proteins, LC3 and p62/SQSTM1 (presently abbreviated as p62). Cytosolic LC3-I is lipidated (LC3-II) during autophagy, becomes associated with the autophagosomal membrane, and serves as a marker for autophagy [31]. p62, a scaffold protein that binds LC3 and ubiquitinated protein aggregates, is degraded during autophagy [32]. Figure 1A shows that the LC3-II/LC3-I ratio, which is a marker of autophagic activation, was significantly higher in ApoE3 astrocytes compared to ApoE4 astrocytes, after 1, 2, and 4 h of starvation (6.5±1.14, 6.2±0.35, and 5.6±0.71 compared to 2.4±0.36, 2.58±0.50, and 1.82±0.02, respectively; p < 0.05). In addition, the levels of p62, which is associated with the degradation steps and is degraded during autophagy, decreased more rapidly following amino acid starvation in ApoE3 astrocytes compared to ApoE4 astrocytes (0.23±0.01, 0.18±0.03, and 0.24±0.03 compared to 0.43±0.04, 0.52±0.13, and 0.28±0.06, respectively; p < 0.05). Taken together, these results imply that induction of autophagy is impaired in ApoE4 astrocytes. Next, we examined autophagy levels in ApoE3 and ApoE4 astrocytes following treatment with rapamycin, an inhibitor of mTOR, which is known to induce autophagy. Utilizing the LC3-II/LC3-I ratio and p62 levels as markers, we found that, similar to the EBSS treated cells, autophagy was significantly lower in ApoE4 astrocytes following treatment with 100 or 150 nM rapamycin. Accordingly, the LC3II/LC3I ratio under these conditions was 2.9±0.40 and 3.3±0.57 in ApoE3 compared to 1.49±0.21 and 1.34±0.23 for the corresponding ApoE4 cells (p < 0.05; Fig. 1B) whereas the corresponding p62 levels were 0.31±0.07 and 0.28±0.04 in ApoE3 compared to 0.49±0.04 and 0.53±0.09 in ApoE4, (p < 0.05; Fig. 1B). In addition, we found that the EBSS/rapamycin treatments inhibited mTOR activity and decreased the levels of its downstream effector pS6K (Fig. 1A, B). Interestingly, LC3-II/LC3-I ratio was significantly higher under basal untreated conditions in ApoE3 cells compared with ApoE4 cells (Fig. 1A, B), suggesting that the basal autophagy levels in untreated cells are also higher in ApoE3 than in the ApoE4 astrocytes.

Autophagy is a dynamic process, in which LC3-II generation is followed by its degradation in the autolysosome; thus, an increase in LC3-II levels can be derived either from de novo autophagy induction or from inhibition of protein degradation by autophagy. These possibilities can be resolved by measurements of autophagic flux, which are performed by comparing LC3-II levels in the presence or in the absence of a drug that blocks the fusion of the autophagosome with the lysosome [33]. Accordingly, the autophagic flux was determined in the ApoE-expressing astrocytes, using amino acid starvation as autophagy inducer and chloroquine, which inhibits the fusion of autophagosome with the lysosome. As can be seen in Fig. 2, chloroquine led to LC3-II accumulation following starvation for 2 h, in ApoE3 and ApoE4 astrocytes. Quantification of these results revealed that LC3-II/actin levels were significantly higher in ApoE3 astrocytes compared with ApoE4 (9.26±1.75 and 4.5±0.16, respectively; p < 0.05), demonstrating that the autophagic flux is lower in ApoE4 astrocytes compared to ApoE3 astrocytes.

Autophagy can be also monitored microscopically by tagging the LC3 protein with GFP [33]. Since lipidated LC3 is attached to the autophagosome membrane, this tag allows monitoring the formation of autophagosomes in the cells (also termed LC3-GFP puncta). Thus, to further confirm the relationship between ApoE isoforms and autophagy levels, we transfected ApoE astrocytes with LC3-GFP vector and measured the autophagosome formation after 1 h amino acids starvation. Representative images are shown in Fig. 3. Using quantitative analysis, we found that the area of LC3-GFP puncta as the percentage of total cell area was significantly higher in ApoE3 compared to ApoE4 astrocytes. The observed differences are in accordance with the immunoblot results and suggest that the induction of autophagy is more prominent in ApoE3 than in ApoE4 astrocytes. These findings were also confirmed by electron microscopy analysis, which revealed that ApoE3 astrocytes are more responsive to induction of autophagy by amino acids starvation than the ApoE4 astrocytes and that the ApoE3 astrocytes contained less electrodense aggregates than the corresponding ApoE4 astrocytes (Fig. 4). Taken together, these results show that ApoE4 mitigates both basal and induced autophagy in mouse astrocytes.

Insoluble Aβ plaques elimination and soluble Aβ uptake are inhibited by ApoE4 and are affected by chloroquine and rapamycin

It has been suggested that autophagy plays a role in the degradation of senile plaques [14]. We therefore examined the extent to which the ApoE4-driven impaired autophagy is associated with reduction of Aβ plaques clearance. This was pursued utilizing an in situ plaque removal model, in which the ability of the astrocytes to clear senile plaques from brain sections of 5XFAD transgenic mice is determined. As shown in Fig. 5, the ApoE3 astrocytes reduced the levels of Aβ containing senile plaques to a residuary level of 52±11.9% (p < 0.001) of the total Aβ plaques area as compared with cell-free medium. In contrast, ApoE4 astrocytes had no effect under these conditions on the Aβ plaque area (Fig. 5A, B). Hence, ApoE3 astrocytes remove Aβ plaques more effectively than ApoE4 astrocytes. The possibility that the impaired ability of the ApoE4 astrocytes to eliminate Aβ plaques is related to its effects on autophagy was investigated next. To explore this effect, the cells were treated with either the autophagy inhibitor chloroquine or the autophagy inducer rapamycin and the resulting effects on the removal of Aβ plaques by the ApoE astrocytes were determined. As shown in Fig. 6, chloroquine completely abolished the ability of ApoE3 astrocytes to remove Aβ plaques. In contrast, rapamycin significantly increased the Aβ plaque removal capacity of ApoE4 cells (residuary of 31.5±3.1% in ApoE3 and 30.1±5.0% in ApoE4 compared with cell-free medium, p < 0.05) (Fig. 7).

Following the finding that ApoE3 astrocytes eliminate Aβ plaques more efficiently than ApoE4 astrocytes, we examined the possibility that ApoE4 also affects the uptake of soluble Aβ_1 - 42. This was performed by flow cytometry analysis of the amount of fluorescent Aβ, which accumulates within the ApoE3 and ApoE4 astrocytes following 2 h incubation. As shown in Fig. 8A, Aβ uptake by ApoE3 cells (2.94±0.07 and 4.33±0.06; fold induction at 0.025 and 0.05μM Aβ, respectively) was significantly higher than the corresponding uptake by ApoE4 cells (1.62±0.01 and 2.04±0.04; fold induction at 0.025 and 0.05μM Aβ, respectively; p < 0.01). Moreover, when cells were co-incubated with chloroquine to inhibit autophagy, the rate of Aβ uptake was significantly reduced in ApoE3 cells (from 3.2±0.32 to 2.2±0.12; p < 0.05) but not in the ApoE4 cells, whose basal rates of Aβ uptake were low and not affected by this treatment (Fig. 8B). Taken together, these findings show that ApoE4 inhibits Aβ plaques elimination and soluble Aβ uptake, and suggest that the impairment of autophagy is related to these effects.

Autophagy and autophagic flux is inhibited in ApoE4 expressing primary astrocytes

To exclude the possibility of a clonal effect of the cultured astrocytes, we prepared primary astrocytes cultures from ApoE3 and ApoE4 transgenic mice. As shown in Fig. 9A, following amino acids starvation, LC3-II levels decreased more rapidly in ApoE3 astrocytes compared to ApoE4 astrocytes (1.23±0.25, 0.71±0.11 and 0.38±0.03 compared to 1.63±0.33, 1.11±0.15 and 0.67±0.11, respectively; p < 0.05). If this decreased levels of LC3-II in ApoE3 astrocytes results from enhanced degradation by autophagy, it may indicate that autophagy is more efficient also in primary ApoE3 astrocytes. Indeed, when autophagic degradation was blocked using chloroquine (Fig. 9B), the reduction in LC3-II in ApoE3 astrocytes was prevented, indicating that the decreased levels resulted from enhanced autophagy. Moreover, the levels of LC3-I also decreased in ApoE3 but not in ApoE4 astrocytes following autophagy induction (1.15±0.47, 0.42±0.16, and 0.19±0.01 compared to 2.84±0.95, 1.82±0.58, and 1.20± 0.31, respectively; p < 0.05; Fig. 9A). These results may suggest that the lipidation of LC3-I is more efficient in ApoE3 astrocytes. In addition, the starvation resulted in a more dramatic decrease of p62 levels in ApoE3 astrocytes compared to ApoE4 astrocytes (0.34±0.06, 0.30±0.08, and 0.28±0.03 compared to 0.52±0.07, 0.55±0.09, and 0.96± 0.27, respectively; p < 0.05; Fig. 9A). Thus, although autophagy kinetics differs in the primary astrocytes compared to the astrocyte cell lines, in both cases the autophagy is more efficient in ApoE3 astrocytes.

Taken together, the results show that ApoE4 inhibits autophagy and the clearance of Aβ plaques and that it increases the extracellular accumulation of Aβ via a chloroquine sensitive mechanism.

DISCUSSION

Autophagy is the major intracellular degradation pathways of cellular constituents including dysfunctional organelles and protein aggregates [34]. Autophagy is characterized by the formation of autophagosomes, which are double-membrane vesicles responsible for delivering the degradation material to the lysosomes [13]. Several studies demonstrate a link between autophagy and AD [4 , 35–39]. Yet, a connection between ApoE genotype and autophagy has not been demonstrated. In the present study we used murine primary and astrocytic cell lines that express either ApoE3 or ApoE4 to examine the effects of ApoE genotype on autophagy. Utilizing the levels of LC3 proteins as a marker of autophagy activation and the levels of p62 as a marker of autophagy-driven protein degradation, we showed that astrocytes expressing ApoE4 exhibit lower autophagic flux compared with those expressing ApoE3, under several autophagy-inducing conditions. Furthermore, these findings were corroborated by ultrastructure studies, which revealed that ApoE4 cells have reduced autophagy compared to ApoE3. To our knowledge, this is the first time a connection between autophagy and ApoE4 was demonstrated.

There is a growing body of evidence suggesting that the lysosomes are affected isoform specifically by ApoE4 and that the latter increases the accumulation of Aβ_1 - 42 in the lysosomes leading to lysosomal leakage and cellular toxicity [40, 41]. Furthermore, inhibition of Aβ degrading enzyme, in ApoE4 but not in ApoE3 mice, induced the accumulation of Aβ_1 - 42 in lysosomes of hippocampal neurons, which in turn triggered neurodegeneration and cognitive impairments [8 , 42–44]. The extent, to which the presently observed effect of ApoE4 on autophagy drives the effect of ApoE4 on lysosomes or vice versa, remains to be studied; however, additional mechanisms also need to be considered. For example, ApoE receptors such as LRP and ApoER2, which play important roles in key cellular processes [45, 46], were shown to be affected differentially by ApoE4 and ApoE3 [47, 48]. It is thus possible that the inhibitory effects of ApoE4 on autophagy are mediated via these receptors. Further studies are required to assess this possibility.

The present findings that ApoE4 astrocytes eliminate Aβ plaques less effectively than ApoE3 astrocytes and are impaired in the uptake of soluble Aβ are consistent with previous findings that clearance of Aβ by endocytosis is impaired by ApoE4 [49]. Endocytosis and autophagy share similar machinery [50, 51]. Furthermore, several studies demonstrate a convergence of endosomes and autophagosomes in the lysosomal degradation axis, and that impaired autophagic activity is involved in the process of Aβ accumulation [52]. Thus, it is possible that the presently observed effects of ApoE4 on Aβ clearance are driven by a common autophagy/endocytosis/lysosomal axis. This assertion is supported by the present finding that under autophagy inhibiting conditions (treatment with chloroquine), the Aβ plaques removal and the Aβ uptake by ApoE3 were inhibited and that induction of autophagy in ApoE4 astrocytes with rapamycin improved their ability to clear Aβ plaques.

Neurodegeneration in AD is associated with changes in the level of autophagy and Aβ deposits [52]. Furthermore, ApoE4 is associated with increased deposition of Aβ in AD and it has been suggested that it is also associated with autophagy impairments [53]. The present cell culture findings that ApoE4 astrocytes exhibit impaired autophagy, impaired plaque elimination and impaired Aβ uptake, are in accordance with the AD-related findings and suggest that autophagy may play an important role in mediating the pathology of ApoE4. Further studies utilizing neuronal and microglial cell cultures and the corresponding in vivo models are required. Taken together, the results presented suggest that the pathological effects of ApoE4 in astrocytes may be mediated by impaired autophagy and by the concomitant impaired ability of the cells to remove Aβ plaques. The availability of pharmacological agents, which activate autophagy, thus may provide a novel approach to the treatment of ApoE4-related brain pathology in AD.

Footnotes

ACKNOWLEDGMENTS

This research was supported in part by The Legacy Heritage Bio Medical Program of the Israel Science Foundation (grant No. 1575/14); the Joseph K. and Inez Eichenbaum Foundation; the Esterson Trust; and the Harold and Eleanore Foonberg Foundation. Danny M. Michaelson is the incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration.

Authors’ disclosures available online ().

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