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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: J Neuroimmune Pharmacol. 2019 Jul 23;16(1):159–168. doi: 10.1007/s11481-019-09862-1

Antiretroviral drugs promote amyloidogenesis by de-acidifying endolysosomes

Liang Hui 1, Yan Ye 1, Mahmoud L Soliman 1, Koffi L Lakpa 1, Nicole M Miller 1, Zahra Afghah 1, Jonathan D Geiger 1, Xuesong Chen 1,*
PMCID: PMC6981001  NIHMSID: NIHMS1535531  PMID: 31338753

Abstract

Antiretroviral therapeutics (ART) have effectively increased the long-term survival of HIV-1 infected individuals. However, the prevalence of HIV-1 associated neurocognitive disorders (HAND) has increased and so too have clinical manifestations and pathological features of Alzheimer’s disease (AD) in people living with HIV-1/AIDS. Although underlying mechanisms are not clear, chronic exposure to ART drugs has been implicated in the development of AD-like symptoms and pathology. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels. All ART drugs are organic compounds that can be classified as being either weak acids or weak bases, and these physicochemical properties may be of central importance to ART drug-induced AD-like pathology because weak bases accumulate in endolysosomes, weak bases can de-acidify endolysosomes where amyloidogenesis occurs, and endolysosome de-acidification increases amyloid beta (Aβ) protein production and decreases Aβ degradation. Here, we investigated the effects of ART drugs on endolysosome pH and Aβ levels in rat primary cultured neurons. ART drugs that de-acidified endolysosomes increased Aβ levels, whereas those that acidified endolysosomes decreased Aβ levels. Acidification of endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aβ levels. Further, ART drug-induced endolysosome deacidification increased endolysosome sizes; effects that were blocked by ML-SA1-induced endolysosome acidification. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and that endolysosome acidification might attenuate AD-like pathology in HIV-1 positive people taking ART drugs that de-acidify endolysosomes.

Keywords: Antiretroviral therapy, Alzheimer’s disease, amyloid beta, endolysosome pH

Graphical Abstract

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Introduction

Antiretroviral therapeutic (ART) drugs are effectively helping HIV-1 infected individuals live almost full life-spans. However, about 50% of people living with HIV-1 including those who are treated with ART drugs suffer from HIV-associated neurocognitive disorders (HAND) and associated with HAND are clinical manifestations and pathological features of Alzheimer’s disease (AD) (Clifford et al., 2009; Ferrell and Giunta, 2014). Although the underlying mechanisms remain elusive, both neurotoxic HIV-1 viral proteins (Chen et al., 2013; Bae et al., 2014; Khan et al., 2015) and ART drugs (Giunta et al., 2011; Lan et al., 2012; Brown et al., 2014; Gannon et al., 2017) have been implicated in amyloidogenesis and the development of AD-like pathology.

Lysosomes were first discovered by De Duve and colleagues over 60 years ago, and their importance in the degradation of endocytosed, phagocytosed, and autophagocytosed macromolecules was soon recognized (De Duve et al., 1955; de Duve, 2005). The endocytic delivery of extracellular macromolecules and plasma membrane components for degradation requires trafficking through early endosomes to late endosomes followed by transient or complete fusion between late endosomes and lysosomes (Bright et al., 2005; de Duve, 2005; Luzio et al., 2007; Huotari and Helenius, 2011). In addition, the endolysosome system captures and degrades intracellular worn-out constituents through autophagy (Settembre et al., 2013). These processes make endolysosomes a complex and dynamic system that functionally interacts with other organelles. An important hallmark of the endolysosome system is the acidic luminal pH, which is maintained by the electrogenic pumping of protons by v-ATPase in conjunction with vesicular chloride transporters that shunt the membrane potential; this allows for a build-up of luminal protons (Huotari and Helenius, 2011; Mindell, 2012). The acidic pH of endolysosomes is critical for the activity of up to 60 different pH-sensitive hydrolytic enzymes including proteases, lipases and nucleases all of which enable endolysosomes to break down a wide range of endogenous and exogenous cargos (de Duve, 2005). In addition to its role as a cellular ‘recycling bin’, the endolysosome system is critical for a range of physiological functions such as plasma membrane repair, cell homeostasis, energy metabolism, nutrient-dependent signal transduction, and immune responses (Settembre et al., 2013; Perera and Zoncu, 2016). Pathologically, morphological and functional changes to endolysosomes have been described for a wide spectrum of diseases including cancer and neurodegenerative diseases (Colacurcio and Nixon, 2016).

For neurons, endolysosomes are especially important, because neurons are mainly long-lived post-mitotic cells that require endolysosomes in turning-over cellular components and obsolete organelles (Nixon and Cataldo, 1995, 2006). Changes in the structure and function of neuronal endolysosomes have been reported to be one of the earliest pathological features of AD (Cataldo et al., 2004; Tate and Mathews, 2006; Arriagada et al., 2007; Boland et al., 2008) and they precede extracellular deposition of amyloid beta (Aβ) protein (Cataldo et al., 2000). With regards to HAND, endolysosome dysfunction may be a pathogenic mechanism because endolysosome dysfunction has been noted in brains of HIV-1 infected individuals, Aβ protein generation and metabolism occurs in endolysosomes, and amyloidogenesis occurs in HIV-1 infected individuals (Gelman et al., 2005; Achim et al., 2009).

Impaired Aβ precursor protein (AβPP) endocytic trafficking (Rajendran and Annaert, 2012; Morel et al., 2013; Jiang et al., 2014) and endolysosome dysfunction (Nixon, 2005; Rajendran et al., 2008; Shimizu et al., 2008; Sannerud et al., 2011) play critical roles in Aβ generation in neurons. Indeed, once internalized into endosomes, AβPP is cleaved by pH-sensitive β- and γ-secretases to form Aβ (Edgar et al., 2015). Such endosome-derived Aβ can be degraded by pH-sensitive cathepsins in lysosomes which are even more acidic than are endosomes (Miners et al., 2011). Importantly, even modest de-acidification of endolysosomes increases amyloidogenesis and decreases Aβ degradation capabilities in endolysosomes and this results in increased intraneuronal accumulations (Braak and Del Tredici, 2004; LaFerla et al., 2007; Zou et al., 2015) and secreted levels of Aβ (Annunziata et al., 2013; Nilsson et al., 2013).

We have shown that endolysosome de-acidification plays a critical role in HIV-1 Tat-and gp120-induced amyloidogenesis as well as the development of AD-like pathology (Chen et al., 2013; Bae et al., 2014). However, it is not known whether endolysosome de-acidification plays a role in ART drug-induced amyloidogenesis. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels but can also be classified as being either weak acids or weak bases, and these physicochemical properties may regulate ART drug-induced AD-like pathology because weak bases such as the anti-malarial drug chloroquine accumulate in endolysosomes and cause endolysosome de-acidification. In the present study, we tested our hypothesis that endolysosome de-acidification plays a role in ART drug-induced increases in Aβ in primary cultured neurons. We tested 12 ART drugs including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (INIs) and found that those ART drugs that de-acidified endolysosomes also increased Aβ levels, whereas those ART drugs that acidified endolysosomes decreased Aβ levels. Further, we found that acidifying endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aβ levels. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis, and that endolysosome acidification might mitigate against AD-like pathology in people living with HIV-1 who are taking ARTs that de-acidify endolysosomes.

Materials and Methods

Reagents

Zidovudine, abacavir, lamivudine, efavirenz, nevirapine, ritonavir, and nelfinavir were obtained from Sigma. Emtricitabine, tenofovir disoproxil fumarate, darunavir, dolutegravir and elvitegravir were obtained from MedChem Express. LysoSensor (DN160) was obtained from Fisher Thermo Scientific. Aβ1–40 and Aβ1–42 ELISA kits were obtained from Wako. ML-SA1 was obtained from Tocris Bioscience.

Primary cultures of rat hippocampal neurons

As previously described (Buscemi et al., 2007; Hui et al., 2012b), primary cultured hippocampal neurons were prepared from Sprague-Dawley rats. Pregnant dams at embryonic day 18 were sacrificed by asphyxiation with CO2. After the fetuses were removed and decapitated, meninges-free hippocampi were isolated, trypsinized, and seeded onto 35-mm2 poly-D-lysine coated glass-bottom tissue culture dishes. Neurons grown in Neurobasal™ medium containing L-glutamine, antibiotic/antimycotic and B27 supplement were maintained in an incubator (37°C, 5% CO2) for 10–14 days, at which time they were taken for experimentation. Neurons were treated with 12 different ART drugs for up to 48 hrs during which time the media was not changed. Measurements of endolysosomal pH, endolysosome sizes, and Aβ levels were conducted using separate dishes of cells. Typically, the purity of the neuronal cultures was more than 95% as determined by immunostaining of neurons with mouse anti-NeuN or goat anti-MAP2 antibodies (Millipore), and of astrocytes with mouse anti-GFAP antibody (Sigma).

SH-SY5Y cells

Human neuroblastoma cells (SH-SY5Y) expressing wild type AβPP were kindly supplied by Dr. Norman Haughey (John Hopkins University). SH-SY5Y cells were cultured in Eagle’s minimum essential medium (MEM) supplemented with 10% FCS, penicillin/streptomycin, nonessential amino acids, and sodium pyruvate (1 mM) at 37°C in 5% CO2. For our experiments, 4 × 106 SH-SY5Y cells were seeded in 60 mm2 dishes and cultured for 48 h at which time the media was changed to serum-free MEM and cells were treated with chloroquine (Sigma) for an additional 48 h.

Measurement of endolysosome pH

Using a method standard to our laboratory (Liu et al., 2008; Hui et al., 2012a), endolysosome pH was measured with LysoSensor (Yellow/Blue DND-160); a dual ratio-metric indicator dye. Neurons were incubated with 2 μM LysoSensor for 5 minutes at 37°C. Light emitted at 520 nm in response to excitation at 340 nm and 380 nm was measured every 10 seconds for 20 min using a filter-based fluorescence imaging system (Zeiss). The 340/380 nm ratios were converted to pH units using a calibration curve established using 10 μM of the H+/Na+ ionophore monensin and 20 μM of the H+/K+ ionophore nigericin dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl; the pH was adjusted between 3.0 and 7.0 with HCl/NaOH.

Quantification of Aβ levels by ELISA

Aβ levels were quantified by ELISA according to the kits’ manufacturer’s protocol (Hui et al., 2012a). Media from cultured neurons was collected and diluted 1:2 for Aβ1–42 and 1:5 for Aβ1–40 and then assayed in duplicate using a calorimetric sandwich ELISA assay (Wako). Cultured neurons were harvested into RIPA buffer, sonicated, and following centrifugation at 14,000 × g for 10 min at 4°C supernatants were taken for total protein determinations using a DC protein assay (BioRad) and Aβ levels using the ELISA assay systems. All data were normalized to total protein content in each sample.

Immunostaining

Levels of Aβ1–42 were determined in neurons by immuno-fluorescence staining and confocal microscopy (LSM800, Zeiss, Germany). Primary cultured rat hippocampal neurons were incubated for 48 h at 37°C with either dolutegravir (1 μM), lamivudine (1 μM), ML-SA-1 (20 μM), dolutegravir (1 μM) plus ML-SA-1 (20 μM), lamivudine (1 μM) plus ML-SA-1 (20 μM), or DMSO (0.1%) as a control. After washing with PBS, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with ice-cold methanol for 10 minutes. Cells were then blocked with 2.5% goat serum at room temperature for 40 minutes and then incubated overnight at 4°C with a rabbit polyclonal antibody against Aβ1–42 at a dilution of 1:100 (Abcam). Cells were washed and incubated with FITC-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) and Aβ1–42 antibody complexes were visualized and quantified by confocal microscope (Zeiss LSM 800) and Image J software.

Live cell imaging of endolysosomes

The morphology of endolysosomes was analyzed using 50 nM LysoTracker Red DND-99 (Invitrogen) and 1 μM calcein AM (Invitrogen). Primary cultured rat hippocampal neurons were incubated with the dyes for 30 min at 37°C. Images were acquired by laser scanning confocal microscopy (LSM800, Zeiss, Germany). Imaging fields were chosen at random, four to five images were used from every treatment group, and images were analyzed using Image J particle-analyzing software. Profiles of endolysosome sizes were analyzed with non-linear regression (Gaussian fit) using Prism GraphPad 8 software.

Statistical analysis

All experiments were repeated at least three times using at least 2 different batches of primary cultured neurons. Data shown represent means and SEM. Statistical significance between two groups was determined with a Student’s t-test, and statistical significance among multiple groups was analyzed with one-way ANOVA plus a Tukey post-hoc test. Detailed ANOVA analysis for each figure were shown in Supplementary Table 1. *p<0.05 was considered to be statistically significant.

Results

Acute effects of ART drugs on endolysosome pH in primary cultured neurons

Using 12 drugs from four major classes of ARTs including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (INIs), we first determined the acute effects (maximal change within the first 20 minutes) of ART drugs on endolysosome pH. We tested 3 concentrations (2 orders of magnitude apart) for each ART drug examined; DMSO was used as a vehicle control and chloroquine was used as a positive control (Supplementary Figure S1). The range of these 3 concentrations covers the known CSF ART drug concentrations and Cmax values in blood (Supplementary Table 2). Although we tested 3 concentrations, we showed only the highest concentrations of ART drugs that affected endolysosome pH. The summary data illustrated in Figure 1A show that 8 out of the 12 tested ART drugs de-acidified endolysosomes including the NRTIs lamivudine and tenofovir disoproxil fumarate, the NNRTIs efavirenz and nevirapine, the PIs ritonavir, nelfinavir and darunavir, and the INI dolutegravir. Of the 12 drugs tested, two ART drugs had no effect on endolysosome pH; the NRTI emtricitabine and the INI elvitegravir. In addition, we found that the NRTIs zidovudine and abacavir acidified endolysosomes.

Figure 1. ART drugs affect endolysosome pH.

Figure 1.

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(A) Acute effects of ART drugs on endolysosome pH were measured ratio-metrically using LysoSensor dye (DND-160) and primary cultured rat neurons. In real-time live imaging, once baseline levels of endolysosome pH were stable, ART drugs were added and endolysosome pH was measured for 10 min. Δ endolysosome pH represents the difference between ART drug-induced endolysosome pH and baseline (n=7–25, *p<0.05, **p<0.01, ***p<0.001). NRTIs: Zidovudine (10 μM) and abacavir (10 μM) significantly decreased endolysosome pH, whereas lamivudine (1 μM) and tenofovir disoproxil (10 μM) significantly increased endolysosome pH. In addition, emtricitabine (0.1 μM) did not affect endolysosome pH. NNTRIs: Efavirenz (1 μM) and nevirapine (1 μM) significantly increased endolysosome pH. PIs: Ritonavir (1 μM), nelfinavir (1 μM), and darunavir (0.1 μM) significantly increased endolysosome pH. INIs: Dolutegravir (1 μM) significantly increased endolysosome pH, but elvitegravir (1 μM) didn’t affect endolysosome pH. (n=15, *p<0.05, **p<0.01, ***p <0.001) (B) Persistent effects of ART drugs were measured 48 h after application of ART drugs using LysoSensor dye (DND-160) and primary cultured rat neurons. Δ endolysosome pH represented the difference of endolysosome pH between ART drugs and DMSO (vehicle controls). Lamivudine (1 μM), emtricitabine (0.1 μM), efavirenz (1 μM), nevirapine (1 μM), darunavir (0.1 μM), dolutegravir (1 μM) and elvitegravir (1 μM) significantly increased endolysosome pH, whereas zidovudine (10 μM) slightly and abacavir (10 μM) significantly decreased endolysosome pH in neurons (n=8–64, *p<0.05, **p<0.01, ***p <0.001).

Persistent effects of ART drugs on endolysosome pH in primary cultured neurons

Next, we determined the persistent effects (maximal effect 48 hours after drug application) of the 12 ART drugs on endolysosome pH (Figure 1B). Compared with the DMSO control, 7 of 12 tested ART drugs significantly de-acidified endolysosomes including the NRTIs lamivudine and emtricitabine, the NNTRIs efavirenz and nevirapine, the PI darunavir, and the INIs dolutegravir and elvitegravir. In addition, 3 of the 12 ART drugs tested did not significantly affect endolysosome pH including the NRTI tenofovir disoproxil fumarate, and the PIs ritonavir and nelfinavir. Furthermore, the NRTI abacavir significantly acidified endolysosomes.

Effects of ART drugs on secreted Aβ levels in primary cultured neurons

We demonstrated previously that endolysosome de-acidification affected AβPP processing and protein expression levels of a number of endolysosome proteins (Hui et al., 2019). Here we found that treatment of SH-SY5Y cells for 48 h with the endolysosome de-acidifying agent chloroquine resulted in enahnced secretion of both Aβ1–40 and Aβ1–42 and increased protein levels of AβPP, BACE1, and cathepsin D (Supplementary Figure S2). Thus, we determined the effects of 48 h treatment of neurons with the 12 ART drugs on secreted levels of Aβ. The use of 48 h incubation for the measurement of Aβ levels was based on our previous published findings that HIV-1 Tat protein, which de-acidifies endolysosomes, increased Aβ levels following 48 h treatment but not 24 h treatment even though 24 h Tat treatment had persistent endolysosome de-acidifying effects. Thus, we used longer time points to produce measureable effects on Aβ levels. Compared to controls, of the 7 ART drugs that significantly de-acidified endolysosomes, 6 ART drugs significantly elevated secreted levels of both Aβ1–40 and Aβ1–42 (Figure 2A and 2B) including the NRTIs lamivudine and emtricitabine, the NNTRIs efavirenz and nevirapine, the PI darunavir, and the INI dolutegravir. Elvitegravir, which de-acidified endolysosomes, significantly elevated secreted levels of Aβ1–40 but not Aβ1–42. The two NRTIs, zidovudine and abacavir that acidified neuronal endolysosomes also significantly decreased secreted levels of Aβ1–40. Three ARTs that significantly de-acidified endolysosomes acutely, but not persistently, produced significant increases in levels of both Aβ1–40 and Aβ1–42; the NRTI tenofovir, and the PIs ritonavir and nelfinavir.

Figure 2. ART drugs affect amyloidogenesis in primary rat neurons.

Figure 2.

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(A) Levels of Aβ1–40 from the media of cultured neurons after ART drug treatment for 48 h were detected by ELISA. Δ secreted Aβ1–40 represented the difference of secreted Aβ1–40 between ARTs and DMSO (vehicle controls). Lamivudine (1 μM), emtricitabine (0.1 μM), tenofovir disoproxil (10 μM), ritonavir (1 μM), nelfinavir (1 μM), darunavir (0.1 μM), efavirenz (1 μM), nevirapine (1 μM), dolutegravir (1 μM) and elvitegravir (1 μM) significantly increased secreted Aβ1–40, whereas zidovudine (10 μM) and abacavir (10 μM) significantly decreased secreted Aβ1–40 (n=4–6, *p<0.05, **p<0.01, ***p<0.001). (B) Levels of Aβ1–42 from the media of cultured neurons after ART drug treatment for 48 h were detected by ELISA. Δ secreted Aβ1–42 represented the difference of secreted Aβ1–40 between ARTs and DMSO (vehicle controls). Compared to controls, lamivudine (1 μM), emtricitabine (0.1 μM), tenofovir disoproxil (10 μM), ritonavir (1 μM), nelfinavir (1 μM), darunavir (0.1 μM), efavirenz (1 μM), nevirapine (1 μM), dolutegravir (1 μM) significantly increased secreted Aβ1–42. Elvitegravir (1 μM) didn’t affect secreted Aβ1–42. In addition, zidovudine (10 μM) and abacavir (10 μM) slightly decreased secreted Aβ1–42. (n=4–6, *p<0.05, **p<0.01, ***p<0.001)

Acidifying endolysosomes with ML-SA1 blocked dolutegravir- and lamivudine-induced changes to endolysosome size

If endolysosome de-acidification-induced effects of ART drugs are critical for enhanced amyloidogenesis then acidifying endolysosomes should prevent ART drug-induced pathological changes. Consistent with our previous findings that activation of TRPML1 l channels acidifies endolysosome pH (Bae et al., 2014), administration of the TRPML1 agonist ML-SA1 (20 μM) to primary cultures of hippocampal neurons quickly and significantly caused endolysosome acidification (Figure 3A). ML-SA1 was used at a concentration of 20 μM because at this concentration ML-SA1 had robust effects in acidifying endolysosome pH and decreasing Aβ levels (Hui et al., 2019). Thus, we tested whether ML-SA1 prevents ART drug-induced endolysosome de-acidification. Here, dolutegravir and lamivudine were used because these two drugs exhert consistent and robust effects in de-acidifying endolysosome pH and increasing Aβ secretion, and because these two ART drugs are part of TRIUMEQ, the current common combination of ART drugs used in USA. We demonstrated that even in the presence of dolutegravir or lamivudine ML-SA1 still signficantly decreased endolysosome pH (Figure 3B, C). Because endolysosome de-acidification is known to change morphological features of endolysosomes, we determined effects of dolutegravir and lamivudine in the absence and presence of ML-SA1 on endolysosome sizes. As anticipated because of their ability to de-acidify endolysosomes, dolutegravir and lamivudine both increased endolysosome sizes in neurons (Figure 3D). When dolutegravir and lamivudine were tested in the presence of ML-SA1, endolysosome sizes were similar to those observed when vehicle alone was added (DMSO) or when ML-SA1 was added in the absence of dolutegravir and lamivudine (Figure 3D).

Figure 3. Acidifying endolysosomes with ML-SA1 prevented endolysosome de-acidifying ART drug-induced enlargement of endolysosomes.

Figure 3.

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(A) ML-SA1 (20 μM) acidified endolysosomes in primary cultured neurons (n=12, **p<0.01). (B) In the presence of dolutegravir (1 μM), ML-SA1 (20 μM) acidified endolysosomes in primary cultured neurons (n=25, ***p<0.001). (C) In the precence of lamivudine (1 μM), ML-SA1 (20 μM) acidified endolysosomes in primary cultured neurons (n=18, **p<0.01). (D) Following 48 h treatment, endolysosomes were labeled with LysoTracker (red). The sizes of endolysosomes were quantified with Image J software and the frequency distribution of different sizes of endolysosomes was determined. Dolutegravir (1 μM, DTG, brown) and lamivudine (1 μM, 3TC, blue) induced a right shift of the frequency distribution curve; an effect that was prevented by co-treating with ML-SA1 (20 μM).

Acidifying endolysosomes prevented de-acidifying ART drug-induced increases in Aβ

With the above results with ML-SA1 in hand, we were then able to determine causal relationships between endolysosome de-acidification and ART drug-induced increases in Aβ levels. The INI dolutegravir and the NRTI lamivudine both significantly increased levels of secreted Aβ1–40 and Aβ1–42 (Figure 4A). When ML-SA1 was administered with either dolutegravir or lamivudine, the ART drug-induced increases in secreted levels of Aβ1–40 and Aβ1–42 were blocked (Figure 4A). Similarly, when primary cultures of hippocampal neurons were tested 48 h after treatment with dolutegravir or lamivudine in the absence or presence of ML-SA-1, ART drug-induced increases in Aβ1–42 were blocked by ML-SA1 (Figure 4B).

Figure 4. ML-SA1 prevented endolysosome de-acidifying ART drug-induced amyloidogenesis.

Figure 4.

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(A) Following 48 h treatment, Aβ levels in neurons were measured by immunostaining with a Aβ1–42 antibody. Dolutegravir (1 μM) and lamivudine (1 μM) markedly increased the immunopositive signal of Aβ1–42, and such effects were prevented by co-treating with ML-SA1 (20 μM) (n=20–26, *p<0.05, scale bar=10 μm). (B) Following 48 h treatment, levels of secreted Aβ1–40 and Aβ1–42 from the media of cultured neurons were detected by ELISA. Compared to DMSO controls, dolutegravir (1 μM) and lamivudine (1 μM) significantly increased secreted levels of Aβ1–40 and Aβ1–42, and these effects were blocked by co-treatment with ML-SA1 (20 μM) (n=4, *p<0.05, **p<0.01, ***p<0.001).

Discussion

This study investigated linkages between ART drug-induced endolysosome de-acidification and amyloidogenesis in primary cultures of rat hippocampal neurons. We tested 12 ART drugs including nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors. In general, we found that those ART drugs that de-acidified endolysosomes also increased Aβ levels, whereas those ART drugs that acidified endolysosomes decreased Aβ levels. We found also that acidifying endolysosomes with the TRPML agonist ML-SA1 blocked ART drug-induced increases in Aβ levels. Furthermore, we found that ML-SA1 was able to block dolutegravir- and lamivudine-induced increases in endolysosome sizes in neurons. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and morphological changes to endolysosomes in neurons.

ART drugs have changed dramatically the clinical outcomes of people living with HIV-1. Clearly, a major benefit of these drugs is that they increase the lifespan of HIV-1 infected individuals to a point where people are now living almost full life-spans. They have also reduced the severity of neurological complications associated with HIV-1/AIDS. But, in this post-ART era there persists a high prevalence (about 50%) of milder forms of neurological complications termed HIV-associated neurological dysfunction (HAND) (Elbirt et al., 2015). Concomitantly and increasingly, people living with HIV-1 including those chronically ingesting ART drugs are experiencing clinical manifestations and pathological features of AD (Clifford et al., 2009; Ferrell and Giunta, 2014). A wide variety of ART drugs continue to be used in treating HIV-1 including nucleoside and non-nucleoside reverse transcriptase inhibitors, HIV-1 protease, fusion, entry and integrase inhibitors, as well as pharmacokinetic enhancers that increase the effectiveness of ART. Unfortunately, there is no cure for HIV-1/AIDS and therefore ART drug treatments are life-long, and this raises the prospect that health might be impacted in other ways.

Mounting evidence suggests that ART drugs are neurotoxic and may contribute to the pathogenesis of HAND and AD (Xu and Ikezu, 2009; Solomon et al., 2017). Many mechanisms have been implicated in HAND pathogenesis including involvement of mitochondria, endoplasmic reticulum, endolysosomes, and other subcellular organelles (Brown et al., 2014; Gannon et al., 2017). ART drugs can be classified physicochemically as being either weak acids or weak bases (Kashuba et al., 1999; Gallicano, 2000). For endolysosome-linked mechanisms, this might be of particular importance to ART drug-induced AD-like pathogenesis because weak bases accumulate in endolysosomes and cause endolysosome de-acidification. Such endolysosome de-acidification can result in elevated levels of Aβ because beta-secretase is mainly endosomal and gamma-secretase is mainly lysosomal while Aβ enzymatic degradation of Aβ occurs mainly in lysosomes (Miners et al., 2011; Vingtdeux et al., 2012; Moreau et al., 2014; Edgar et al., 2015). Even modest de-acidification of endolysosomes can increase the genesis and decrease the degradation of Aβ; the result being increased intraneuronal accumulations of and secreted levels of Aβ. As an example, the anti-malarial drug chloroquine, a well-known weak base, is known to de-acidify and change the morphology of endolysosomes, and to increases Aβ levels (Chu et al., 1998).

Weak acids and bases can be characterized using dissociation constants such as pKa, pKBH and pKBH+. However, limited data is available on the ART drugs tested and therefore it was difficult to know a priori whether an ART drug was a weak base or weak acid. Thus, we measured directly the ability of the ART drugs to affect endolysosome pH using a ratio-metric dye-based method. We found that 8 of 12 tested ART drugs de-acidified endolysosomes acutely and for all but one of these 8 drugs such de-acidifying effects persisted even 48 h after the drugs were applied. There are changes in the acute effects and persistent effects of ART drugs on endolysosome pH. For instance, emtritabline and elvitegravir did not de-acidfiy endolysosomes acutely but significantly de-acidifies endolysosome following 48 h treatment, whereas tenofovir, ritonavavir, and nelfinavir lost the acute endolysosome de-acidifying effects following 48 h treatment. We suspect that the metabolism of ART drugs might lead to such differences because metabolism of ART drugs could lead to gain-of-function or loss-of-function on ART drug-mediated endolysosome de-acidifying effects. These effects of ART drugs on endolysosome pH were independent of the mechanism by which the ART drugs affect HIV-1; endolysosome de-acidification was observed for ART drugs from all 4 categories tested and these include the NRTIs lamivudine and emtricitabine, the NNTRIs efavirenz and nevirapine, the PI darunavir, and the INIs dolutegravir and elvitegreavir. The endolysosome de-acidifying effects were similar to those observed with the weak base chloroquine, however we cannot exclude other possible mechanisms. Currently, we do not know how zidovudine and abacavir acidified neuronal endolysosomes, but it does not seem to simply relate to their physiochemical properties, because weak acids have no apparent effect on endolysosome pH.

Endolysosome de-acidification changes morphological features of endolysosomes. Accordingly, we tested effects of two ART drugs that were found to de-acidify endolysosomes on endolysosome sizes; dolutegravir and lamivudine. As anticipated because of their ability to de-acidify endolysosomes, dolutegravir and lamivudine both increased endolysosome sizes in neurons. When dolutegravir and lamivudine were tested in the presence of ML-SA1, endolysosome sizes were similar to those observed when vehicle alone was added (DMSO) or when ML-SA1 was added in the absence of dolutegravir and lamivudine. These effects of ART drugs on endolysosome morphology are consistent with findings of others showing that impairing endolysosome degradation leads to increased sizes (Chen et al., 2013). However, in addition, we also observed ART drug-induced increased numbers of smaller endolysosomes, and this might suggest an induction of endolysosome biogenesis possibly due to stress responses or autophagy inhibition.

In general, we found that ART drugs that de-acidified endolysosomes increased levels of Aβ and that ART drugs that acidified endolysosomes decreased levels of Aβ. Six of the seven endolysosome de-acidifying ART drugs significantly elevated secreted levels of Aβ1–40 and Aβ1–42. However, the integrase inhibitor elvitegravir that mildly de-acidified endolysosomes significantly increased levels of Aβ1–40 but not Aβ1–42. It is not currently clear why elvitegravir affected the secretion of Aβ1–40 and Aβ1–42 differently. However, we speculate that this difference might result from the degree of ART drug-induced endolysosome de-acidification. AβPP exhibits a pH-dependent conformational change (Hoefgen et al., 2015), and thus subtle differences in changes of pH could affect its processing, leading to the generation of different forms of Aβ. The activity of γ-secretase, which is responsible for generation of different forms of Aβ (Aβ38–42) (Acx et al., 2014), can be also influenced by pH (McLendon et al., 2000; Quintero-Monzon et al., 2011), and thus subtle differences in changes of pH could affect activity of γ-secretase differently, leading to the generation of different forms of Aβ. On the other hand, the activity of cathepsins, which have been implicated in degradation of Aβ (Miners et al., 2011; Saido and Leissring, 2012), is also dependent on pH, and thus subtle differences in changes of pH could affect activity of cathepsins differently, leading to the accumulation of different form of Aβ.

In contrast, endolysosome acidifying ART drugs zidovudine and abacavir, decreased levels of Aβ1–40 and Aβ1–42. Upon further exploring the causal relationship, we demonstrated that an endolysosome acidifying agent ML-SA1 blocked endolysosome de-acidifying ART drug-induced increases in Aβ, indicating that endolysosome de-acidification plays a critical role in ART drug-induced amyloidogenesis. These findings are certainly consistent with the concept that endolysosome pH plays an important role in ART drug-induced amyloidogenesis. We did not test the effects of the ART drugs in the presence of the endolysosome de-acidifying HIV-1 viral proteins Tat and gp120 (Chen et al., 2013; Bae et al., 2014). However, when tested together synergistic effects might be observed.

Although, our findings suggest that endolysosome de-acidification plays a critical role in ART drug-induced amyloidogenesis, endolysosome de-acidification may not be the only mechanism that leads to amyloidogenesis. We found that one NRTI tenofovir and two protease inhibitors, nelfinavir and ritonavir did not significantly affect endolysosome pH but significantly increased Aβ levels. These findings are consistent with a recent report showing that certain protease inhibitors can promote amyloidogenesis via ER stress (Gannon et al., 2017). Thus, endolysosome de-acidification appears to play an important role in the effects of HIV-1 Tat and gp120 (Chen et al., 2013; Bae et al., 2014) as well as the effects of ART drugs on amyloidogenesis. Combined ART is now recommended for all HIV patients and the treatment of PLWH is life-long, and current recommended treatment strategy consists of two NRTIs in combination with a third active ART drug from one of three drug classes: an INSTI, a NNRTI, or a PI with a pharmacokinetic enhancer. Thus, future in vitro and in vivo studies on combined ART drugs on endolysosome pH and amyloidogenesis are warranted. In future studies, we would combine those individual drugs that do not robust de-acidify endolysosome or combine those ART drugs that de-acidify endolysosomes and determine their effects on amyloidogenesis. Findings from current studies and future studies may help provide guidance on current clinical selection of ART drugs and on future development of HIV therapies in people living with HIV-1 experiencing AD-like symptoms.

Supplementary Material

11481_2019_9862_MOESM1_ESM

Acknowledgments

Funding: This study was funded by the National Institute of General Medical Sciences (P30GM100329 and U54GM115458), the National Institute of Mental Health (R01MH100972 and R01MH105329), the National Institute of Neurological Diseases and Stroke (R01NS065957), and the National Institute of Drug Abuse (2R01DA032444).

Footnotes

Conflicts of interest: Liang Hui declares that he has no conflict of interest. Yan Ye declares that she has no conflict of interest. Mahmoud Soliman declares that he has no conflict of interest. Koffi L. Lakpa declares that he has no conflict of interest. Nicole M. Miller declares that she has no conflict of interest. Zahra Afghah declares that she has no conflict of interest. Jonathan D. Geiger declares that he has no conflict of interest. Xuesong Chen declares that he has no conflict of interest.

Compliance with Ethical Standards

Ethical approval (Animals): All procedures performed in studies involving animals were in accordance with the ethical standards of the University of North Dakota Animal Care and Use Committee adherent with the Guide for the Care and Use of Laboratory Animals (NIH publication number 80–23).

Ethical approval (Humans): This article does not contain any studies with human participants performed by any of the authors

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

References

  1. Achim CL, Adame A, Dumaop W, Everall IP, Masliah E (2009) Increased accumulation of intraneuronal amyloid beta in HIV-infected patients. J Neuroimmune Pharmacol 4:190–199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Acx H, Chavez-Gutierrez L, Serneels L, Lismont S, Benurwar M, Elad N, De Strooper B (2014) Signature amyloid beta profiles are produced by different gamma-secretase complexes. J Biol Chem 289:4346–4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Annunziata I, Patterson A, Helton D, Hu H, Moshiach S, Gomero E, Nixon R, d’Azzo A (2013) Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-beta secretion via deregulated lysosomal exocytosis. Nat Commun 4:2734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arriagada C, Astorga C, Atwater I, Rojas E, Mears D, Caviedes R, Caviedes P (2007) Endosomal abnormalities related to amyloid precursor protein in cholesterol treated cerebral cortex neuronal cells derived from trisomy 16 mice, an animal model of Down syndrome. Neurosci Lett 423:172–177. [DOI] [PubMed] [Google Scholar]
  5. Bae M, Patel N, Xu H, Lee M, Tominaga-Yamanaka K, Nath A, Geiger J, Gorospe M, Mattson MP, Haughey NJ (2014) Activation of TRPML1 clears intraneuronal Abeta in preclinical models of HIV infection. J Neurosci 34:11485–11503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J Neurosci 28:6926–6937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braak H, Del Tredici K (2004) Alzheimer’s disease: intraneuronal alterations precede insoluble amyloid-beta formation. Neurobiol Aging 25:713–718; discussion 743–716. [DOI] [PubMed] [Google Scholar]
  8. Bright NA, Gratian MJ, Luzio JP (2005) Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol 15:360–365. [DOI] [PubMed] [Google Scholar]
  9. Brown LA, Jin J, Ferrell D, Sadic E, Obregon D, Smith AJ, Tan J, Giunta B (2014) Efavirenz promotes beta-secretase expression and increased Abeta1–40,42 via oxidative stress and reduced microglial phagocytosis: implications for HIV associated neurocognitive disorders (HAND). PLoS One 9:e95500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Buscemi L, Ramonet D, Geiger JD (2007) Human immunodeficiency virus type-1 protein Tat induces tumor necrosis factor-alpha-mediated neurotoxicity. Neurobiol Dis 26:661–670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA (2000) Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer’s disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol 157:277–286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cataldo AM, Petanceska S, Terio NB, Peterhoff CM, Durham R, Mercken M, Mehta PD, Buxbaum J, Haroutunian V, Nixon RA (2004) Abeta localization in abnormal endosomes: association with earliest Abeta elevations in AD and Down syndrome. Neurobiol Aging 25:1263–1272. [DOI] [PubMed] [Google Scholar]
  13. Chen X, Hui L, Geiger NH, Haughey NJ, Geiger JD (2013) Endolysosome involvement in HIV-1 transactivator protein-induced neuronal amyloid beta production. Neurobiol Aging 34:2370–2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chu T, Tran T, Yang F, Beech W, Cole GM, Frautschy SA (1998) Effect of chloroquine and leupeptin on intracellular accumulation of amyloid-beta (A beta) 1–42 peptide in a murine N9 microglial cell line. FEBS Lett 436:439–444. [DOI] [PubMed] [Google Scholar]
  15. Clifford DB, Fagan AM, Holtzman DM, Morris JC, Teshome M, Shah AR, Kauwe JS (2009) CSF biomarkers of Alzheimer disease in HIV-associated neurologic disease. Neurology 73:1982–1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Colacurcio DJ, Nixon RA (2016) Disorders of lysosomal acidification-The emerging role of vATPase in aging and neurodegenerative disease. Ageing Res Rev 32:75–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. de Duve C (2005) The lysosome turns fifty. Nat Cell Biol 7:847–849. [DOI] [PubMed] [Google Scholar]
  18. De Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F (1955) Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J 60:604–617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Edgar JR, Willen K, Gouras GK, Futter CE (2015) ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-beta accumulation. J Cell Sci 128:2520–2528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Elbirt D, Mahlab-Guri K, Bezalel-Rosenberg S, Gill H, Attali M, Asher I (2015) HIV-associated neurocognitive disorders (HAND). Isr Med Assoc J 17:54–59. [PubMed] [Google Scholar]
  21. Ferrell D, Giunta B (2014) The impact of HIV-1 on neurogenesis: implications for HAND. Cell Mol Life Sci 71:4387–4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gallicano K (2000) Antiretroviral-drug concentrations in semen. Antimicrob Agents Chemother 44:1117–1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gannon PJ, Akay-Espinoza C, Yee AC, Briand LA, Erickson MA, Gelman BB, Gao Y, Haughey NJ, Zink MC, Clements JE, Kim NS, Van De Walle G, Jensen BK, Vassar R, Pierce RC, Gill AJ, Kolson DL, Diehl JA, Mankowski JL, Jordan-Sciutto KL (2017) HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via beta-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation. Am J Pathol 187:91–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gelman BB, Soukup VM, Holzer CE 3rd, Fabian RH, Schuenke KW, Keherly MJ, Richey FJ, Lahart CJ (2005) Potential role for white matter lysosome expansion in HIV-associated dementia. J Acquir Immune Defic Syndr 39:422–425. [DOI] [PubMed] [Google Scholar]
  25. Giunta B, Ehrhart J, Obregon DF, Lam L, Le L, Jin J, Fernandez F, Tan J, Shytle RD (2011) Antiretroviral medications disrupt microglial phagocytosis of beta-amyloid and increase its production by neurons: implications for HIV-associated neurocognitive disorders. Mol Brain 4:23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hoefgen S, Dahms SO, Oertwig K, Than ME (2015) The amyloid precursor protein shows a pHdependent conformational switch in its E1 domain. J Mol Biol 427:433–442. [DOI] [PubMed] [Google Scholar]
  27. Hui L, Chen X, Geiger JD (2012a) Endolysosome involvement in LDL cholesterol-induced Alzheimer’s disease-like pathology in primary cultured neurons. Life Sci 91:1159–1168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hui L, Chen X, Bhatt D, Geiger NH, Rosenberger TA, Haughey NJ, Masino SA, Geiger JD (2012b) Ketone bodies protection against HIV-1 Tat-induced neurotoxicity. J Neurochem 122:382–391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hui L, Soliman ML, Geiger NH, Miller NM, Afghah Z, Lakpa KL, Chen X, Geiger JD (2019) Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis. J Alzheimers Dis 67:393–410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Huotari J, Helenius A (2011) Endosome maturation. EMBO J 30:3481–3500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jiang S, Li Y, Zhang X, Bu G, Xu H, Zhang YW (2014) Trafficking regulation of proteins in Alzheimer’s disease. Mol Neurodegener 9:6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kashuba AD, Dyer JR, Kramer LM, Raasch RH, Eron JJ, Cohen MS (1999) Antiretroviral-drug concentrations in semen: implications for sexual transmission of human immunodeficiency virus type 1. Antimicrob Agents Chemother 43:1817–1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Khan MB, Lang MJ, Huang MB, Raymond A, Bond VC, Shiramizu B, Powell MD (2015) Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Abeta secretion in SH-SY5Y neural cells. J Neurovirol. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. LaFerla FM, Green KN, Oddo S (2007) Intracellular amyloid-beta in Alzheimer’s disease. Nat Rev Neurosci 8:499–509. [DOI] [PubMed] [Google Scholar]
  35. Lan X, Kiyota T, Hanamsagar R, Huang Y, Andrews S, Peng H, Zheng JC, Swindells S, Carlson GA, Ikezu T (2012) The effect of HIV protease inhibitors on amyloid-beta peptide degradation and synthesis in human cells and Alzheimer’s disease animal model. J Neuroimmune Pharmacol 7:412–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liu J, Lu W, Reigada D, Nguyen J, Laties AM, Mitchell CH (2008) Restoration of lysosomal pH in RPE cells from cultured human and ABCA4(−/−) mice: pharmacologic approaches and functional recovery. Invest Ophthalmol Vis Sci 49:772–780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Luzio JP, Pryor PR, Bright NA (2007) Lysosomes: fusion and function. Nat Rev Mol Cell Biol 8:622–632. [DOI] [PubMed] [Google Scholar]
  38. McLendon C, Xin T, Ziani-Cherif C, Murphy MP, Findlay KA, Lewis PA, Pinnix I, Sambamurti K, Wang R, Fauq A, Golde TE (2000) Cell-free assays for gamma-secretase activity. FASEB J 14:2383–2386. [DOI] [PubMed] [Google Scholar]
  39. Mindell JA (2012) Lysosomal acidification mechanisms. Annu Rev Physiol 74:69–86. [DOI] [PubMed] [Google Scholar]
  40. Miners JS, Barua N, Kehoe PG, Gill S, Love S (2011) Abeta-degrading enzymes: potential for treatment of Alzheimer disease. J Neuropathol Exp Neurol 70:944–959. [DOI] [PubMed] [Google Scholar]
  41. Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O’Kane CJ, Wechsler DS, Rubinsztein DC (2014) PICALM modulates autophagy activity and tau accumulation. Nat Commun 5:4998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Morel E, Chamoun Z, Lasiecka ZM, Chan RB, Williamson RL, Vetanovetz C, Dall’Armi C, Simoes S, Point Du Jour KS, McCabe BD, Small SA, Di Paolo G (2013) Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system. Nat Commun 4:2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S, Tanaka M, Iwata N, Saito T, Saido TC (2013) Abeta secretion and plaque formation depend on autophagy. Cell Rep 5:61–69. [DOI] [PubMed] [Google Scholar]
  44. Nixon RA (2005) Endosome function and dysfunction in Alzheimer’s disease and other neurodegenerative diseases. Neurobiol Aging 26:373–382. [DOI] [PubMed] [Google Scholar]
  45. Nixon RA, Cataldo AM (1995) The endosomal-lysosomal system of neurons: new roles. Trends Neurosci 18:489–496. [DOI] [PubMed] [Google Scholar]
  46. Nixon RA, Cataldo AM (2006) Lysosomal system pathways: genes to neurodegeneration in Alzheimer’s disease. J Alzheimers Dis 9:277–289. [DOI] [PubMed] [Google Scholar]
  47. Perera RM, Zoncu R (2016) The Lysosome as a Regulatory Hub. Annu Rev Cell Dev Biol 32:223–253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Quintero-Monzon O, Martin MM, Fernandez MA, Cappello CA, Krzysiak AJ, Osenkowski P, Wolfe MS (2011) Dissociation between the processivity and total activity of gamma-secretase: implications for the mechanism of Alzheimer’s disease-causing presenilin mutations. Biochemistry 50:9023–9035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rajendran L, Annaert W (2012) Membrane trafficking pathways in Alzheimer’s disease. Traffic 13:759–770. [DOI] [PubMed] [Google Scholar]
  50. Rajendran L, Schneider A, Schlechtingen G, Weidlich S, Ries J, Braxmeier T, Schwille P, Schulz JB, Schroeder C, Simons M, Jennings G, Knolker HJ, Simons K (2008) Efficient inhibition of the Alzheimer’s disease beta-secretase by membrane targeting. Science 320:520–523. [DOI] [PubMed] [Google Scholar]
  51. Saido T, Leissring MA (2012) Proteolytic Degradation of Amyloid beta-Protein. Cold Spring Harb Perspect Med 2:a006379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sannerud R, Declerck I, Peric A, Raemaekers T, Menendez G, Zhou L, Veerle B, Coen K, Munck S, De Strooper B, Schiavo G, Annaert W (2011) ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1. Proc Natl Acad Sci U S A 108:E559–568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Settembre C, Fraldi A, Medina DL, Ballabio A (2013) Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat Rev Mol Cell Biol 14:283–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shimizu H, Tosaki A, Kaneko K, Hisano T, Sakurai T, Nukina N (2008) Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production. Mol Cell Biol 28:3663–3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Solomon IH, De Girolami U, Chettimada S, Misra V, Singer EJ, Gabuzda D (2017) Brain and liver pathology, amyloid deposition, and interferon responses among older HIV-positive patients in the late HAART era. BMC Infect Dis 17:151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tate BA, Mathews PM (2006) Targeting the role of the endosome in the pathophysiology of Alzheimer’s disease: a strategy for treatment. Sci Aging Knowledge Environ 2006:re2. [DOI] [PubMed] [Google Scholar]
  57. Vingtdeux V, Sergeant N, Buee L (2012) Potential contribution of exosomes to the prion-like propagation of lesions in Alzheimer’s disease. Front Physiol 3:229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Xu J, Ikezu T (2009) The comorbidity of HIV-associated neurocognitive disorders and Alzheimer’s disease: a foreseeable medical challenge in post-HAART era. J Neuroimmune Pharmacol 4:200–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zou C, Montagna E, Shi Y, Peters F, Blazquez-Llorca L, Shi S, Filser S, Dorostkar MM, Herms J (2015) Intraneuronal APP and extracellular Abeta independently cause dendritic spine pathology in transgenic mouse models of Alzheimer’s disease. Acta Neuropathol 129:909–920. [DOI] [PMC free article] [PubMed] [Google Scholar]

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