Extracellular amyloid-β 42 oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells

Abstract

Background

The levels of soluble amyloid-β (Aβ)₄₂ oligomers in the brains of patients with Alzheimer's disease (AD) are well-known to correlate with the extent of synaptic loss. However, the abnormal role of these oligomers in disrupting the balance of the endo-lysosomal pathways for substance degradation in AD brains and cellular models remains unclear.

Objective

We aimed to investigate whether extracellular Aβ₄₂ oligomers alter the composition, distribution, and identity of vesicular components and impact the processing of substances involved in the endo-lysosomal pathways of protein degradation.

Methods

We overexpressed Rab7, Lamp-1, and β-1,4-galactosyltransferase-1 proteins in nondifferentiated SH-SY5Y cells. We then incubated these cells with extracellular Aβ₄₂ oligomers, and evaluated the effects on the morphology, composition, and distribution of vesicular components in the endo-lysosomal pathway using super-resolution confocal microscopy. Additionally, we assessed the effects of Aβ₄₂ oligomers incubation on the degradation and processing of endocytosed fluorescent transferrin in vivo.

Results

Our findings revealed that Aβ₄₂ oligomers alter the distribution and identity of Rab7- and Lamp-1-coated vesicles, as well as the Golgi apparatus. This alteration resulted in the formation of disorganized vesicles carrying distinct surface markers of the endo-lysosomal pathway, without evident changes to the cytoskeleton. In vivo evaluation showed a delayed degradation of endocytosed fluorescent transferrin after incubation with Aβ₄₂ oligomers.

Conclusions

Aβ₄₂ oligomers may contribute to neuronal toxicity by inducing changes in the identity, distribution and balance of vesicular components associated with the endo-lysosomal pathway. This disruption impacts the processing and degradation of various materials that accumulate in the cytoplasm

Keywords

Alzheimer’s disease amyloid-β oligomers confocal microscopy endosomes intracellular trafficking lysosomes Rab7

Introduction

Accumulation of insoluble protein aggregates is a histopathological feature observed in the brains of Alzheimer's disease (AD) patients. Abnormally processed tau proteins aggregate intracellularly; meanwhile, amyloid-β (Aβ) peptides of 1–42 amino acids in length, which are the products of sequential cleavage of amyloid-β protein precursor (APP) by β- and γ-secretases, accumulate in the extracellular space in the form of distinct Aβ plaques.^1,2 Regarding the pathogenic role of the Aβ₄₂ peptide in AD, it has long been recognized that the filamentous forms of this peptide that accumulate in insoluble plaques are the primary cause of neuronal degeneration in the brains of individuals affected by the disease. However, subsequent evidence has supported the idea that the early occurrence of oligomeric entities in the brains of AD patients^3–5 produces more toxic and physiological alterations in the normal functioning of neurons. When Aβ is released to the extracellular space and adopts an oligomeric state, it can be endocytosed by neighbor neurons, altering several intracellular processes as mitochondrial stress, among others, and leading to metabolic imbalance and toxicity.

It is widely accepted that the levels of soluble Aβ oligomers in the brain are strongly correlated with the severity of the disease and the extent of synaptic loss, rather than the accumulation of fibrillar Aβ plaques.^6,7 In addition, it has been reported that in AD cases, there is a reduction in the number of synapses, as well as a decrease in the expression of presynaptic (synaptophysin) and postsynaptic (synaptopodin and PSD-95) proteins, compared to healthy controls, which has been partially attributed to the accumulation of Aβ.⁸ Coincidentally, in animal models of AD, synaptic loss has been confirmed to occur long before the development of typical neurofibrillary lesions.⁹

The cellular and molecular mechanisms by which Aβ oligomers exert toxicity are varied and involve alterations in the permeability of the plasma membrane, dysregulation of intracellular calcium homeostasis, interactions with receptors, impairment of signaling cascades, functional alterations in the mitochondria and endoplasmic reticulum, increased phosphorylation of tau by activation of various kinases, and an increase in the amount of free radicals, among others.^10–17 Other studies have also reported that exposure to these oligomers alters vesicular and mitochondrial transport in axons of differentiated primary hippocampal neurons.¹⁶ There is evidence that Aβ oligomers can affect the intracellular dynamics of synaptic vesicles by directly interacting with various vesicular components and interfering with the mechanisms that regulate the trafficking, docking, and fusion of vesicles for the release of synaptic proteins.^18–21

Other abnormalities that occur in AD include the aberrant accumulation of misfolded proteins and failure in their transport and degradation.^22,23 As a cellular response to this accumulation, early studies in the brains of AD patients have documented a significant upregulation of lysosomal activity, as determined by increased synthesis of acid hydrolases and an elevated number of abnormal lysosomes in the affected areas, which also accumulate Aβ deposits.^24–27 Moreover, alterations in the intracellular trafficking of substances in neurons can also be associated with disturbances in the expression and regulation of the activity of Rab GTPases.²⁸ In coordinated interactions with other effector proteins (e.g., COPI, kinesins, dyneins, Golgins, HOPS complex, SNARESs, etc.),^29,30 Rab GTPases participate in the regulation of intracellular vesicular trafficking.

In 2007, Scheper et al.³¹ reported increased expression of Rab6 in the brains of patients who developed AD. This protein participates in retrograde trafficking connecting the Golgi apparatus to the endoplasmic reticulum. Several other GTPases groups participate in vesicular trafficking that constitutes the endo-lysosomal pathway, with Rab5 being the main early endosome effector and Rab7 being a fundamental constituent of late endosomes.²⁸ Rab4, Rab5, and Rab7 have been reported to be overexpressed in AD brain tissue, as well as in animal and cell models representative of this pathology, and are involved in endocytosis along with early and late endosomal formation.^32–34 When a Rab5 antibody was used to identify this GTPase, which modulates the transport kinetics between the plasma membrane and early endosomes, a marked increase in the volume and size of early endosomes was observed in AD brain tissue.²⁵ Selective up-regulation of Rab5 and Rab7 in the hippocampus of individuals with mild cognitive impairment and AD cases has also been demonstrated.^35,36 Recent research involving C6 glial cells has demonstrated that endocytosed Aβ oligomers physically hinders the interaction between Rab7 and the Rab-interacting lysosomal protein (RILP). This inhibition affects the maturation process of early to late endosomes, which is essential for the recycling of the miRNA ribonucleoprotein complexes (miRNPs) and their binding to cytokine-encoded mRNAs that need to be degraded.³⁷

From all the evidence, Aβ-induced dysregulation of the expression and activity of Rab GTPases involved in the endo-lysosomal pathway may promote aberrant endosomal signaling, leading to disturbances in the mechanisms of protein processing and degradation, which in the long run may lead to neurodegeneration.

In the present study, we aimed to evaluate whether extracellular Aβ₄₂ oligomers can alter the distribution of Rab7- and Lamp-1-coated vesicles, components of the endo-lysosomal pathway, as well as their activities in the processing of substances inside cultured neuroblastoma cells.

Based on the current research, we propose that an additional mechanism of toxicity of Aβ₄₂ oligomers on neurons may include changes in the identity and localization balance of the vesicular components of the endo-lysosomal pathway, affecting their functionality and, therefore, the processing and degradation of distinct materials accumulated in the cytoplasm of the cells.

Methods

SH-SY5Y cell culture and transfection

SH-SY5Y has long been used by our group and others to transiently express proteins and evaluate distinct physiological and cellular processes under various stressor conditions. In their undifferentiated state, these cells are successfully transfected by distinct plasmids with minor alteration in their viability. Despite their undifferentiated phenotype, where neurites are almost absent compared to mature neurons, their spindle and polyhedral shapes facilitate the analysis of intracellular components in a larger cytoplasmic area.

SH-SY5Y human neuroblastoma cells were obtained from the American Type Culture Collection (ATCC CRL-226) (Manassas, VA, USA) and cultured in DMEM-F12 medium supplemented with 5% (v/v) Fetal Bovine Serum (FBS) (Invitrogen Life Technologies-GIBCO, Carlsbad. CA, USA), 100 U/mL Penicillin, 100 µg/mL Streptomycin, and maintained at 37°C in a humidified atmosphere of 5% CO₂. The culture medium was replaced every two days. In this study, SH-SY5Y cells were maintained without growth factors in DMEM-F12 medium to avoid their differentiation. For passages, cells were detached by incubation in 0.25% trypsin for 2 min at 37°C.

When the growing cells reached 70–80% confluence, they were transiently transfected with the pmRFP-C3 plasmid, which codifies for the Red Fluorescent Protein (RFP)-tagged to Rab7 protein (provided by Ari Helenius; Addgene plasmid #14436; http://n2t.net/addgene: 14436; RRID: Addgene_14436).³⁸ In some cases, it was co-transfected with the mEmerald-lysosomes-20 plasmid codifying a fluorescent Emerald-tagged to Lamp-1 protein (provided by Michael Davidson; Addgene plasmid #54149; http://n2t.net/addgene: 54149; RRID: Addgene_54149) or the plasmid pmTurquiose2-Golgi, which codifies a fluorescent Turquiose2-N1 tagged to β-1–4 galactosyltransferase-1 (provided by Dorus Gadella; Addgene plasmid #36205; http://n2t.net/addgene: 36205; RRID: Addgene_36205).³⁹ Lipofectamine 2000 was used as a transfecting agent following the manufacturer's instructions (Invitrogen Life Technologies-GIBCO). These proteins were selected because they are key components of the endo-lysosomal trafficking process and reside on the surface of vesicular components for more accurate localization.

As a transfection control, neuroblastoma cells were co-transfected with plasmids codifying for Rab7 and the APP (provided by Dennis Selkoe & Tracy Young-Pearse; Addgene plasmid #30154; http://n2t.net/addgene:30154; RRID: Addgene 30154) for 72 h. The viability of SH-SY5Y cells, subjected or not to different conditions, was evaluated using the MTT (3-(4,5-diMethylThiazol-2-yl)-2, 5-diphenyl Tetrazolium bromide) assay (Sigma-Aldrich, St Louis, MO, USA), as previously described.⁴⁰

Preparation of Aβ₄₂ oligomers and cell incubation

Oligomeric Aβ₄₂ was prepared according to Klein.⁴¹ In brief, lyophilized Aβ₄₂ peptide (AnaSpec AS20276) was dissolved in 1,1,1,1,3,3,3-hexafluoruro-2-propanol (HFIP) (Sigma, St Louis, MO) at a concentration of 1 mM (1 mg/0.22 mL) to keep the peptide in the monomeric state and maintained for 30 min in the dark at 37°C. Afterwards, the solution was aliquoted, the HFIP was evaporated by applying Speed Vac for 15 min, and the peptide film was stored at −80°C. Prior to cell incubation, oligomerization of Aβ₄₂ peptides was achieved by incubating Aβ₄₂ aliquots in a polymerization buffer containing 1% Dimethyl sulfoxide (DMSO) (Sigma) in phosphate-buffered saline (PBS) (pH 7.4) at 37°C for 12 h and then sonicated.

Specific concentrations of Aβ₄₂ oligomers were obtained for cell incubation by diluting the primary stock in SFB-complemented DMEM-F12 medium.

Cells attached to 0.1% (diluted in PBS) collagen pre-covered coverslips were incubated in wells containing Aβ₄₂ oligomers diluted in FBS-complemented DMEM-F12 medium at different times and washed in DMEM-F12 alone at the end of the treatment. As control experiments, cells were also exposed to Aβ₁₋₄₀ peptide (AnaSpec, AS24235), and the same parameters were evaluated after 72 h of incubation. Additional experiments in transferrin-loaded neuroblastoma cells (Invitrogen Life Technologies-GIBCO, T2871) included the exposure to Chloroquine (Sigma, C6628) to inhibit autophagy.

Electrophoresis and western blotting

The oligomeric form of Aβ₄₂ was corroborated by polyacrylamide gel electrophoresis (PAGE) and immunoblotting using an Aβ antibody (Table 1). Based on the initial concentration of the Aβ₄₂ oligomer stock solution, samples were diluted one-to-one in sample buffer containing only 100 mM Tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCL, Sigma) pH 6.8 0.2% Bromophenol blue (Sigma) and 20% Glycerol (J.T. Baker, Xálostoc, Edo. Mex., México). Oligomeric forms of Aβ₄₂ were separated on a 12% Sodium Dodecyl Sulfate (SDS)-polyacrylamide gel (BIO-RAD Laboratories, Inc, Hercules, CA, USA) and transferred to a nitrocellulose membrane for immunoblot analysis. The membranes were blocked overnight at 4°C in 5% nonfat dried milk in PBS-t (PBS-0.1% Tween 20, Sigma). The mouse monoclonal antibody to Aβ was diluted in PBS-t, added to the membranes, and incubated for 1 h at room temperature (RT). After washing in PBS-t, the membrane was incubated with the corresponding peroxidase-conjugated secondary antibody to mouse IgG (1:20,000; ZYMED Invitrogen, Carlsbad, CA, USA) in PBS-t for 1 h. Bands of immunoreactive oligomeric forms of Aβ₄₂ were visualized after membrane incubation in Western Lightning Plus Ehanced Cemiluminescence Ragent (ECL, PerkinElmer Inc. Waltham, MA, USA) and were visualized on autoradiography films (Kodak Medical X-ray, general purpose-blue, Eastman Kodak Company, Rochester, NY, USA) according to the manufacturer's instructions.

Table 1.

Antibodies and fluorescent dyes used in this study.

Antibody	Epitope	Host	Purpose	Dilution	Source
Anti- amyloid-β	13–28*	Mouse IgG	IF, WB	1:1000	Sigma Aldrich A8978
Anti- amyloid-β	13–28*	Mouse IgG	IF, WB	1:2000	Sigma Aldrich A8978
Anti-α-tubulin	α-tubulin	Mouse IgG	IF	1:500	Santa Cruz sc-32293
Anti-Rab7	Rab7	Rabbit IgG	IF, WB	1:500	AB clonal A12784
Anti-Flag	Flag	Rabbit IgG	IF	1:3000	Sigma Aldrich F7425
Hoechst-33258	nuclei	Excitation/ emission 340/461	IF	1:3000	Invitrogene 33258
Rhodamine-Phalloidin	Actin-F cytoskeleton	540/565	IF	1:500	Invitrogene R415
Fluorescein- Phalloidin	Actin-F cytoskeleton	496/516	IF	1:500	Invitrogene F432
Fluorescein-Transferrin	-	495/518	IF	1:500	Invitrogene T72871

*amino acid sites. IF: immunofluorescence; WB: western blotting.

To obtain cell lysates, Aβ₄₂-treated and non-treated cells were washed twice with cold PBS, scraped, and lysed in radioimmunoprecipitation buffer (RIPA) containing a protease inhibitor cocktail (150 mM NaCl, 50 mM Tris pH 8.0, 1.0% Triton X-100, 0.5% sodium deoxycholate, 1 mM PMSF, 100 mM NaF, 1 mM Na3VO 4.2 g/mL Complete; (Roche, Indianapolis, IN, USA)). Subsequently, they were centrifuged for 10 min at 14,000 g. The supernatant was collected, and the final protein concentration was determined using the Bio-Rad protein assay reagent. Electrophoretic separation was performed following the parameters described above. The same procedure was conducted for transfected cells according to the abovementioned methodology.

Immunofluorescence and high-resolution confocal microscopy

Transfected and non-transfected undifferentiated SH-SY5Y cells incubated with or without Aβ₄₂ oligomers were subjected to multiple immunofluorescent labeling. Cells were fixed with 4% paraformaldehyde for 15 min at RT and permeabilized for 10 min in 0.1% Triton X-100 PBS. Prior to immunolabeling, cells were blocked with 0.5% gelatin and 1.5% fetal bovine serum in PBS for 1 h at RT.

For multiple labeling, cells expressing fluorescent-tagged proteins were co-stained with specific primary antibodies and/or fluorescent cell diffusible dyes to identify specific intracellular components (See Table 1). Cells were incubated for 1 h at RT for antibody co-labeling in a humidity chamber. An antibody to mouse IgG tagged with fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) was used as the secondary antibody by incubating for 1 h at RT in PBS (1:200). Multiple labeling was achieved in some experiments by adding the fluorescent nuclear marker Hoescht 33258 (Molecular Probes Invitrogen Life Technologies, Grand Island, NY, USA) for 3 min.

Fluorescent samples were analyzed in a Leica-TCP-SP8 confocal laser scanning microscope with a 63x (NA: 1.4) OIL PH3 CS2 HC PL APO lens (Leica Microsystems, Heidelberg, Germany). The resulting images were obtained and analyzed using Leica Confocal-LAS-AF Lite version 2.x software. A novel HyD hybrid detector (included in the setup) was used to analyze the association between fluorescent markers critically. It reduces low signal levels and high noise during the capture process and increases their sensitivity to produce sharp, detailed images. Moreover, the high-resolution images were obtained using a super-resolution Lightning module, overcoming the optical limits of less than 200 nm. An optical channel was applied using Differential Interference Contrast (DIC) as a comparative background to corroborate cell morphology and locate fluorescent signals.

Quantitative determination of colocalization

Quantitative colocalization was evaluated using the colocalization tool included in the LAS-X software (LAS-X quantification tool) from high-resolution single optical sections and selected regions of interest. To determine the dispersion of fluorescent proteins and their colocalization along the principal longitudinal axis of the cell, a linear vector was traced from the nucleus to the distal part of the principal cell process. We divided the cytoplasmic space along this vector into three major regions: centrosomal, medial, and distal. At least four regions of interest were randomly selected from each region, and colocalization values between two fluorescent markers were obtained. At least fifteen cells were analyzed for any specific combination of fluorescent markers.

The significance of the colocalization values was validated by Pearson's correlation analysis⁴² and is presented in two ways, as a heat map displaying the converted pseudo-color values of all the individual determinations, or as the mean of Pearson's values and their corresponding standard errors. In addition, the spatial distribution of the two signals was also analyzed using single optical sections and fluorescence intensity profiles of linear vectors created from the centrosomal region to the distal part of the cells. Finally, 2-D and 3-D projections of the entire stack of optical sections were also obtained using the LAS-X 3D viewer.

Live cell analysis

To analyze the endocytosis and processing of proteins via the endo-lysosomal pathway, transfected and non-transfected cells were cultured in thin glass chambers (101350, SLP Life Sciences, Gyeonggi-do, Korea), which were designed to reach the optimal focus distance when viewing the samples through an inverted microscope. Cells treated or untreated with Aβ₄₂ oligomers were transferred to a Ludin box-DMi incubation chamber connected to a confocal microscope and observed in a stable environment for varying periods.

To evaluate the functional alteration of the endo-lysosomal pathway of protein degradation by incubation of Aβ₄₂ oligomers in Rab7-expressing neuroblastoma cells, the time course of in vivo degradation of fluorescent transferrin, added exogenously and traced for several hours by intracellular endocytosis, was analyzed. In parallel, coverslip-attached cells were also subjected to the same treatments and fixed and processed for confocal microscopy analysis.

Results

Exposure to Aβ₄₂ oligomers alters the distribution of Rab7-coated vesicles of neuroblastoma cells

As reported by Klein,⁴³ Aβ₄₂ oligomers were prepared by incubating monomeric Aβ₄₂ (42 amino acid residues) in polymerization buffer for 6–24 h. High molecular weight species (∼250 KDa) could be sufficiently produced by 12 h of incubation (Figure 1A) and used to expose non-differentiated SH-SY5Y cells to these oligomeric forms of Aβ₄₂. Initially, the cell toxicity of Aβ₄₂ oligomers was corroborated by performing a survival analysis of neuroblastoma cells exposed to increased concentrations of these Aβ₄₂ aggregates. As shown in Figure 1B, incubation with 15 µM Aβ₄₂ oligomers for 72 h reduced cell viability to 60% compared to untreated cells, and almost to 50% with 25 µM Aβ₄₂ oligomer exposure. The typical cell morphology of non-treated neuroblastoma cells was not severely affected by cell incubation in 15 µM Aβ₄₂ oligomers for 72 h, as shown by the distribution of the F-actin cytoskeleton depicted in Figure 1C and 1D. Treated cells preserved an assembled F-actin cytoskeleton (Figure 1E, F). The tubulin cytoskeleton was also unaffected, preserving its assembled distribution in the cytoplasmic space under Aβ₄₂ oligomer incubation (Figure 1E, F). Furthermore, nuclear morphology was unaffected, showing a typical round or oval profile (Figure 1C-F). Apoptotic nuclei were not detected under this condition. Since few morphological changes were observed in neuroblastoma cells incubated with 15 µM of Aβ₄₂ oligomers, this concentration was used uniformly in subsequent experiments.

Figure 1.

Reduction in viability of non-differentiated SH-SY5Y cells by incubation with Aβ₄₂ oligomers. (A) Observations on the kinetics of oligomerization of monomeric Aβ₄₂ (42 amino acid residues) by western blot analysis using a monoclonal antibody against the Aβ peptide. High-molecular-weight species (∼250 KDa) were formed after 6 h of incubation in polymerization buffer. (B) Evaluation of the viability of SH-SY5Y cells incubated for 72 h with several concentrations of Aβ₄₂ oligomers using the MTT method.⁴⁰ A population of around 15 thousand cells was initially seeded for each condition. One-way ANOVA, **p < 0.01, ***p < 0.001. Mean and Standard Error of the Mean (SEM) are shown. Immunofluorescence and confocal microscopy of control (C, D) and Aβ₄₂ oligomers-treated cells (E, F) showing the aspect of the F-actin cytoskeleton (second column) detected with Phalloidin-Rhodamine and the microtubule organization (third column) as visualized using a monoclonal antibody against α-tubulin. Cells were counterstained with Hoescht-33258 (HS) to visualize the nuclei. No profile of apoptotic nuclei was observed. DIC: Differential Interference Contrast Microscopy. Scale bars: 33 µm in C, 13 µm in D, 33 µm in E, 17 µm in F.

In Aβ₄₂ oligomer-treated neuroblastoma cells, we analyzed the distribution of these entities by immunofluorescence in incubation times of as long as 72 h. We found that variable-size Aβ₄₂ oligomer aggregates were commonly distributed over the cell surface, with no localization in the cytoplasm (Supplemental Figure 1A, B), as the resolution of confocal microscopy defined it. Aβ₄₂ oligomer-treated cells were washed and extracted for analysis using PAGE and western blotting, which confirmed that Aβ₄₂ oligomers and complexes were absent from the cells (Supplemental Figure 1C). From here onwards, all the Aβ₄₂ -oligomer effects we observe are suspected to be caused by primary contact on the extracellular side of the plasma membrane.

On the other hand, to visualize Rab7-coated vesicles, we initially evaluated this protein's basal expression in non-differentiated neuroblastoma cells by immunofluorescence and western blot using an antibody to Rab7. However, the Rab7 expression was too weak to chase its distribution by immunofluorescence in variable-size vesicular components (Figure 2A). To overcome this limitation, we decided to overexpress Rab7 in neuroblastoma cells by conducting transient transfection with a plasmid codifying for RFP-tagged Rab7 protein (Figure 2A, B). According to previous reports, when the Rab7 plasmid is used, the protein is expressed in its native form, and its activation will depend on the conditions of the cell. Figure 2C(a) and 2C(c) show that Rab7-coated vesicles of different sizes are predominantly distributed in the juxtanuclear region (arrows) and few elements remain distal to this localization. However, upon incubation with Aβ₄₂ oligomers (Figure 2C(b), C(d)), Rab7-coated vesicles delocalized from the perinuclear area to more anterograde sites in the cytoplasm and commonly accumulated in distal regions of the cytoplasmic process (arrowheads in Figure 2C(b), C(c)).

Figure 2.

The incubation with Aβ₄₂ oligomers produced alterations in the distribution of Rab7-coated vesicles in the cytoplasm. Cells were processed for immunofluorescence and confocal microscopy using an antibody to detect endogenous expression of Rab7 (Table 1). As shown in (A), the fluorescent signal of endogenous Rab7 is reduced compared to cells that were transfected with a plasmid coding for this molecule attached to the RFP protein. Extracts of the same groups of cells were obtained and resolved by PAGE and western blotting using the same antibody to Rab7 (B). The low expression of endogenous Rab7 (∼23 kDa) is confirmed in both groups of cells, and an enriched band of ∼50 kDa corresponding to RFP-attached Rab7 protein is observed in the transfected cells group. (C) Transfection of control SH-SY5Y cells with a plasmid encoding RFP-tagged Rab7 protein and fluorescence counterstaining of the F-actin and tubulin cytoskeletons. Rab7-coated vesicles are mainly distributed in the juxtanuclear region (arrows in C(a) and C(c)). Following incubation of cells with 15 µM Aβ₄₂ oligomers, a redistribution of Rab7-coated vesicles into more anterograde regions is observed (C(b) and C(d)). Arrowheads indicate the distal relocalization of Rab7-coated vesicles. Scale bars: 20 µm in A, C(a), C(c), and C(d); 18 µm in C(b).

As control experiments, we prepared Aβ₁₋₄₀ (Aβ₄₀) in the same polymerization conditions, and Rab7-expressing neuroblastoma cells were incubated with this mixture for 72 h. No changes in the juxtanuclear localization of Rab7-coated vesicles were observed upon this condition (Supplemental Figure 2). Moreover, as a control of transfection, we also co-expressed Rab7 and APP in neuroblastoma cells, and compared to non-APP-expressing cells, no change in the distribution of Rab7-coated vesicles was observed (Supplemental Figure 3).

Aβ₄₂ oligomers disturb the association of Rab7-coated vesicles with lysosomes

Neuroblastoma cells were double-transfected with plasmids encoding RFP-tagged Rab7 and mEmerald-tagged Lamp-1, a well-known marker for lysosomes (Figure 3A, B), and the pattern of association between these markers in vesicular components was evaluated via quantitative colocalization using super-resolution images obtained by confocal microscopy (Figure 3C-H). Low-magnification images (Figure 3A, B) showed the distribution of Rab7- and Lamp-1-coated vesicles, which were predominantly localized around the nuclear and centrosomal regions (arrows); however, a certain amount of Lamp-1 vesicles was also observed in the remaining cytoplasm. At higher magnification (Figure 3C-F), colocalization between the two markers was observed in many vesicles near the centrosomal region (arrows). Nonetheless, a further number of vesicles were also observed to show only one of each marker. By tracing a linear vector in a single section from the nuclear area to a distal point in the cytoplasm (Figure 3G), the intensities of FITC and TRITC emission were determined. The line-scan plots show that both signals increase concomitantly over the nuclear region, while remaining low at the distal site of the cell (plots in panel I). This strong association between these two markers can be clearly observed in a single high-resolution optical section (Figure 3H). Many vesicles co-carrying Rab7 and Lamp-1 are distinguished by yellow pseudocolor.

Figure 3.

Alteration in the Rab7 and lamp-1-coated vesicle distribution in SH-SY5Y cells by exposure to Aβ₄₂ oligomers. Normal neuroblastoma cells were co-transfected with plasmids encoding RFP-tagged Rab7 protein and mEmerald-tagged Lamp-1, a conspicuous lysosomal marker. (A) and (B) show panoramic views of neuroblastoma cells expressing Rab7 and Lamp-1 using a DIC contrast channel to better trace the fluorescent signal in the cytoplasmic space. Arrows indicate the sites of colocalization of both markers. One representative cell is shown in (C-F), evidencing the colocalization of diverse populations of vesicles carrying Rab7 and Lamp-1, predominantly in the juxtanuclear region (arrows). (G) and (H) correspond to a single optical section of super-resolved images obtained from the same cell in (C-F), where colocalization can be distinguished. Quantitative analysis of the fluorescence signal peaks for each marker was obtained along a linear vector traced from the nucleus to a distal region of the cell (G). The graph in (I) illustrates the fluorescent intensity of Rab7 and Lamp-1 in relation to the distance between the nucleus and a distal point of the cell, indicating a predominant localization of both markers to the centrosomal region, which progressively diminishes in the cell towards the distal point. (J) and (K) are panoramic views of cells treated with extracellular Aβ₄₂ oligomers. Arrows indicate the abnormal redistribution of Rab7 and Lamp-1-coated vesicles to the distal regions of the cells. Amplification of one representative cell (L-Ñ) shows the colocalization of both markers at the distal points of the cell processes (arrows). Single sections showed in (O) and (P) evidenced a reduced colocalization of both markers in the centrosomal region. The graph in (Q) illustrates the fluorescent intensity of Rab7 and Lamp-1 versus the distance between the nucleus and a distal point of the representative cell shown in (O), now indicating a predominant localization of both markers to the distal region of the cell. Scale bars: 25 µm in A-B; 10 µm in C-F; 6 µm in G; 5 µm in H; 36 µm in J-K; 15 µm in L-Ñ; 12 µm in O; 4 µm in P.

When double-transfected cells were incubated with Aβ₄₂ oligomers, a large number of vesicles relocalized to the distal extremes of the cells (arrows in Figure 3J-K), and the colocalization of both markers, visible as yellow pseudo-color at the distal point of the cell, is evident in higher-magnification images (arrows in Figure 3L-Ñ). When the fluorescence signal from centrosomal to distal regions was analyzed linearly (Figure 3O), the opposite profile was observed, displaying high fluorescence values for both markers at the distal point of the cell (Figure 3Q). A reduced amount of double-labeled vesicles was observed in a high-resolution optical section of the centrosomal region (Figure 3P).

Colocalization between Rab7 and Lamp-1 in vesicular elements of Aβ₄₂ oligomer-treated and untreated cells was also calculated using confocal microscopy software by analyzing fluorograms and determining Pearson's correlation coefficients.⁴² Cells were anatomically subdivided into three major areas to determine anterograde vesicle dispersion: centrosomal, medial, and distal. The fluorescent signal of vesicles was obtained from regions of interest in these sectors and compared between Aβ oligomer-treated and untreated cells (Figure 4). To be more accurate in the analysis of vesicle dispersion, cells displaying polyhedral or spindle-shaped morphology were selected, leaving out of this determination those small cells with a rounded profile, which mostly correspond to recently divided cells.

Figure 4.

Quantitative determination of colocalization between Rab7- and Lamp-1-coated vesicles in Aβ oligomer-treated and untreated cells. Neuroblastoma cells are anatomically subdivided into centrosomal, medial, and distal regions, and the fluorescence signals of vesicles were obtained from the regions of interest (ROI) in these sectors and compared between Aβ₄₂ oligomer-treated and untreated cells. (A) Heat map of Pearson's correlation values (between +1 and 0). The centrosomal region was found to have higher colocalization values than the distal zone in untreated control cells. (B) Conversely, higher correlation values were found for the distal region in Aβ₄₂ oligomer-treated cells compared to the centrosome area. Fifteen cells from 4 separate experiments of treated and non-treated cell groups were included in this analysis—sixty total cells for each group. (C) Plotting and comparing significant differences in Pearson's correlation values between Aβ₄₂ oligomer-treated and untreated cells in a specific cell region. Notably, inverted colocalization values were found between centrosomal and distal regions of the cells. One-way ANOVA, **p < 0.01, **** p < 0.0001. Mean and SEM are shown.

The heat map of Pearson's correlation values (between +1 and 0) in Figure 4A shows higher colocalization values in the centrosomal region compared with the distal zone of control untreated cells. In contrast, this distribution was inverted in Aβ₄₂ oligomer-treated cells, with higher correlation values in the distal region (Figure 4B). Analyzed by region, colocalization between Rab7- and Lamp-1-coated vesicles was significantly reduced in the centrosomal region of treated cells (Figure 4C). Due to the dispersion of vesicles into the distal region in cells incubated with Aβ₄₂ oligomers, in this region, colocalization values between Rab7- and Lamp-1- coated vesicles were significantly increased compared to untreated cells (Figure 4C).

Aβ₄₂ oligomers induce disassembly and fragmentation of the Golgi apparatus

The Golgi apparatus is an essential hub for processing, packaging and sorting newly formed proteins and for trafficking. Since it cannot be excluded from being affected by stress factors that produce neuronal alterations in AD, we aimed to analyze whether Aβ₄₂ oligomers also alter the structure of this membranous organelle compared to the distribution of vesicular elements containing Rab7 proteins. To this end, the SH-SY5Y cells were co-transfected with a plasmid carrying the B4GALT sequence (a conspicuous protein associated with the transverse side of the Golgi apparatus, tagged to Turq2) and the plasmid encoding Rab7-RFP mentioned above.

As shown in Figure 5A, in control cells the Golgi apparatus was localized as a compact structure in the vicinity of the nucleus, demonstrating coexistence but not strong colocalization with Rab7-containing vesicles (arrows in Figure 5A). Dispersion of B4GALT and Rab7 was observed in cells after Aβ₄₂ oligomer treatment (Figure 5B). Vesicles carrying Golgi bodies and Rab7 were detected in the distal region of the cells (arrows in Figure 5B), which were visualized in an intensity profile analysis along a linear vector from the nucleus to the distal region of the cells (compare the plots of Figure 5E and 5F). To compare the specificity of the effect of Aβ₄₂ oligomers over the distribution of Golgi apparatus and Rab7-coated vesicles in neuroblastoma cells, treatment was performed with brefeldin-A, a drug known to disrupt the stacking organization of the Golgi apparatus in lamellae. As shown in Figure 5C, incubation with brefeldin-A resulted in severe fragmentation of the Golgi apparatus (arrows), but there was no significant redistribution of Rab7-coated vesicles (asterisk). This effect can be visualized in the fluorescence intensity profile depicted in Figure 5G.

Figure 5.

Disassembly and fragmentation of the Golgi apparatus are produced by incubation with Aβ₄₂ oligomers. (A) Co-transfection of SH-SY5Y cells with plasmids encoding Rab7 and BAGALT, a conspicuous protein associated with the trans side of the Golgi apparatus, tagged with Turquoise2-N1. Arrows indicate the juxtanuclear localization of the Golgi apparatus and Rab7-coated vesicles. (B) Co-transfected cells treated with Aβ oligomers. Arrows indicate dispersed vesicles of Rab7-coated vesicles and fragmented lamellae of the Golgi apparatus. (C) Co-transfected cells treated with brefeldin-A. The asterisk denotes the centrosomal localization of Rab7-coated vesicles. Arrows indicate finely dispersed vesicles of the Golgi apparatus. (D) Comparative colocalization analysis by plotting Pearson's correlation evaluation between Aβ₄₂-treated and untreated cells. One-way ANOVA, *p < 0.05, ** p < 0.01. Mean and SEM are shown. Pairwise comparisons are presented for the centrosomal, medial, and distal regions of the cells. (E-G) Profiles of fluorescence intensity for both markers along a linear vector from the centrosomal to distal regions of the cells. Profiles were obtained from single optical sections of confocal super-resolved images. Scale Bars: 43 µm in A1; 25 µm in A4; 24 µm in B2; 17 µm in B5; 24 µm in C3; 18 µm in C6.

Remarkably, there was a significant increase in colocalization between Rab7 and dispersed Golgi vesicles in the distal region of the cell (Figure 5D), representing an abnormality and, therefore, a nonsensical mixture of membrane markers in the vesicular components. Moreover, differences in the size of dispersed vesicles of the Golgi apparatus were found when cells were incubated with either Aβ₄₂ oligomers or brefeldin-A (Supplemental Figure 4). The typical morphology of the Golgi apparatus was observed as a compact group of lamellae in the centrosomal region of the cell (Supplemental Figure 4A). When cells were subjected to incubation with Aβ₄₂ oligomers, the Golgi lamellae were disorganized and dispersed in small vesicles with a diameter of 0.7 µm (Supplemental Figures 4B, D). In contrast, brefeldin-A treatment of cells produced more dispersed and significantly smaller vesicles of Golgi lamellae, with a diameter range of 0.32 µm (Supplemental Figure 4C, D). The disturbance of the Golgi apparatus structure by Aβ₄₂ oligomers seems to be selective and may involve alterations in specific regulators of lamellae stability.

Exposure to Aβ₄₂ oligomers alters endo-lysosomal degradation of transferrin in vivo

To evaluate the functional alteration of the endo-lysosomal pathway of protein degradation by Aβ₄₂ oligomers, the temporal course of fluorescent transferrin degradation was evaluated in vivo in Rab7-expressing neuroblastoma cells. For this purpose, transfected cells were incubated with Aβ₄₂ oligomers for 48 h to induce alterations in the localization of Rab7-coated vesicles. Cells were then pre-loaded with exogenous fluorescein-tagged transferrin for 3 h to warrant endocytosis of this protein (Figure 6B). After transferrin washing, a group of cells was fixed and the cells were analyzed by super-resolution confocal microscopy (time 0, Figure 6A2). The other groups of cells were washed and fixed after incubation with Aβ₄₂ oligomers for 24 (Figure 6A4), 48 (Figure 6A6), and 72 h (Figure 6A8). At the same time, the same experiment was performed on the cells, but in the absence of Aβ₄₂ oligomers, which were also fixed and used as control groups (Figure 6A1, A3, A5, A7).

Figure 6.

In vivo, incubation with Aβ₄₂ oligomers alters the processing and degradation of endocytosed transferrin in Rab7-expressing SH-SY5Y cells. (B) Protocol for evaluating transferrin degradation in neuroblastoma cells with or without Aβ₄₂ oligomer incubation. (A) Fluorescence and DIC overlap images of RFP-Rab7-expressing cells pre-loaded with fluorescent transferrin (Time 0). Panels also correspond to Aβ₄₂ oligomer-treated and untreated control cells after 24, 48, and 72 h of incubation. After 24 h, most control cells have degraded fluorescent transferrin, whereas in Aβ₄₂ oligomer-treated cells, some amount of this protein remains in the cytoplasm (arrows). Note that in these cells, during 72 h of treatment, Rab7-coated vesicles are evenly dispersed and redistributed to the distal points of the cells (arrowheads). (C) Quantitative determination of fluorescent transferrin in cells treated or not with Aβ₄₂ oligomers. At 24 h, normal untreated cells had degraded a significant amount of fluorescent transferrin, whereas in Aβ₄₂ oligomer-treated cells, the protein was retained in the cytoplasm for more than 48 h. Scale Bars: panel 1: 29 µm; panel 2: 19 µm; panels 3 and 4: 31 µm; panel 5: 27 µm; panel 6: 31 µm; panel 7: 24 µm; panel 8: 34 µm. DIC: Differential Interference Contrast microscopy.

Figure 6A1 and 6A2, show that Rab7-expressing neuroblastoma cells were successfully loaded with fluorescent transferrin in similar amounts after 3 h of incubation between Aβ₄₂-treated and untreated cells. By 24 h, control cells (Figure 6A3) contained cells that were almost entirely processed for fluorescent transferrin, and very few cells preserved their fluorescent signal (asterisks). In contrast, transferrin was still detected in the cytoplasm of many cells in the Aβ₄₂-treated group (Figiure 6A4, white arrows in the green channel). The Rab7-coated vesicles (RFP signal) are dispersed into the distal regions of the Aβ₄₂-treated cells (arrowheads in Figure 6A4, A6, A8), as shown in previous figures. After 48 h of incubation with Aβ₄₂ oligomers, fluorescent transferrin was still visualized in the cytoplasm of some cells (Figure 6A6).

By contrast, in control cells, Rab7-coated vesicles remained in the centrosomal region. Meanwhile the fluorescent signal of transferrin almost disappeared (Figure 6A5, A7). When cells were incubated with Aβ₄₂ oligomers for 72 h of observation, the fluorescent signal of transferrin was barely visible in very few cells (Figure 6A8). Quantitative analysis of the fluorescence signal of cytoplasmic transferrin indicated that Aβ₄₂ exposure significantly reduced the capability of cells to process transferrin (Figure 6C), indicating that this peptide interferes with the endo-lysosomal pathway for protein processing and degradation.

To confirm that, under normal conditions, endocytosed transferrin is degraded via the autophagy process, we exposed Rab7-expressing neuroblastoma cells to Chloroquine for 24 h, which is a well-known inhibitor of lysosomal degradation (Figure 7). Subsequently, cells were incubated with fluorescent-transferrin for 3 h, and after washing the cells were maintained for 6 h under fresh cultured media. At the end of this time the cells were fixed and analyzed using confocal microscopy. As shown in the Figure 7, in control non-treated cells, transferrin is almost eliminated from the cytoplasm (Figure 7A). However, in Chloroquine-treated cells, a significant amount of transferrin remains in the cytoplasm of the cells (Figure 7B, C). Note that Chloroquine treatment did not affect the centrosomal localization of Rab7-coated vesicles.

Figure 7.

Inhibition of autophagy by chloroquine produced a delay in the degradation of transferrin in Rab7-expressed SH-SY5Y cells. Rab7-expressing neuroblastoma cells were treated with 50 µm chloroquine dissolved in DMEM-F12 medium for 24 h. After this, the cells were washed and incubated with fluorescent transferrin (25 mg/mL) for 3 h. The cells were rewashed and maintained in fresh DMEM-F12 medium for 6 h. Finally, the cells were fixed, and the signals of both fluorescent markers were analyzed by confocal microscopy. As shown in (A), after 6 h, control non-treated cells have processed the endogenous transferrin. By contrast, in Chloroquine-inhibited autophagy cells (B and C), some amount of fluorescent transferrin remained in the cytoplasmic space. In these cells, the centrosomal distribution of Rab7-coated vesicles is not affected. Scale bars: 42 µm in A, 20 µm in B, and 12 µm in C. DIC: Differential Interference Contrast Microscopy.

Discussion

Our study employs a novel approach to analyze the effects of Aβ₄₂ oligomers on intracellular trafficking. It is widely accepted that the oligomeric form of Aβ₄₂ is the entity that produces the most significant pathological consequences for neurons exposed to its accumulation in AD. Results derived from the study of the human brain, transgenic animals, and in vitro systems suggest that these oligomeric forms affect neurons and glial cells through abnormal activation of various proinflammatory cascades, increased oxidative stress, mitochondrial dysfunction, and dysregulation of intracellular signaling pathways associated with impairment on synaptic plasticity and axonal transport.^44–46 Given that intracellular trafficking is a fundamental mechanism for proper functioning of neurons, our work aims to analyze abnormalities in the intracellular distribution and functionality of the vesicular components of the endo-lysosomal pathway in a cell model subjected to extracellular exposure to Aβ₄₂ oligomers.⁴³ In addition, we analyzed the composition of vesicular components using super-resolution confocal microscopy. This technique allowed us to observe these components at diameters smaller than 200 nm, providing a more accurate representation of the entire vesicle population. This is important because there are few studies that have quantitatively examined the organization of vesicular elements in different cells incubated with monomeric or oligomeric Aβ.^37,47

Notably, alterations in the distribution and association of vesicles coated with Rab7, Lamp-1, and the Golgi apparatus were found, as well as effects on the processing and degradation of transferrin via the endo-lysosomal pathway. Previous reports have shown that exposure to Aβ oligomers is cytotoxic and leads to alterations in various cellular processes, which, depending on the experimental conditions, may lead to cell death and damage to various intracellular components.^48,49

Despite previous data indicating that some changes in the actin cytoskeleton were produced by exposing cells to Aβ oligomers,⁵⁰ we did not find strong evidence of alteration of this filamentous component under our experimental conditions. Although no disorganization of cytoskeleton assembly was found in our cellular model, it is known that its functionality and the mechanisms that regulate intracellular trafficking based on the integrity of these elements and regulatory proteins may be affected in the aging brain and AD.⁵¹ Rab proteins, which belong to the Ras superfamily, are markers of endosomes that recruit motor proteins and anchoring factors to facilitate vesicle transport and membrane fusion.⁵² The expression of these Rabs proteins has been reported to be dysregulated in AD, which may alter the endo-lysosomal system.⁵³

Although deregulation of the endo-lysosomal system during its early stages has been proposed, there are few reports on AD-related changes in Rab7 expression and distribution.^25,54 Furthermore, alterations in the expression of proteins and mRNAs for intracellular trafficking markers, including Rab5 and Rab7, have been described.^35,36,55 However, despite increased expression of genes associated with autophagy and lysosome function, the accumulation of autophagic vacuoles in axons and dendrites containing improperly degraded material suggests a crucial defect in the functionality of these organelles. All of this evidence suggests that the expression and regulation of the endosomal pathway, particularly Rab7, may undergo various alterations during AD. Our research went further than the existing evidence, offering more insight into the functional effects associated with the identity and distribution of vesicular components in the endo-lysosomal pathway, which are affected by the presence of extracellular Aβ oligomers.

In our cellular system, when Rab7 was expressed, the molecule was observed to be associated with vesicular elements distributed mainly in the centrosomal area of the cells (Figure 2C(a), C(c)). These vesicles corresponded to late endosomes, whose centrosomal localization was remarkably changed upon incubation with Aβ₄₂ oligomers. The occurrence of endo-lysosomes was evidenced when we co-expressed Lamp-1 protein, a lysosomal surface marker.^56,57 Clearly, in Figure 3A–H, a variable population of vesicles consisting of late endosomes is visible as deduced by the evident colocalization between Rab7 and Lamp-1.⁵⁸ By quantitative high-resolution confocal microscopy, a close relationship and distribution of vesicle-carrying Rab7 and Lamp-1 was determined.

After incubation with Aβ₄₂ oligomers, the lysosome population also changed its distribution, delocalizing towards the distal regions of the cells (Figure 3J–P). Interestingly, as shown in the heat diagrams of colocalization signals by zones (Figure 4A, B), there was an inverse distribution of endo-lysosomes between the centrosomal area and the distal region of the cells under the actions of the Aβ₄₂ oligomers. In those vesicles that were abnormally redistributed toward the distal regions of the cells, colocalization between Rab7 and Lamp-1 increased more in cells subjected to the action of Aβ₄₂ oligomers (Figure 4). These results suggest that there may be changes in the localization of vesicular components, as well as alterations in their molecular identity, as indicated by the colocalization patterns. These changes could potentially lead to abnormalities in the processing and degradation of materials within cells. The effects produced by Aβ₄₂ oligomers are specific and depend on both the sequence of the peptide and its aggregative oligomeric state. Incubation with the 1–40 amino acids Aβ variant did not produce the same effects (Supplemental Figure 2), as it did not form the pathological oligomers needed for these outcomes.

On the other hand, a little coexistence was found between membrane elements of the Golgi apparatus and late endosomes in the juxtanuclear region of the cells (Figure 5). However, under the action of Aβ₄₂ oligomers, a dispersion of the lamellae of the Golgi apparatus occurred, generating a population of vesicles of various diameters dispersed in anterograde direction, and abnormally showing an increased colocalization with Rab7-coated vesicles. This indicates a lack of organization in the composition and distribution of vesicular elements within the cells.

The accumulation of autophagic vacuoles due to impaired fusion of autophagosomes with late endosomes and lysosomes as well as defective degradation of dysfunctional lysosomes has been described in postmortem brain tissues from AD patients.^59,60 From the top to the bottom, our results show more comprehensively an alteration in the composition and distribution of vesicular trafficking components produced by Aβ₄₂ oligomers in cultured neuroblastoma cells. In the long run, this action can have lethal consequences to the cells. It is noteworthy that various studies have reported the internalization of Aβ₄₂ oligomers, however. in our experimental system, we have no evidence of internalization of Aβ₄₂ oligomers incubated in cells, at least as evaluated by high-resolution confocal microscopy. We cannot dismiss the possibility that unresolved entities, observed through optical microscopy, may be incorporated into the cytoplasmic space.

Thus, most of the explanations we can offer regarding the effects of these entities are based on secondary effects. In this sense, the mechanisms that explain the effect found in our system may involve the interference of distinct signaling pathways, which are secondary actions resulting from the interaction of Aβ₄₂ oligomers with different elements of the plasma membrane.

Mechanisms underlying the alteration of vesicular components of the endo-lysosomal pathway by oligomeric Aβ₄₂ exposure

The functional alterations produced by neuronal exposure to Aβ oligomers are diverse, and different mechanisms of toxicity have been proposed based on the study model, including autopsy brain tissue from AD patients, transgenic animals, and in vitro cultured cells. It is reasonable to assume that microtubules and associated motor proteins, in addition to providing structure, act as guiding elements for the transport of vesicular components,^61,62 and could be indirectly affected when the cells are exposed to Aβ oligomers. However, this assumption is contradictory because previous studies reported varying alterations in the stability of microtubules due to the action of Aβ oligomers.^63,64 In our study, we found no significant changes in the organization of the tubulin cytoskeleton when incubated with Aβ₄₂ oligomers, which suggests that structural rails for transporting vesicles were not compromised.

Another body of evidence suggests that localization and transport of Rab7-carrying vesicles relay on the motor protein complex dynein-dynactin,^65–67 and that the GTP-bound form of Rab7 facilitates the attachment of late endosomes to these dynein-dynactin complexes.^68,69 It was later reported that FYCO1 (FYVE and coiled-coil [CC] domain containing 1) also interacts with Rab7 and its associated dynein/dynactin complex to regulate the traffic of vesicles and autophagic flux.⁷⁰ According to these reports, we can hypothesize that the mislocalization of Rab7 found in the present study may be due to an indirect effect on dynein activity produced by extracellular Aβ₄₂ oligomers. In future experiments, it would be interesting to evaluate the expression and localization levels of dynein and other mechanoenzymes in our experimental model of Aβ₄₂ toxicity.

In AD brains, an increased level of ceramide has been detected in the early stages of the disease.^71,72 Ceramide accumulation, which may alter the late endocytic process based on Rab7⁷³ was observed when cultured neurons were exposed to Aβ,⁷⁴ suggesting that this condition may alter the continuity of endocytic trafficking. Thus, the abnormalities in Rab7 endocytic trafficking that we observed in the cell system may be associated with dysregulation of the amount of ceramide formation induced by Aβ-oligomer exposure. This abnormality is something that can be explored in future experiments.

Similarly, in our cellular model, the positioning and stability of the Golgi apparatus, underwent fragmentation in the form of round vesicles dispersed in the anterograde direction, when exposed to extracellular Aβ₄₂ oligomers. This action may occur due to changes in the activity of motor proteins, such as dynein, or proteins involved in the positioning and stability of the Golgi apparatus. Key proteins in this context include GM130 (Golgi matrix protein 130) and GRASP65.⁷⁵ Phosphorylation of GM130 and GRASP65 produced by Cyclin-dependent kinase 5 (CdK5), has been reported to alter the organization of the Golgi apparatus.⁷⁶ This modification was produced by Aβ oligomers incubation.⁷⁶

On the other hand, it is interesting to note that in our cell model, regardless of its Aβ₄₂ oligomer-induced Golgi apparatus dispersion, there is also an abnormal interaction of this organelle with Rab7-coated vesicles, wherein the levels of colocalization between B4GALT and Rab7 are increased. This action may translate into an imbalance in identity and directionality between the secretory and endocytic pathways of material processing. To compare the specificity of this effect, cells co-expressing BAGALT and Rab7 were subjected to the action of brefeldin-A. This drug, which is known to alter vesicular flow between the lamellae of the Golgi apparatus,⁷⁷ produced fragmentation of this structure in very fine vesicles throughout the cytoplasm (Figure 5C and 5G and Supplemental Figure 4), whereas no change was found in the centrosomal distribution of Rab7-coated vesicles.

It is interesting to note that in our experiment the formation of vesicles from fragmented Golgi apparatus, which have a smaller diameter produced by Brefeldin-A, can occur through various mechanisms that involve changes in the normal fusion of the proteins that maintain the structure of this organelle and the endoplasmic reticulum. Additionally, it has been suggested that Brefeldin-A treatment triggers the ADP-ribosylation of NADPH and BARS-50, both of which are essential for maintaining the normal structure of the Golgi apparatus.⁷⁸ The fragmentation of the Golgi apparatus into larger, more morphologically complex vesicular components, caused by incubation with Aβ₄₂ oligomers (Supplemental Figure 4), may result from effects on other molecular components that provide stability to its cisternal structure. However, until now, little attention has been given to the morphological characteristics of the fragmented Golgi induced by Aβ₄₂ oligomers, and further experiments are needed to better understand this abnormal process. This result suggests that under the exposure of Aβ₄₂ oligomers, changes in the positioning and distribution of the various components of intracellular trafficking are specific and may arise through the influence of specific mechanisms.

There is also evidence that Aβ oligomers interact with components of the plasma membrane to affect the functioning and signal transduction of diverse molecules associated with this organelle. Aβ oligomers have been demonstrated to interact with some membrane receptors (e.g., NMDA receptors) to inhibit long time-range potentiation (LTP),⁷⁹ induce calcium influx and increase neuronal oxidative stress,⁸⁰ as well as activation of glycogen synthase kinase-3β (GSK-3β) which has been reported to impair kinesin-1-based transport.^81,82 The increase in intracellular ROS levels, a condition that can alter intracellular transport, has been reported in neurological disorders. Finally, although our results describe a specific effect on a particular physiological state in neuroblastoma cells, the effects of this peptide in the brains of AD patients depend on several factors associated with the involvement of different cell lineages, including glial cells. While astrocytes attempt to compensate for the pathological brain environment by eliminating abnormal Aβ and tau aggregates, they can also suffer from the abnormal effects of these peptides’ accumulation. Using primary cultures of astrocytes donated by AD patients and healthy subjects, which were exposed to oligomeric Aβ oligomers, Ristori et al. (2025) found that the effects of this peptide can vary depending on the astrocyte group, with those from the AD group being more susceptible to changes associated with a senescence-associated secretory phenotype that produces neurotoxicity. These changes were not observed in the astrocytes of healthy subjects, even though they had endocytosed these pathological entities. While Aß has cytotoxic effects, it is essential to consider that healthy astrocytes in the brain play a crucial role in mitigating the negative impacts of this peptide.⁴⁶

With all this body of evidence and based on the various levels and mechanisms of Aβ toxicity, the effect observed in our work may represent the result of several of these deregulatory mechanisms that indirectly affect the distribution of components of the endolysosomal pathway. The effect on motor proteins and the alteration of regulatory kinases, as well as products of lipid metabolism, can alter the synchronized assembly and association of various molecules in the membrane of transport vesicles, such as Rabs proteins, which finely regulate the directionality of these elements in the intracellular space.

Functional significance of the alteration in the distribution of Rab7 and the endo-lysosomal pathway

The positioning of late endosomes and lysosomes within the perinuclear region is essential for the proper breakdown of materials through the endo-lysosomal pathway.⁸³ Consequently, any changes in this process may impact the functionality of neurons. To confirm in vivo that extracellular Aβ₄₂ oligomers affect the functionality of the endo-lysosomal pathway in our model, we evaluated under these conditions the endocytosis and degradative processing of fluorescein-tagged transferrin, which is generally eliminated by this pathway in the cells.⁸⁴

Interestingly, we found that the abnormal redistribution of Rab7- and Lamp-1-carrying vesicles did not impede the ability of cells to endocytose transferrin in the presence of Aβ₄₂ oligomers. However, the elimination process of this molecule was prolonged compared with control cells (Figure 6), thus corroborating that functional alterations in this pathway are associated with alterations in the distribution of late endosomes, lysosomes, and endo-lysosomes. As a control experiment of lysosomal degradation of transferrin, transferrin-loaded cells were treated with Chloroquine, an inhibitor of autophagy, and a delay in the processing of these cells was observed compared to non-treated cells (Figure 7). Based on these similarities, our results indicate that the incubation with Aβ₄₂ oligomers may also affect the degradation pathways of molecules via endo-lysosomes.

It has been reported that in transgenic animals mimicking AD, defects in the acidification of lysosomes are caused by a reduction in the activity of V-ATPase, which reduces the degradation capacity of the vacuoles and leads to their accumulation.⁸⁵ Based on this information, it is feasible that in the redistribution of Rab7-coated vesicles and lysosomes produced by exposure to Aβ oligomers, the acidification process of late endosomes and lysosomes is altered, thereby decreasing the efficiency of their degradative activity. It is relevant to mention that the degradation process upon incubation with Aβ₄₂ oligomers was not completely interrupted; therefore, as previously reported, other degradation mechanisms could compensate for the failure of this system if the cells are not severely affected by these oligomeric entities.⁸⁶ Our data suggest that Aβ₄₂ oligomers may affect the organization of different structures and proteins that regulate the endo-lysosomal degradation pathway. The identification of different populations of vesicular components with a mixed set of surface markers is a significant discovery. This finding indicates not only a change in their localization but also a loss of molecular identity and directionality during intracytoplasmic processing of vesicular components (Figure 8). If this occurs in AD brains, this action may contribute to the buildup of misfolded proteins that trigger toxic processes and neuronal death.

Figure 8.

Alteration of the endo-lysosomal process by extracellular Aβ₄₂ oligomers. In neuroblastoma cells, exposure to Aβ₄₂ oligomers disrupts the normal functioning of the endo-lysosomal pathway, which is responsible for processing various substances within the cell. Under normal circumstances, vesicles coated with Rab7 and LAMP-1 are typically located in the juxtanuclear region, where they collaborate to form endo-lysosomes. However, Aβ₄₂ oligomers interfere with this process through an unclear extracellular mechanism, altering the molecular identity of these vesicles and resulting in an abnormal mixture of surface markers. Consequently, this disruption causes the Golgi apparatus and other cellular components to relocate to more distal regions of the cell. These abnormalities are linked to significant changes in the degradation of cellular substances.

Conclusion

One of the early mechanisms of toxicity in AD may involve changes in the distribution and function of the various vesicular components within the endocytic pathways mediated by Rab7, as shown in this study. These changes may also affect secretion and other forms of intracellular transport, which involve different Rab proteins. Such alterations could occur early in neurons affected by AD due to the accumulation and exposure to oligomeric forms of Aβ₄₂, even before the characteristic fibrillary pathology of AD appears. Addressing these initial effects on neurons in the brain of patients with early-stage AD may improve their functionality and reduce physiological changes that occur at the onset of the disease.

Supplemental Material

sj-docx-1-alz-10.1177_13872877261422415 - Supplemental material for Extracellular amyloid-β₄₂ oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells

Supplemental material, sj-docx-1-alz-10.1177_13872877261422415 for Extracellular amyloid-β₄₂ oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells by Graciela Mendoza-Franco, Fanny Rodríguez-Cruz, Sheyla Saraí Estrada-Modesto, Enrique O Hérnandez González, Antony A Boucard, Marco Antonio Meraz Ríos, Gustavo Basurto-Islas and Francisco Garcia-Sierra in Journal of Alzheimer's Disease

Footnotes

Acknowledgements

Thanks to Dorus Gadella, Ari Helenius, and Michael Davidson for providing the pmTurquiose2-Golgi, pmRFP-C3, and mEmerald-lysosomes-20 plasmids, respectively, through Addgene acquisition. Confocal microscopy facilities were provided by the Confocal Microscopy Unit at the Cell Biology Department of CINVESTAV-IPN (CONAHCyT-Mexico grant: 300062). Proofread by Modern Manuscript Editing Services. Thanks to María de Jesús García-Sierra for artwork in .

ORCID iD

Francisco Garcia-Sierra

Ethical considerations

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Author contribution(s)

Graciela Mendoza-Franco: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft.

Fanny Rodríguez-Cruz: Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft.

Sheyla Saraí Estrada-Modesto: Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft.

Antony A Boucard: Conceptualization; Formal analysis; Investigation; Methodology; Supervision; Validation; Visualization; Writing – original draft.

Marco Antonio Meraz Ríos: Conceptualization; Formal analysis; Investigation; Supervision; Validation; Visualization; Writing – original draft.

Gustavo Basurto-Islas: Conceptualization; Investigation; Methodology; Supervision; Validation; Visualization; Writing – original draft.

Francisco Garcia-Sierra: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Project administration; Resources; Supervision; Validation; Visualization; Writing – original draft.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research was partially supported by Secretaría de Educación, Ciencia, Tecnología e Innovación (SECTEI-Mexico) (grant 3785c24 to Francisco Garcia-Sierra). Graciela Mendoza Franco received scholarships from Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT-Mexico) (grant 752629), and from SECTEI-Mexico (grant 3785c24 to Francisco Garcia-Sierra).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study.

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Extracellular amyloid-β 42 oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells

Abstract

Background

Objective

Methods

Results

Conclusions

Keywords

Introduction

Methods

SH-SY5Y cell culture and transfection

Preparation of Aβ42 oligomers and cell incubation

Electrophoresis and western blotting

Immunofluorescence and high-resolution confocal microscopy

Quantitative determination of colocalization

Live cell analysis

Results

Exposure to Aβ42 oligomers alters the distribution of Rab7-coated vesicles of neuroblastoma cells

Aβ42 oligomers disturb the association of Rab7-coated vesicles with lysosomes

Aβ42 oligomers induce disassembly and fragmentation of the Golgi apparatus

Exposure to Aβ42 oligomers alters endo-lysosomal degradation of transferrin in vivo

Discussion

Mechanisms underlying the alteration of vesicular components of the endo-lysosomal pathway by oligomeric Aβ42 exposure

Functional significance of the alteration in the distribution of Rab7 and the endo-lysosomal pathway

Conclusion

Supplemental Material

sj-docx-1-alz-10.1177_13872877261422415 - Supplemental material for Extracellular amyloid-β42 oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells

Footnotes

Acknowledgements

ORCID iD

Ethical considerations

Consent to participate

Consent for publication

Author contribution(s)

Funding

Declaration of conflicting interests

Data availability statement

Supplemental material

References

Supplementary Material

Preparation of Aβ₄₂ oligomers and cell incubation

Exposure to Aβ₄₂ oligomers alters the distribution of Rab7-coated vesicles of neuroblastoma cells

Aβ₄₂ oligomers disturb the association of Rab7-coated vesicles with lysosomes

Aβ₄₂ oligomers induce disassembly and fragmentation of the Golgi apparatus

Exposure to Aβ₄₂ oligomers alters endo-lysosomal degradation of transferrin in vivo

Mechanisms underlying the alteration of vesicular components of the endo-lysosomal pathway by oligomeric Aβ₄₂ exposure

sj-docx-1-alz-10.1177_13872877261422415 - Supplemental material for Extracellular amyloid-β₄₂ oligomers alter endo-lysosomal trafficking of Rab7- and Lamp-1-coated vesicles in cultured neuroblastoma cells