LRP::FLAG Rescues Cells from Amyloid-β-Mediated Cytotoxicity Through Increased TERT Levels and Telomerase Activity

Abstract

Alzheimer’s disease (AD) represents the most common form of neurodegenerative disorders with only palliative treatments currently available. Amyloid plaque formation caused by amyloid-β (Aβ) aggregation and neurofibrillary tangle formation caused by hyperphosphorylated tau are hallmarks for the development of AD. The 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in AD and tools blocking or downregulating LRP/LR impede amyloid plaque formation in vitro and in vivo. We have recently shown that LRP::FLAG enhances telomerase activity with a concomitant reduction of senescent markers. Here, we overexpressed LRP::FLAG in HEK293 and SH-SY5Y cells, which resulted in an increase in hTERT levels as well as increased telomerase activity and increased cell viability in the presence of cytotoxic levels of exogenous Aβ. LRP::FLAG overexpression decreased Aβ shedding and intracellular Aβ levels in HEK293 cells. This suggests that LRP::FLAG rescues cells from Aβ-induced cytotoxicity through increased telomerase activity. This study recommends LRP::FLAG as a novel alternative therapeutic for AD treatment through activation of telomerase activity.

Keywords

Alzheimer’s disease amyloid-β 37 kDa/67 kDa laminin receptor (LRP/LR)telomerase TERT

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of progressive neurodegenerative disorders afflicting in excess of 47 million people globally. This form of dementia predominantly affects the aging population and is a significant socio-economic burden, whereby in 2015, the global cost was estimated at 818 billion USD [1]. There is a considerable knowledge gap which exists in the understanding of the disease-causing mechanism and as a result, only palliative therapeutic options are currently available. This highlights the necessity for disease-modifying treatment strategies for neurodegenerative disorders such as AD. AD is defined by the accumulation of the neurotoxic amyloid-β 42 (Aβ₄₂) peptide, resulting in the development of extracellular Aβ plaques [2, 3] and the aggregation of hyperphosphorylated tau (a microtubule associated protein), producing intracellular neurofibrillary tangles [4]. Collectively, it is these factors and their associations that cause the progressive and devastating behavioral and cognitive dysfunction seen in those suffering from AD.

The 4 kDa Aβ peptide is the candidate etiological cause for AD, expressly, it is the 42-amino acid (aa) proteolytic cleavage product which amasses to cause the neurotoxic effects present in AD [5]. Generation of the Aβ peptide occurs via the amyloidogenic pathway, when the amyloid-β protein precursor (AβPP) is sequentially cleaved by β-secretase and γ-secretase, respectively. AD occurs when the amyloidogenic pathway is inappropriately favored and when Aβ₄₂ degradation is reduced, thus leading to the accumulation of these peptides [6]. AD pathology arises as a consequence of Aβ-mediated distortions in the neural morphology. These distortions impede neurotransmission and, correspondingly, cause neural dysfunction [7]. Aβ neurotoxicity has furthermore been linked to Aβ-induced neuronal apoptosis through disruption of intracellular membranes [8, 9] as well as oxidative stress, both of which contribute to mitochondrial dysfunction [10] and DNA damage [11, 12]. The mechanism by which Aβ prompts the characteristic neuronal loss is due to its direct interaction with receptors on the cell surface [13] or through indirect interactions, such as incorporation into cell organelles and lipid membranes [14]. The interaction between Aβ and cell surface receptors, such as with the 37 kDa Laminin Receptor Precursor/67 kDa high affinity Laminin Receptor (LRP/LR) [15], prompts internalization of Aβ₄₂, thus causing the intracellular accumulation of Aβ₄₂, and subsequent neurotoxic effects [13 , 16–18].

LRP/LR, otherwise known as LamR1, RPSA, and p40, is a multifunctional type II transmembrane receptor consisting of 295aa and is principally found within lipid raft regions of the plasma membrane in addition to the cytoplasm and the nucleus [19 –21]. LRP/LR is associated with numerous diseases. These include, but are not limited to, cancer, prion disorders, aging, and AD. In cancer, LRP/LR is known to be highly upregulated, leading to enhanced adhesion and invasion and ultimately, metastasis, as well as angiogenesis and the inhibition of apoptosis [22 –30]. In addition, LRP/LR is involved in the aging process due to an interaction with telomerase [31, 32]. Furthermore, LRP/LR is implicated in AD, through its interaction with Aβ on the cell surface, as well as its role in Aβ shedding, due to its interaction with the AD-related proteins: AβPP, γ-secretase, and β-secretase [33, 34]. LRP/LR is involved in the internalization of Aβ₄₂, which causes accumulation of Aβ₄₂ intracellularly [34] and ultimately Aβ₄₂-mediated cytotoxicity [15]. Furthermore, the underlying mechanism of the Aβ₄₂-induced neuronal cytotoxicity is an indirect result of an interaction between the cellular prion protein (PrP^c) and Aβ₄₂ [35]. Antibody (IgG1-iS18) and small hairpin RNA (shRNA) technologies employed to target LRP/LR, to block the receptor and downregulate its expression, respectively, revealed a significant reduction in Aβ-mediated cytotoxicity and Aβ₄₂ shedding [15, 33, 36].

Recently, an additional protein found to be associated with AD is telomerase, a ribonucleoprotein with reverse transcriptase activity. Telomerase is principally found in highly proliferative cells and has a key function in protecting DNA from degradation, through the addition of telomeric (TTAGGG) repeats to the ends of telomeric DNA [37, 38]. Telomerase is a multi-subunit protein with two key components, TERT, the catalytic reverse transcriptase and TERC, the RNA component [39]. Telomerase activity has an important role in cellular senescence and immortalization, whereby it is involved in the aging process and the cancerous state [40]. Additionally, TERT has a role in numerous extra-telomeric processes, predominantly for the conservation of cell viability [41]. TERT protects and aids in the functioning of the mitochondria [42 –44]. When reactive oxygen species (ROS) are present and conditions become hypoxic, TERT translocates to and protects the mitochondria against DNA (mtDNA) damage, as well as apoptosis [45]. Moreover, telomerase has an essential role in regulation of DNA repair and DNA damage responses by which it is involved in the recruitment of DNA repair proteins [41]. It has been shown that telomerase is implicated in the pathological processes of AD [46 –48]. Evidence shows that AD patients have shorter telomere lengths in their neuronal and T cells [47]. Furthermore, in vitro studies have demonstrated that telomerase activity is inhibited by Aβ_42, through the binding of Aβ oligomers to the telomeric DNA-RNA template complex of telomerase [48], representing an antagonistic relationship between telomerase and Aβ within neurons. It has been proposed that upregulation of telomerase provides a potential therapeutic strategy for the treatment of AD, as the resulting overexpression of TERT protects neuronal cells against Aβ-induced apoptosis [46]. A discovery by Otgaar et al. [32] elucidated that overexpression of LRP::FLAG [49] resulted in an increase in TERT expression, telomerase activity, telomere length as well as a reduction in senescent markers [32]. Altogether indicating that LRP/LR has a role in AD and in the regulation of telomerase activity and TERT expression [31 , 36].

Therefore, this study focused on stably transfecting HEK293 and SH-SY5Y cells with the pCIneo-moLRP::FLAG construct, to assess the effect of overexpression of LRP::FLAG on Aβ₄₂ levels, TERT expression, telomerase activity, and cell viability, in an in vitro AD setting.

METHODS AND MATERIALS

Tissue culture

Non-tumorigenic, human embryonic kidney cells (HEK293) were used due to their detectable levels of telomerase activity and their common use as an AD model. Human neuroblastoma cells (SH-SY5Y) were used as the secondary AD model, due to their low levels of TERT. HEK293 and SH-SY5Y cell lines were cultured in 1 : 1 Dulbecco’s modified eagle medium (DMEM) and Ham’s F12 nutrient mixture, supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. Cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere.

Cell transfection

Transfection of HEK293 and SH-SY5Y cells with the pCIneo-moLRP::FLAG plasmid was carried out, following the Clontech Xfect™ transfection protocol, once the cells had reached 50–70% confluency. The transfected subconfluent cell culture was incubated for 48 h. Thereafter, all growth media was replenished with fresh complete growth media and subsequently treated with 800 ng/ml Geneticin, as a selective treatment for transfected cells. Thereafter, cells were treated with 400 ng/ml Geneticin to maintain the transfected cell population.

Western blotting

Western blotting was used to confirm expression of LRP::FLAG and to detect total protein levels of LRP and hTERT, post-transfection with pCIneo-moLRP::FLAG, β-actin was used as the loading control. Briefly, cell lysates were prepared with 1X RIPA buffer and protein levels quantified via the bicinchoninic acid assay (BCA). Protein was then resolved on a 12% SDS-PAGE gel for 45–55 min at 150 V per gel. Subsequently, proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane at 350 mV for 50 min, using a semi-dry transferring apparatus. Membranes were thereafter blocked in 3% BSA (Amresco) in 1X PBS and 0.1% Tween 20 (PBST) for 1 h. After blocking, the blots were incubated with the appropriate primary antibody overnight with gentle shaking at 4°C. The membranes were subsequently washed in PBST and further incubated for 1 hour in the dark, in the respective secondary antibody. The membranes were then washed as outlined above. The proteins were visualized with Clarity™ Western ECL Blotting Substrate (Biorad) and the ChemiDoc™ Imaging System (Biorad). Densitometric analysis was performed with Image Lab 5.1 software (Biorad), whereby all values were further normalized against the β-actin loading control. For the list of all antibodies and dilutions, refer to (Supplementary Table 1).

Confocal microscopy with Airyscan™

Cells were seeded at a density of 1.2×10⁵ cells/well onto cover slips (Labocare – 18×18 mm; 0.19 mm thick) and incubated overnight. All subsequent steps were performed with gentle shaking. Cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature. Cells were washed thrice with 1X PBS and thereafter permeabilized with 0.25% Triton X-100 for 30 min. Coverslips were washed 3 times with 1X PBS and blocked with 0.5% BSA in 1X PBS for 30 min. Cells were incubated with the relevant primary antibody solution (diluted 1 : 200 in 0.5% BSA-PBS) overnight at 4°C. Hereafter, cells were washed 3 times in 0.5% BSA-PBS. Cells were then incubated with the respective secondary antibody diluted (1 : 500) in 500 μl 0.5% BSA-PBS for 2 h in the dark, at room temperature. Coverslips were then washed thrice, in the dark and subsequently incubated in 0.05 μg/ml DAPI diluted in 1X PBS for 5 min in the dark. Coverslips were further rinsed 4 times and mounted onto glass microscope slides with 20 μl Fluoromount (Sigma) and allowed to set in the dark at room temperature for 1.5 h. Thereafter, slides were stored in the dark at 4°C until viewed. Slides were viewed using the Zeiss LSM 780 confocal microscope with the addition of Airyscan™. Subsequently, images were analyzed with Zen 2010 imaging software v2.1. Controls were prepared as above, with the exception of primary antibodies. For the list of all antibodies used for confocal microscopy, (see Supplementary Table 2).

Aβ₄₂-ELISA

Total human Aβ_1–42 was quantified with the use of the Human Amyloid β (aa 1–42) Quantikine ^® ELISA kit by R&D Systems. The manufacturer’s instructions were followed. Briefly: The plate was washed twice with Wash Buffer immediately preceding use. Human Amyloid β (aa1–42) standard, together with extracted protein samples were added to the wells (100 μl/well) and incubated for 2 h at 4°C. Thereafter, each well was aspirated and washed four times with Wash Buffer. Cold Human Amyloid β (aa1–42) conjugate (200 μl/ well) was added and incubated for a further 2 h at 4°C. The plate was subsequently washed four times with Wash Buffer and 200 μl /well of Substrate Solution was added. Following this, the plate was incubated for 30 min at room temperature in the dark, after which 50 μl/ well of Stop Solution was added. The optical density was determined using an ELISA microtiter plate reader at 450 nm with wavelength correction at 540 nm.

Telomerase activity

The TRAPeze^® RT Telomerase Detection Kit (Merck), was used to determine the effect of LRP::FLAG overexpression on telomerase activity. Telomerase activity was quantified as per the manufacturer’s instructions. Briefly, cells were harvested and lysates prepared by resuspension in CHAPS Lysis Buffer ((3-((3-cholamidopropyl) dimethylammonio)-1- propanesulfonate)). Protein and RNA fractions were collected in the supernatant, where after protein concentrations were quantified with the NanoDrop^® ND-1000 (Thermo Scientific) and standardized to 500 ng/μl for all experimental and control reactions. All samples were analyzed via qPCR with the Roche LightCycler LC480, with the following cycling parameters applied: one cycle of 37°C for 30 min, 95°C for 2 min, and 45 cycles of 95°C for 15 s, 59°C for 60 s, and 45°C for 10 s. Telomerase activity was thereafter calculated from the standard curve generated by 1 : 10 serial dilutions (20–0.0002 amoles) of the provided TSR8 control template as per Merck Millipore instructions. All controls were included: a minus telomerase control (CHAPS Lysis Buffer), a no template control (Nuclease free/PCR Grade Water) and a heat-treated telomerase negative control, whereby 10 μl of each 500 ng/μl sample was incubated at 85°C for 10 min prior to detection. A telomerase positive cell extract was provided as a positive control and made up as per the protocol. The data was analyzed with LightCycler1 Software version 1.5.1.

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium bromide (MTT) cell viability assay

The MTT assay was used to assess cell viability following transfection and after treatment with cytotoxic levels of Aβ₄₂. Briefly, non-transfected and LRP::FLAG transfected HEK293 and SH-SY5Y cells were seeded at 1×10⁵ cells/well in a 48 well plate and incubated overnight under normal conditions. Following this, the cells were treated with 500 nM Aβ₄₂ (to mimic Aβ-mediated cytotoxicity) for 48 h. Furthermore, all untreated controls and positive controls (cells incubated with 8 mM protocatechuic acid- PCA) were included for all cell lines. Post 48 h incubation, 100 μl of 1 mg/ml MTT was added to each well and subsequently incubated for 2 h at 37°C. Following incubation, all culture media was aspirated, and the resultant formazan crystals were dissolved in 200 μl dimethyl sulfoxide (DMSO). Subsequently, the optical density was determined using an ELISA plate reader at 570 nm. All experimental and control samples were assayed in triplicate and the percentage cell viability was calculated, relative to the untreated controls.

Statistical analysis

Statistical analysis was performed using Microsoft Excel 365 (Microsoft Corporation) and QuickCalcs Outlier Calculator ©2017 GraphPad Software which employs the Grubbs’ test (extreme studentized deviate). All experiments were performed with a minimum of three biological repeats and error bars represent standard deviation. The Student’s t-test was performed at a 95% confidence interval; where values ^* p < 0.05 were considered statistically significant and values ^** p < 0.01 and ^*** p < 0.001 were considered highly significant.

RESULTS

This study investigated the effect of overexpressing LRP::FLAG on TERT and Aβ protein levels, as well as the resultant effect on telomerase activity and cell viability in the HEK293 and SH-SY5Y AD cell culture models. Here, we show that, LRP::FLAG is exclusively expressed in the transfected HEK293 (HEK293T) (Fig. 1A) and SH-SY5Y (SH-SY5YT) (Fig. 1B) cells, post-transfection of HEK293 and SH-SY5Y cells with the pCIneo-moLRP::FLAG construct. Furthermore, we reveal a resultant increase in TERT levels and telomerase activity as well as a significant reduction in Aβ levels in the HEK293 and SH-SY5Y cells overexpressing LRP::FLAG.

Fig.1

Western blot analysis confirms that LRP::FLAG is overexpressed in HEK293 and SH-SY5Y cells. HEK293 and SH-SY5Y cells were confirmed to be overexpressing LRP::FLAG after stable transfection with pCIneo-moLRP::FLAG. A) LRP::FLAG is detected in HEK293 transfected cells and not in non-transfected HEK293 cells. B) LRP::FLAG is detected in SH-SY5Y transfected cells and not in non-transfected SH-SY5Y cells. β-actin is used as the loading control. C) Densitometric analysis indicates a significant 58% increase in LRP protein level after LRP::FLAG overexpression in HEK293 cells. D) Densitometric analysis in SH-SY5Y transfected cells indicates LRP::FLAG overexpression significantly increases LRP protein levels by 165% Error bars represent standard deviation, n = 3 biological repeats. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001; Student’s t-test.

Overexpression of LRP::FLAG increases hTERT levels with a concomitant decrease in Aβ levels in SH-SY5Y cells

Since we confirmed transfection in SH-SY5Y cells, confocal microscopy with the addition of Airyscan™ was employed to assess the effects on the levels and respective co-localization of LRP, Aβ₄₂ and hTERT. In non-transfected SH-SY5Y cells, LRP localizes on the cell surface and intracellularly (Fig. 2A), as we previously reported [19]. Under the same conditions, Aβ tends to localize to the cell surface as has been shown previously [50] and to a lesser extent in the cytoplasm (Fig. 2B). Although Aβ levels are low in SH-SY5Y cells, co-localization (Fig. 2E) was observed between LRP and Aβ on the cell surface and to a lesser extent in the cytoplasmic regions (Fig. 2D–F). This is conducive to what was previously reported [15]. Upon transfection of SH-SY5Y cells with pCIneo-moLRP::FLAG (Fig. 2H), there is a clear increase in total levels of LRP (Figs. 1D and 2G), attributed to the endogenous LRP as well as the LRP::FLAG proteins, with a concomitant decrease in Aβ levels, whereby levels are almost undetectable (Fig. 2I) in comparison to the non-transfected cells (Fig. 2B). In SH-SY5Y cells, hTERT localizes uniformly across the nuclear and cytosolic regions (Fig. 2N), whereby co-localization occurs between LRP and hTERT (Fig. 2P–R). However, upon overexpression of LRP::FLAG, the SH-SY5YT cells exhibit a clear increase in hTERT levels and cytosolic localization, as observed in (Fig. 2T), which is further shown by the increase in fluorescence intensity indicated in (Fig. 2X).

Fig.2

LRP::FLAG overexpression decreases Aβ levels and increase hTERT levels in SH-SY5Y cells. A–L) Intracellular localization and co-localization of LRP (FITC-Green) and Aβ (APC-Red) as well as LRP::FLAG (FITC-Green) and Aβ in SH-SY5Y and SH-SY5YT cells. M–X) Intracellular localization and co-localization of LRP (FITC-Green) and hTERT (APC-Red) as well as LRP::FLAG (FITC-Green) and hTERT in SH-SY5Y and SH-SY5YT cells. A, M) Endogenous LRP levels in SH-SY5Y cells. B) Endogenous Aβ levels in SH-SY5Y cells. G) LRP levels are increased in SH-SY5YT cells. H, S) LRP::FLAG expression is confirmed in SH-SY5YT cells. I) Aβ expression becomes almost undetectable upon overexpression of LRP::FLAG. C, J, O, U) Nuclei are stained with DAPI [Blue]. Co-localization occurs between LRP and Aβ (D, K) and between LRP and hTERT (P, V) represented by yellow fluorescence in merged images, as white areas (E, Q, W) and as fluorescence in the third quadrant of the 2D cytofluorograms (F, L, R, X). All images are at 630 X magnification and a resolution of 140 nm (Airyscan™). Scale bars represent 10 μm.

Since we know of an antagonistic relationship between TERT/telomerase and Aβ in AD, as previously reported [48], confocal microscopy with the addition of Airyscan™ was employed to determine whether these two proteins co-localize in the cell. Here, we observe that hTERT and Aβ do indeed co-localize, predominantly in the cytoplasmic regions, as indicated by white areas in Fig. 3E and as detectable fluorescence in quadrant 3 of (Fig. 3F). This furthermore suggests an interaction and a possible association between the two proteins.

Fig.3

Immunofluorescence microscopy in SH-SY5Y cells suggests co-localization of hTERT and Aβ. hTERT [APC-Red] (A) and Aβ [FITC-Green] (B) co-localize on the cell surface and intracellularly in SH-SY5Y cells. Nuclei are stained with DAPI [Blue] (C). Co-localization is represented by yellow fluorescence in the merged image (D), as white areas (E) and as fluorescence in the third quadrant of the 2D cytofluorogram (F). All images are at 630 X magnification and a resolution of 140 nm (Airyscan™).

LRP::FLAG overexpression significantly decreases intracellular Aβ₄₂ levels and Aβ₄₂ shedding in HEK293 and SH-SY5Y cells

An Aβ₄₂-ELISA was performed on both the HEK293 and SH-SY5Y cells to further analyze Aβ₄₂ levels. Analysis of intracellular Aβ₄₂ levels in HEK293 and HEK293T cells, showed a significant reduction of 53.7 % in intracellular Aβ₄₂ in the HEK293T cells (p = 0.0009) (Fig. 4A). No significant change was observed on analysis of intracellular Aβ₄₂ levels in both untreated SH-SY5Y and SH-SY5YT cells (Fig. 4C). Moreover, intracellular Aβ₄₂ levels in the SH-SY5Y and SH-SY5YT cells were almost undetectable without Aβ₄₂ treatment (Fig. 4C). Therefore, immunofluorescent labelling was used to quantify Aβ₄₂ levels in the SH-SY5Y cells and is described as above (Fig. 2A–L). Furthermore, since LRP/LR is involved in the shedding of Aβ₄₂ [51], extracellular Aβ₄₂ levels were assessed by Aβ₄₂-ELISA to determine whether overexpression of LRP::FLAG would cause an increase in Aβ₄₂ shedding. Surprisingly, we observed a significant reduction in extracellular Aβ₄₂ levels (p = 0.0105) in the HEK293T cells, when compared to HEK293 cells (Fig. 4B). This indicates a reduction in Aβ shedding in the HEK293T cells overexpressing LRP::FLAG.

Fig.4

LRP::FLAG overexpression decreases intracellular Aβ₄₂ levels and Aβ₄₂ shedding in HEK293 cells. A significant decrease in total intracellular Aβ₄₂ levels is observed in HEK293T cells (A). A significant decrease in Aβ₄₂ shedding is seen in HEK293T cells (B). Aβ₄₂ concentration is expressed as a % pg Aβ₄₂ per/mg total protein. Non-transfected HEK293 were set to 100% Error bars represent standard deviation, n = 3 biological repeats. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001; Student’s t-test.

Overexpression of LRP::FLAG significantly increases telomerase activity in HEK293 and SH-SY5Y cells

Multiple studies have reported that an increase in TERT expression induces an increase in telomerase activity [52, 53], therefore, subsequent to observing an increase in hTERT levels post- LRP::FLAG overexpression (Fig. 2T), relative telomerase activity was quantified via qPCR. In addition, since Aβ₄₂ is known to have a relationship with hTERT [48], telomerase activity was assayed in both the presence and absence of cytotoxic levels of Aβ₄₂. Here, we affirm that, in the absence of Aβ₄₂ (0 nM), HEK293T (p = 0.0067) and SH-SY5YT (p = 0.0025) cells exhibited significantly higher telomerase activity when compared to non-transfected HEK293 and SH-SY5Y cells, respectively (Fig. 5). This is conducive to what we previously reported in HEK293 cells [32]. Upon treatment with cytotoxic levels of Aβ₄₂ (500 nM), we observed a significant decrease in relative telomerase activity in both the non-transfected HEK293 (p = 0.0056) and SH-SY5Y (p = 0.0005) cells, as well as in HEK293T (p = 1.81E–06) cells. Surprisingly, however, both transfected cell lines displayed significantly higher telomerase activity in comparison to non-transfected HEK293 (p = 0.03) and SH-SY5Y (p = 8.62E–05) cells after treatment with Aβ₄₂ (500 nM). This suggests that overexpression of LRP::FLAG increases telomerase activity, despite the presence of high levels of Aβ.

Fig.5

LRP::FLAG overexpression significantly increases telomerase activity in HEK293 and SH-SY5Ycells. Relative telomerase activity (RU = relative units) is increased in HEK293T compared to non-transfected HEK293 cells (A) and in SH-SY5YT cells compared to non-transfected SH-SY5Y cells (B) both before and after treatment with cytotoxic levels of synthetic Aβ₄₂ (500 nM). All values were normalized against the negative controls; all negative control values were subtracted from the signal of each sample. This data is representative of the mean value obtained across all biological repeats for each cell line and each treatment. Error bars represent standard deviation, n = 3 biological repeats. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001; Student’s t-test.

Overexpression of LRP::FLAG rescues HEK293 and SH-SY5Y cells from Aβ₄₂ – mediated cytotoxicity

We have previously shown that LRP/LR is a receptor for Aβ₄₂, whereby it is involved in the internalization and therefore, the accumulation of Aβ₄₂ intracellularly, ultimately inhibiting cell proliferation and inducing apoptosis [51]. Since we revealed that LRP::FLAG overexpression increased hTERT levels with a concomitant decrease in Aβ₄₂ levels, an MTT assay was performed to determine whether LRP::FLAG overexpression would also influence the viability of the transfected HEK293 and SH-SY5Y cells in the presence of cytotoxic levels of synthetic Aβ₄₂ peptides (Fig. 6). We observed that treatment with 500 nM Aβ₄₂, reduced cellular viability significantly in both non-transfected HEK293 (p = 0098) and SH-SY5Y (p = 1.31E–07) cells, with a decrease of 32.68% and 63.33% seen in comparison to untreated HEK293 and SH-SY5Y cells, respectively. (Fig. 6). Simultaneously, we investigated the effect of the 500 nM synthetic Aβ₄₂ treatment on cell viability in HEK293T and SH-SY5YT cells (Fig. 6). Interestingly, we revealed that these cells exhibited a significant 62.62 % (p = 0.0011) and 91.44 % (p = 7.75E–06) less cell death, respectively, when compared to the non-transfected counterparts treated under the same conditions. Taken together, these data indicate the novel finding that LRP::FLAG overexpression rescues both HEK293 and SH-SY5Y cells from Aβ₄₂-induced cytotoxicity.

Fig.6

LRP::FLAG overexpression rescues HEK293 and SH-SY5Y cells from Aβ₄₂ induced cytotoxicity. Cellular viability of (A) HEK293 and HEK293T as well as (B) SH-SY5Y and SH-SY5YT cells, as determined by MTT assay. Cell viability was assessed 48 h post-treatment with 500 nM synthetic Aβ₄₂ and the untreated set to 100%. PCA was used as a positive control. Error bars represent standard deviation. n = 3 biological repeats ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001; Student’s t-test.

DISCUSSION

We have recently shown that 5XFAD transgenic mice treated with the anti-LRP/LR specific antibody, IgG1-iS18, exhibited an improvement in memory, a decrease in Aβ plaque formation, with a significant reduction in both soluble and insoluble Aβ₄₂ levels. This was suggested to be a neuroprotective effect as attributed to a concomitant and significant increase in mTERT levels observed [36]. Despite the previously explained role of LRP/LR in the pathological process of disease, we recently elucidated that overexpression of LRP::FLAG [49] resulted in an increase in TERT expression, telomerase activity, telomere length as well as a reduction in senescent markers [32]. Altogether indicating that LRP/LR has a role in the regulation of telomerase activity and TERT expression [31 , 36]. Furthermore, since telomerase plays a role in AD [46 –48], we therefore wanted to determine the effect of overexpressing LRP::FLAG in an AD setting. This present study demonstrates that overexpression of LRP::FLAG increases TERT levels and concurrently reduces intracellular Aβ₄₂ levels and Aβ₄₂ shedding in HEK293 cells. In addition, we reveal a concomitant increase in telomerase activity and rescue from Aβ₄₂-mediated cytotoxicity.

It was previously reported that LRP/LR both co-localizes and interacts with hTERT in HEK293 and MDA-MB231 cells [31, 32]. Here, it is seen that LRP and hTERT do indeed co-localize in the SH-SY5Y cells (Fig. 2P, R), as determined by confocal microscopy and that co-localization occurs in the nuclear and cytosolic regions. In addition, we observed a substantial increase in hTERT levels in the SH-SY5YT cells after LRP::FLAG overexpression. Interestingly, we found that the hTERT was localized more abundantly in the cytoplasmic regions, than in the nucleus. Studies have reported that TERT translocates from the nucleus to the mitochondria, where it plays a role in decreasing ROS levels, DNA damage, and apoptosis [42 –44]. Spilsbury et al. [54] revealed that TERT preferentially localizes to the mitochondria in neurons upon AD pathology; here, they suggested that TERT decreases ROS levels and improves respiratory chain function. It is well-reported that AD is a multifactorial disease, where many components of the cell play a role; however, it has recently been described that mitochondrial dysfunction is one of the earliest observed pathogenic alterations, which occurs well before the accumulation of amyloid plaques [55, 56]. Numerous studies have reported impairment of mitochondrial function as well as increased production of ROS in AD transgenic mouse models and AD patients [57 –59]. Although the mechanism behind mitochondrial dysfunction and AD is not completely understood, it is known that there is a cumulative effect between mitochondrial dysfunction and Aβ accumulation [60].

Since it was previously observed that LRP co-localizes with hTERT and with Aβ₄₂, respectively, it was of interest to assess whether hTERT and Aβ₄₂ co-localize. We observed co-localization between hTERT and Aβ₄₂ in the cytoplasm of the SH-SY5Y cells (Fig. 3), thus, suggesting an interaction between these proteins. Interestingly, we observed a significant decrease in intracellular Aβ₄₂ levels in the HEK293T cells, as well as the SH-SY5YT cells overexpressing LRP::FLAG, when compared to the non-transfected cells, respectively. Furthermore, a significant reduction in extracellular Aβ₄₂ levels was observed in the transfected HEK293 cells, when compared to the non-transfected HEK293 cells, thereby, representing a significant reduction in Aβ₄₂ shedding. Since Aβ₄₂ levels were undetectable in the SH-SY5Y cells by Aβ₄₂-ELISA, we were unable to assess whether there was a change in Aβ₄₂ shedding upon overexpression of LRP::FLAG in these cells. Wang et al. [48] proposed that there is an antagonistic relationship between telomerase and Aβ₄₂ within neurons, since Aβ₄₂ is involved in the inhibition of telomerase activity, through the binding of the telomeric DNA/RNA template of telomerase. Since we saw an increase in hTERT protein levels, as well as localization to the cytoplasm, we propose this concomitant decrease in Aβ₄₂ levels seen in both the SH-SY5Y (Fig. 2) and the HEK293 (Fig. 4A) cells, as well as the reduction in Aβ₄₂ shedding (Fig. 4B), is owed to the interaction between TERT and Aβ.

It has been reported by multiple studies, that an increase in hTERT expression causes an increase in telomerase activity, and that TERT behaves as the limiting factor for telomerase activity [52, 53]. Therefore, as we observed an increase in hTERT levels and a decrease in Aβ₄₂ levels, we determined the resulting effect on telomerase activity in both the absence and presence of cytotoxic levels of Aβ₄₂. We confirmed a significant increase in telomerase activity in both HEK293 (Fig. 5A) and SH-SY5Y (Fig. 5B) cells overexpressing LRP::FLAG. Furthermore, this follows the trend we previously reported in HEK293 cells [32]. Although telomerase expression is a known hallmark of cancer, it has been shown that ectopic expression of telomerase is not associated with malignancy [61]. It was reported that the introduction of telomerase to fibroblasts did not alter other growth parameters. The telomerase-expressing cells showed no other changes in growth patterns or genomic instability, and these cells did not bypass cell-cycle induced check point controls typical of cancer cells [61]. Indeed, The HEK293 and SH-SY5Y cells overexpressing LRP::FLAG maintained a dependence on standard serum concentrations and exhibited contact inhibition.

Treatment with cytotoxic levels of Aβ₄₂, thereby mimicking Aβ₄₂-mediated cytotoxicity, caused a consequential reduction in telomerase activity in both transfected and non-transfected HEK293 and SH-SY5Y cells. Since we know Aβ₄₂ induces apoptosis [46] and inhibits telomerase activity [48], it is proposed that this reduction in telomerase activity is observed as a combination of these two factors. Remarkably, however, telomerase activity was significantly higher in both the HEK293T and SH-SY5YT cells when treated with synthetic Aβ₄₂ peptides and compared to their non-transfected counterparts treated under the same conditions (Fig. 5). Additionally, we performed an MTT assay, subsequent to a 48 h exogenous treatment with 500 nM synthetic Aβ₄₂ peptides, to assess the effect on cell viability. Interestingly, less cell death was observed in both the HEK293T (Fig. 6A) and the SH-SY5YT (Fig. 6B) cells, post treatment with cytotoxic levels of Aβ₄₂ peptides, when compared to the corresponding non-transfected cells. Thus, these novel findings suggest overexpression of LRP::FLAG significantly increases telomerase activity, despite the presence of Aβ₄₂ and furthermore rescues cells from Aβ-mediated cytotoxicity. This observation is proposed to be attributable to the fact that the increase in hTERT levels and the resulting increase in telomerase activity after overexpression of LRP::FLAG, is occurring prior to the manifestation of Aβ₄₂-mediated cytotoxicity.

The pathological agent of AD, Aβ, has a functional role in mitochondrial dysfunction by disrupting calcium homeostasis through the formation of ion-permissible channels in the cell membrane, as well as causing oxidative stress [10]. In addition, ROS-induced alterations in mitochondrial membrane potential have been shown to be caused by p53, thereby, promoting apoptosis [62]. The “mitochondrial cascade hypothesis” can be used to explain sporadic, late-onset AD. It explains how a decline in the efficiency of mitochondrial activities occurs as a result of age-related effects on genetic factors (nuclear and mitochondrial DNA), thus causing an increase in ROS, thereby triggering production of Aβ [63]. Telomerase is responsible for maintaining telomere length [64] and the loss of telomerase activity is pivotal in cellular senescence, whereby it is involved in the aging process [40]. Furthermore, AD sufferers exhibit shorter telomeres in their neuronal cells [47], which is indicative of acceleration of this process. Vera et al. [65] suggested that the rescuing effect of telomerase on short telomeres is sufficient to restore cell and organismal viability as well as genomic stability. Therefore, we propose that the increase in hTERT levels can compensate for the adverse effects caused by the high levels of Aβ₄₂, by increasing telomerase activity. Through the increase in telomerase activity, we suggest the age-related effects on the mitochondria are decreased, thereby lowering mitochondrial dysfunction and reducing the production of Aβ. In conjunction with this, the increased levels of hTERT seen in the cytoplasm could have an additive effect, to protect the mitochondria from both the internalization of Aβ, as well as Aβ-induced mitochondrial impairment, as previously described. It is reported that p53 is present at elevated levels in neurons affected by amyloid plaques [66], as well as in transgenic mice overexpressing the Aβ peptide [67] Fu et al. [68] elucidated that TERT can prevent the activation of caspases, prior to mitochondrial dysfunction and through an interaction with the tumor suppressor, p53 [62]. Therefore, it is likely that TERT is furthermore exerting a protective effect in the cytoplasm, against the aforementioned detrimental effects of Aβ, through the modulation of p53-dependent mechanisms. Thus, we suggest the increase in TERT levels and telomerase activity is playing a protective function against apoptotic stresses caused by Aβ and mitochondrial perturbations, which influence each other in a deadly cycle and is thereby, providing a neuroprotective function. However, further studies are being performed to investigate the effect of overexpressing LRP::FLAG on ROS production and mitochondrial function.

This study has shown that LRP::FLAG overexpression increases hTERT expression, concomitantly increases telomerase activity, and concurrently decreases intracellular Aβ₄₂ levels and shedding in HEK293 and SH-SY5Y AD cell culture models. Together, our results suggest that the telomerase protein, TERT, seems to be a protective factor against AD pathology and may offer resistance against pathological Aβ. We therefore propose that LRP::FLAG overexpression is serving as a telomerase activating compound to reduce AD pathology and is resulting in neuroprotective processes as indicated by the rescue from Aβ₄₂ -induced cytotoxicity observed in this study. Thus, overexpression of LRP::FLAG could form the basis for a powerful, novel preventative treatment for neurodegenerative disorders, such as AD.

Footnotes

ACKNOWLEDGMENTS

We thank Affimed Therapeutics GmbH, Heidelberg, Germany for providing the IgG1-iS18 antibody.

This work is based upon research supported by the National Research Foundation (NRF), the Republic of South Africa (RSA). Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s), and therefore, the National Research Foundation does not accept any liability in this regard thereto. Financial support was further received from the South African Medical Research Council (MRC) under a self-initiated grant awarded to SFTW. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s), and therefore, the MRC does not accept any liability in this regard thereto.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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LRP::FLAG Rescues Cells from Amyloid-β-Mediated Cytotoxicity Through Increased TERT Levels and Telomerase Activity

Abstract

Keywords

INTRODUCTION

METHODS AND MATERIALS

Tissue culture

Cell transfection

Western blotting

Confocal microscopy with Airyscan™

Aβ42-ELISA

Telomerase activity

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium bromide (MTT) cell viability assay

Statistical analysis

RESULTS

Overexpression of LRP::FLAG increases hTERT levels with a concomitant decrease in Aβ levels in SH-SY5Y cells

LRP::FLAG overexpression significantly decreases intracellular Aβ42 levels and Aβ42 shedding in HEK293 and SH-SY5Y cells

Overexpression of LRP::FLAG significantly increases telomerase activity in HEK293 and SH-SY5Y cells

Overexpression of LRP::FLAG rescues HEK293 and SH-SY5Y cells from Aβ42 – mediated cytotoxicity

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Aβ₄₂-ELISA

LRP::FLAG overexpression significantly decreases intracellular Aβ₄₂ levels and Aβ₄₂ shedding in HEK293 and SH-SY5Y cells

Overexpression of LRP::FLAG rescues HEK293 and SH-SY5Y cells from Aβ₄₂ – mediated cytotoxicity