Abstract
The neuronal perturbations in Alzheimer’s disease are attributed to the formation of extracellular amyloid-β (Aβ) neuritic plaques, composed predominantly of the neurotoxic Aβ42 isoform. Although the plaques have demonstrated a role in synaptic dysfunction, neuronal cytotoxicity has been attributed to soluble Aβ42 oligomers. The 37kDa/67kDa laminin receptor has been implicated in Aβ42 shedding and Aβ42-induced neuronal cytotoxicity, as well as internalization of this neurotoxic peptide. As the cellular prion protein binds to both LRP/LR and Aβ42, the mechanism underlying this cytotoxicity may be indirectly due to the PrPc-Aβ42 interaction with LRP/LR. The effects of this interaction were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assays. PrPc overexpression significantly enhanced Aβ42 cytotoxicity in vitro, while PrP–/– cells were more resistant to the cytotoxic effects of Aβ42 and exhibited significantly less cell death than PrPc expressing N2a cells. Although anti-LRP/LR specific antibody IgG1-iS18 significantly enhanced cell viability in both pSFV1-huPrP1-253 transfected and non-transfected cells treated with exogenous Aβ42, it failed to have any cell rescuing effect in PrP–/– HpL3-4 cells. These results suggest that LRP/LR plays a significant role in Aβ42-PrPc mediated cytotoxicity and that anti-LRP/LR specific antibodies may serve as potential therapeutic tools for Alzheimer’s disease.
INTRODUCTION
Alzheimer’s disease (AD) is a progressive and devastating neurodegenerative disorder characterized symptomatically by behavioral and cognitivedysfunction [1]. As the most prevalent neurodegenerative disorder affecting the aging population, with over 38 million people being afflicted worldwide and economic costs estimated to run into millions of dollars, scientific interest remains prioritized on the development of novel therapeutic strategies [2].
The neuronal perturbations in AD are attributed to the formation of extracellular amyloid-β (Aβ) neuritic plaques, composed predominantly of the neurotoxic Aβ42 isoform [3] and intracellular neurofibrillary tangles, which are aggregations of hyperphosphorylated tau protein, a microtubule associated protein [4]. Aβ40 - 42 generation occurs via the proteolytic cleavage of the amyloid-β protein precursor (AβPP) byβ-secretase, also termed BACE1 (β-site AβPP cleaving enzyme-1) and the presenilin-containing γ-secretase complex thereby leading to release of the sAβPPβ fragment and Aβ peptide [5]. Although this amyloidogenic pathway has a normal physiological function, it has been suggested that it is the misappropriate favoring of this pathway or the decline in Aβ42 clearance or degradation that leads to the accumulation of Aβ42 peptides and thus the development of AD [6].
Although Aβ plaques and neurofibrillary tangles have been reported to play a role in synaptic dysfunction, the neuronal loss characteristic of AD has been largely attributed to the formation of soluble Aβ42 oligomers [7, 8] and recently, a 3D model of AD has provided definitive proof for Aβ as the etiological agent in AD [9]. A common thread seen in Aβ-induced cell cytotoxicity is the requirement for an interaction between these toxic oligomers and cellular components [10, 11]. Owing to their hydrophobic nature soluble Aβ42 oligomers are incorporated into the plasma membrane resulting in membrane distortion and ion channel formation [12]. This might facilitate a large influx of cytotoxic Ca2 + ions [13]. The progression of AD appears to correlate with an accumulation of intracellular Aβ42 oligomers [14, 15], which may arise from the increased uptake of Aβ42 oligomers by cell surface receptors. The internalized Aβ42 oligomers and aggregates cause cellular dysfunction, aberrant cell signaling, and cellular damage leading to cell death [10]. One such receptor to which Aβ42 binds resulting in adverse effects is the cellular prion protein [16–18].
The cellular prion protein (PrPc) is a glycosylphosphatidylinisotol (GPI) anchored cell surface receptor expressed by most tissues. It is present in particularly high concentrations in neuronal cells, where its physiological functions include protection against oxidative stress, synaptic transmission, and copper homeostasis. In 2007, Parkin et al. demonstrated that a negative feedback loop exists between PrPc and β-secretase [19]. PrPc binding was shown to inhibit β-secretase cleavage of AβPP thus reducing Aβ production and shedding [19]. However, since Aβ42 oligomers bind with high affinity to PrPc, internalization of the Aβ42-PrPc complexes may reduce the levels of cell surface PrPc resulting in less β-secretase inhibition and enhanced Aβ shedding. PrPc has also been shown to bind to the 37kDa/67kDa laminin receptor (LRP/LR) [20]. This receptor, which is also known to bind with high affinity to laminin-1, is a multifunctional protein involved in cell survival, proliferation, cell adhesion, invasion as well as the internalization and intracellular recycling of PrPc [21–25]. LRP/LR has been shown to play a central role in metastatic cancer. Anti-LRP/LR specific antibodies reduced cell adhesion and invasion and limited the induction of angiogenesis in numerous cancer cell types [26–30]. An interaction between laminin-1 and Aβ42 oligomers has been demonstrated in previous studies [31]. As LRP/LR and Aβ42 share the aforementioned mutual binding partners (PrPc and laminin-1), it has been proposed that LRP/LR may be implicated in the pathogenesis of AD. Several studies established that LRP/LR not only affects Aβ processing [32], but is also seen to interact with the secretases responsible for the generation of Aβ as well [33].Furthermore, recent studies have implicated LRP/LR as a key player in the cellular internalization of Aβ42 [34] and in Aβ42-induced cytotoxicity [35]. However, owing to the binding of PrPc to both LRP/LR and Aβ, the mechanism by which Aβ induces neuronal cytotoxicity may be indirectly due to the PrPc-Aβ interaction with LRP/LR. The aim of this study was to examine the effect of PrPc in Aβ42-induced cell cytotoxicity and to determine the role of the PrPc-LRP/LR interaction in Aβ mediated cytotoxicity. The determination of the role played by PrPc and more importantly, the PrPc-LRP/LR interaction in Aβ42-induced cytotoxicity may have potentially important implications for understanding the pathogenic mechanism of AD and may thus contribute to the development of effective therapeutic strategies.
METHODS AND MATERIALS
Tissue culture
HEK293 and HpL3-4 (mouse hippocampal PrPc null) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), while Neuro2a (N2a) (mouse neuroblastoma cells) were grown in EMEM. All media was supplemented with 10% Fetal Calf Serum (FCS) and 1% Penicillin/Streptomycin solution. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere.
Immunofluorescence confocal microscopy
N2a, HpL3-4, and HEK 293 cells were seeded onto microscope coverslips and cultured until a confluence of 50–70% was obtained. The cells were fixed with 4% Paraformaldehyde in PBS for 10 min at 4°C, rinsed thrice with 1xPBS and blocked with 0.5% BSA in PBS (5–10 min at room temperature). Coverslips wereadditionally washed with 1xPBS and placed such that the cell-free side of the coverslip lay flush with the slide. A 100 μl volume of primary antibody solution (diluted in 0.5% BSA-PBS) containing differing combinations of the following antibodies: 1:150 anti-PrPc 8H4 (mouse, Sigma Aldrich), 1:100 anti-β-amyloid (22–35) (rabbit, Sigma Aldrich) or 1:150 IgG1-iS18 (human) [26] was administered to the cells. Post an overnight incubation at 4°C in moist containers, coverslips were again washed thrice in 0.5% BSA-PBS and placed on clean slides. A 100 μl volume of secondary antibody solution containing the corresponding secondary antibodies, namely 1:300 goat anti-mouse APC (Abcam), 1:300 goat anti-mouse FITC, 1:300 goat anti-rabbit APC (Abcam) or 1:300 goat anti-human FITC (Abcam) were administered to cells and incubated for 1 h in the dark. Coverslips were washed thrice with 0.5% BSA-PBS and once in PBS. Coverslips were then mounted using a 50 μl volume of Fluoromount (Sigma Aldrich). Slides were viewed using the Zeiss LSM 780 confocal microscope and captured using the AxioCam MRm camera. Images were subsequently analyzed using Zen 2010 imaging software. Controls were prepared using the above-mentioned protocol with the omission of primary antibodies to test for non-specific binding of the secondary antibodies.
HEK293 transfection methodology
HEK293 cells were seeded to attain 50% confluence within 24 h and incubated in 5% CO2 humidified atmosphere at 37°C. Post incubation, cells were transfected with the pSFV1-huPrP1-253 plasmid using the TransIT®-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions. Cells were incubated in a 5% CO2 humidified atmosphere at 37°C for 48 h.
Flow cytometry
N2a, HpL3-4, and pSFV1-huPrP1-253 plasmid [20] transfected and non-transfected HEK923 cells were fixed (4% Paraformaldehyde in PBS, 10 min, 4°C) centrifuged at 1500 g for 10 min and resuspended in 1 ml 1xPBS. Half of each cell sample was treated with 30 μg/ml anti-PrPc antibody 8H4 (Sigma Aldrich), while the other half of the cell sample was incubated in 1xPBS. Cells were then incubated at 4°C overnight. Post incubation the cells were washed three times in 1xPBS subsequent to centrifugation at 1700 g (room temperature). All cells were treated with 20 μg/ml of the corresponding goat anti-mouse APC coupledsecondary antibody (Abcam) and incubated for 1 h at room temperature. The cells were washed a further three times as above. The samples were analyzed using the BD Accuri C6 flow cytometer whereby 10,000 events were recorded using the FL-4 detector.
3-(4,5-dimethylthiazol-2-Yl-2,) 5-diphenyltetrazolium bromide (MTT) cell viability assay
Cells were seeded in a 96 well plate as to attain 50% confluence within 24 h and incubated in 5% CO2 humidified atmosphere at 37°C. HEK293 cells were then transfected with or without the pSFV1-huPrP1-253 plasmid using the TransIT®-LT1 Transfection Reagent (Mirus) and incubated at 5% CO2 humidified atmosphere at 37°C for 24 h. Post incubation, synthetic amyloid beta (Aβ42) peptide (Sigma Aldrich) was administered to the cells in varying concentrations (100 nM, 200 nM, and 500 nM) to determine the effect thereof on cell viability. HpL3-4 and N2a cells were treated with 500 nM Aβ42 24 h post seeding into 96 well plates (as they did not require transfection). In addition untreated controls (cells incubated in DMEM) as well as positive controls (cells incubated with 8 mM protocatechuic acid (PCA), an apoptosis inducing agent) were included for all cell lines. Furthermore pSFV1-huPrP1-253 transfected HEK293 and non-transfected HEK293, HpL3-4, and N2a cells were additionally co-incubated with Aβ42 (at 500 nM) and 50 μg/ml IgG1-iS18 antibody or 50 μg/ml anti- chloramphenicol acetyltransferase (CAT) antibody (Sigma Aldrich). Treated cells were incubated for 48 h (37°C, 5% CO2), following which 20 μl of 1 mg/ml MTT was added to each well and the cells subsequently incubated (37°C, 5% CO2) for 2 h. After incubation, culture media was aspirated and 180 μl of DMSO was added to each well to lyse the cells and dissolve the formazan crystals formed within the cells. The absorbance was recorded at 570 nm using an ELISA microtitre plate reader and the percentage of cell viability, relative to the untreated controls, calculated. Each experiment was performed in triplicate, three times.
Aβ preparation
Amyloid beta fragment 1-42 (Sigma Aldrich) was resusupended in DMSO to a concentration of 1 mg/ml. It was then aliquoted and stored at –20°C. The preparations were thawed at room temperature and diluted according to experimental requirements.
Statistical evaluation
Student’s t-tests were used to analyze the data and obtain p values. Significance was reported where p ≤ 0.05. All statistical evaluations were performed using GraphPad Prism (version 5.03) software.
RESULTS
PrPc levels
Flow cytometry was employed to ascertain the relative percentages of cells from the tested cell line populations expressing PrPc on their surface. N2a cells, being of neuronal origin express endogenously high levels of PrPc as evidenced by the flow cytometric plot in Fig. 1A, where 99.40% of the N2a cells stained positively for PrPc expression. HpL3-4 cells, which were generated from the hippocampi of Rikn PrP-knockout mice lacking the entire PrP open reading frame [36, 37], are not expected to express PrPc.Figure 1B confirms that the HpL3-4 cells indeed lack PrPc expression as shown by the lack of positively stained cells. HEK293 cells overexpressing PrPc due to transfection with the pSFV1-huPrP1-253 plasmid reveal greater cell surface staning for this protein in comparison to non-transfected HEK293 cells expressing PrPc endogenously (Fig. 1C).
LRP/LR and Aβ42 co-localize on the cell surface only in the presence of PrPc
Co-localization studies were performed in order to visualize the location of LRP/LR, PrPc, and Aβ within the cells of interest. Previous attempts to reconstitute HpL3-4 cells with PrPc revealed that they behave very similarly to N2a cells [38]. However, it is difficult to establish reconstituted HpL3-4 cells that have normal levels of expression as they usually tend to overexpress PrPc, which is most likely due to the non-endogenous promoter activity. We therefore chose to use N2a cells as controls since they are derived from mouse neuronal tissue (as are the HpL3-4 cells) and are known to express PrPc endogenously. Ifco-localization is observed between proteins, the close proximity may indicate possible interactions. Since the interaction between LRP/LR and PrPc is well established [20, 39], the main focus of this part of the study was to observe the co-localization between LRP/LR and Aβ in the presence (N2a) and absence (HpL3-4) of PrPc. The absence of PrPc expression in HpL3-4 cells was confirmed by the lack of detection of this protein using confocal microscopy. Interestingly, in these cells, LRP/LR and Aβ showed no signs of co-localization as evidenced by the lack of the expected yellow color which would result if the APC (red) and FITC (green) fluorescences overlapped. Furthermore, there is a complete lack of a diagonal in the 2D cytofluorogram (plot showing the co-distribution of the red and green fluorescence) for the Aβ and LRP/LR merge image (Fig. 2). However in the N2a cells, in which endogenous PrPc detection is readily detected, co-localization was seen between PrPc and LRP/LR, as well as between PrPc and Aβ (Fig. 3). As shown previously [35], co-localization between LRP/LR and Aβ is visible in these cells in contrast to the HpL3-4 cells where this co-localization did not occur. This suggests that PrPc is a pre-requisite for the co-localization between Aβ and LRP/LR. HEK293 cells displayed the same staining patterns as N2a cells (Supplementary Figure 1).
Overexpression of PrPc shows no effect on cell viability
The MTT cell viability assay was employed to assess the effects of PrPc overexpression on the viability of HEK293 cells. The lipofection transfection methodology significantly reduced cell viability when compared to the untransfected control cells (Fig. 4A). However PrPc overexpression did not affect cell viability when compared to the mock-transfected control.
Overexpression of PrPc significantly enhances the cytotoxic effect of exogenous Aβ42 treatment
The MTT viability assay was employed to assess the cytotoxic effect of synthetic Aβ42 at various concentrations on HEK293 cells (Fig. 4B). Exogenous treatment with 100 nM, 200 nM, and 500 nM of synthetic Aβ42 significantly reduced cell viability in HEK293 cells, which confirms previous data [35]. pSFV1-huPrP1-253 transfected and mock transfected cells were subsequently treated with 100 nM, 200 nM, and 500 nM of exogenous Aβ42. PrPc overexpression significantly enhanced cell death induced by the Aβ42 treatments when compared to similarly treated mock transfected cells (Fig. 4C). 8 mM PCA solution, an apoptosis inducing agent, was employed as a positive control.
Anti-LRP/LR specific antibody IgG1-iS18 rescues cells from Aβ42-PrPc mediated cytotoxicity
The MTT viability assay was employed to determine the effect of the anti-LRP/LR specific antibody IgG1-iS18 on cell viability of pSFV1-huPrP1-253 transfected and mock transfected HEK293 cells when treated with 200 nM and 500 nM of exogenous Aβ42. Treatment with IgG1-iS18 at 500 nM Aβ42 in both the pSFV1-huPrP1-253 transfected and mock transfected HEK293 cells showed a significant increase in cell viability (Fig. 4D). Antibody treatment alone, in the absence of Aβ42 administration, lacked any effect on cell viability (Fig. 4E).
HpL3-4 cells are more resistant to the cytotoxic effects of Aβ42 than N2a cells
N2a and HpL3-4 cells were exposed to various treatments to determine the effects of exogenous Aβ42 addition. Control untreated cells were set to 100% cell viability. DMSO was used as a vehicle control, as it is the solvent for the Aβ42 peptides and cell viability for these cells did not differ significantly from the untreated controls (Fig. 5A, B). Cells treated with the apoptosis inducing PCA showed significant reductions in cell viability. From the HEK293 data (Fig. 4), 500 nM Aβ was the most cytotoxic, therefore only this concentration was used for the N2a and HpL3-4 experiments. When 500 nM Aβ42 was exogenously added to the N2a and HpL3-4 cells, both cell lines underwent a significant reduction in cell viability in comparison to untreated and DMSO treated cells (Fig. 5A, B). However, there was a significant difference between the two cell lines; the HpL3-4 cells were more resistant to the cytotoxic effects of Aβ42 (Fig. 5C).
Anti-LRP/LR specific antibody IgG1-iS18 significantly rescues cells from Aβ-induced cytotoxicity in a PrPc dependent manner
Figures 5A and B indicate that Aβ42 is able to induce significant cytotoxicity in both PrPc expressing N2a and PrP–/– HpL3-4 cells, albeit to different extents. The above experiments were repeated with the addition of 50 μg/ml of either anti LRP/LR antibody (IgG1-iS18) or anti-CAT antibody (control) 24 h post Aβ42. Figure 5D reveals that in N2a cells the blockage of LRP/LR with IgG1-iS18 was able to rescue the cells from Aβ-induced cytotoxicity, as the viability of cells treated with both Aβ and IgG1-iS18 did not differ significantly from that of untreated controls. There are, however, significant differences when comparing the Aβ + IgG1-iS18 treated cells to those treated with Aβ and those treated with Aβ + anti-CAT antibody treatments (Fig. 5D). Human neuroblastoma SH-SY5Y cells revealed a similar pattern of cell death upon exposure to Aβ and increased cell survival in response to IgG1-iS18 (Supplementary Figure 2). In contrast, the HpL3-4 cells did not show any recovery when treated with IgG1-iS18 post Aβ incubation and showed no significant differences to control cells treated with Aβ alone and Aβ + anti-CAT (Fig. 5E). In Fig. 5F, the results from the N2a and HpL3-4 cells are combined, clearly illustrating the drastic difference in IgG1-iS18 rescuing ability between the N2a and HpL3-4 cells.
DISCUSSION
Since the discovery that PrPc served as a receptor for neurotoxic Aβ oligomers [16], a wealth of reports have further substantiated these claims and proposed mechanisms by which this interaction elicits neurotoxic effects. The binding of Aβ oligomers to PrPc results in the downstream activation of one of the Src family kinases, namely Fyn kinase [40, 41]. This kinase in turn phosphorylates the NR2B subunit of N-methyl-D-aspartate resulting in synaptic and dendritic spine loss, characteristic of the neurodegeneration seen in AD [42]. The membrane receptor metabotropic glutamate receptor mGluR5 was found to be responsible for the activation of fyn kinase upon the formation of Aβ-PrPc complexes [43]. Furthermore, Rushworth et al. found that lipid raft membrane localization was crucial to this pathogenic interaction between Aβ-PrPc and the subsequent activation of Fyn. They also found that this complex was internalized into endosomes via low-density lipoprotein 1 (LRP1, not to be confused with LRP/LR) [44]. Further highlighting the pathogenic role of PrPc in AD was the finding that the interaction between PrPc and Aβ inhibits the fibrillation of Aβ oligomers, resulting in PrPc “trapping” Aβ in their most neurotoxic conformation, i.e., Aβ oligomers [45].
LRP/LR is a well-recognized cellular receptor for PrPc [20, 39]. The interaction between these two proteins has been previously characterized in numerous cell lines originating from various species [46]. In this study, we sought to examine the function of LRP/LR in the interaction between Aβ and PrPc, given its significant role in binding and internalizing PrPc. To this end, we employed two neuronal cell lines, one of which lacks PrPc expression (HpL3-4). Furthermore, HEK293 cells were utilized for the overexpression of PrPc due to their ease of transfectability (attempts to overexpress PrPc in N2a cells were unsuccessful; data not shown). Flow cytometry was employed in order to confirm the cell surface expression of PrPc in the cell lines used for this study. A high endogenous PrPc expression was noted in the vast majority of the N2a neuroblastoma cells examined (Fig. 1A), whileHpL3-4 cells completely lacked expression of this protein as was to be expected (Fig. 1B). HEK293 cells that had been transfected with the PSFV1huPrP1-253 plasmid revealed a higher expression of PrPc, compared to the endogenous levels in untransfected cells(Fig. 1C).
The interaction between LRP/LR and PrPc has been previously characterized, as has the Aβ interaction with PrPc. Recently, a report showed LRP/LR also co-localized with Aβ in both HEK293 and N2a cells [35]. In this present study we sought to determine whether this co-localization is dependent on the presence of PrPc. Co-localization studies revealed that in HpL3-4 cells, which do not express PrPc (Fig. 2), co-localization between Aβ and LRP/LR is completely abolished. In N2a cells, however, Aβ and LRP/LR are seen to co-localize, which demonstrates the necessity of PrPc in facilitating this co-localization (Fig. 3).
Since a co-localization between Aβ and LRP/LR was seen in the presence of PrPc, we sought to establish whether LRP/LR may also play a role in the observed cytotoxicity that Aβ elicits via its interaction with PrPc. Prior to assessing the role of PrPc, it was necessary to confirm that the Aβ42 concentrations employed were cytotoxic. MTT cell viability assays confirmed the cytotoxic effect of exogenous Aβ42 on HEK293 cells at varying concentrations (Fig. 4B). Moreover, the results suggest a direct correlation between Aβ42 concentration and cell cytotoxicity once a threshold concentration has been achieved. Although the threshold of synthetic Aβ42 has been previously documented to be a 100–200 nM concentration [47], disruption of long-term potentiation [47] and significant decrease in cell viability are largely observed at 500 nM Aβ concentrations [48]. The results presented here mirror those previously described, where 500 nM Aβ42 was also the most cytotoxic concentration. It is important to note that the 500 nM concentration employed is below the critical concentration required for fibril formation in vitro [49]. MTT cell viability assays were employed to examine the role of the Aβ42-PrPc interaction in Aβ42-induced cell cytotoxicity once PrPc had been overexpressed. Cells overexpressing PrPc displayed significantly lower cell viability upon Aβ42 treatment when compared to similarly treated mock-transfected controls (Fig. 4C). This suggests that the Aβ42-induced cell cytotoxicity observed here is enhanced through the Aβ42-PrPc interaction. These results correlate with those of previous studies [44]. Cell viability was not compared to untreated controls owing to the detectable toxicity of the transfection methodology (Fig. 4A). When the cells were submitted to antibody treatment post Aβ42 incubation, we saw a dramatic reduction in cell death in the cells treated with the anti LRP/LR specific antibody IgG1-iS18, in both the non-transfected and PrPc overexpressing cells, in comparison to those that had been treated with anti-CAT antibody (Fig. 4D). This reveals that LRP/LR plays a significant role in PrPc mediated Aβ-induced cytotoxicity, as a blockade of this receptor ameliorates the detrimental effect of PrPc overexpression on cell viability upon exposure to exogenous Aβ. Antibody treatments alone had no significant effect on cell viability (Fig. 4E), suggesting that it is the direct blockage of LRP/LR in the presence of Aβ and PrPc that contributes to the rescuing effect seen in Fig. 4D.
These results were further verified in N2a cells, where a significant reduction in cell viability is seen upon exposure to exogenous Aβ42 (Fig. 5A). Since there are a number of reports which highlight the ability of Aβ to elicit cytotoxic events that are independent of PrPc [50–52], it is unsurprising that the HpL3-4 cells still undergo cell death upon exposure to 500 nM Aβ (Fig. 5B). However, HpL3-4 cells are more resistant to Aβ-induced cytotoxicity than the N2a cells, as would be expected due to the lack of PrPc expression (Fig. 5C).
Upon co-incubation with Aβ42 and IgG1-iS18, N2a cells underwent significantly less cell death as is seen in Fig. 5D. The cells that were treated with the anti-LRP/LR antibody had significantly higher cell viability in comparison with the Aβ only and Aβ + anti-CAT controls, signifying that the blockage of LRP/LR somehow impairs the mechanism responsible for Aβ-induced cytotoxicity. An insight into this mechanism was provided by the results of antibody treatment of HpL3-4 cells exposed to Aβ42. No cell rescuing effect is observed in the cells treated with IgG1-iS18 (in combination with Aβ), mimicking the results observed with Aβ only and Aβ + anti-CAT antibody (Fig. 5E). The findings from experiments with both N2a and HpL3-4 cells (Fig. 5F) showing that LRP/LR blockade has a strong cell rescuing effect only in the presence of PrPc strongly suggests that this receptor is involved in mediating the cytotoxic relationship between Aβ and PrPc.
The interaction between LRP/LR and PrPc is well documented and LRP/LR is known to be the cellular receptor that facilitates the internalization of PrPc into endosomes [53]. More recently, Da Costa Dias et al. have reported on the interaction between LRP/LR and Aβ and have also shown how this cellular receptor functions in internalizing Aβ into the cell. However, these studies were all performed in cell lines which endogenously express PrPc [34]. The present study provides insight into these interactions and a possible mode of action for LRP/LR in AD. When exogenous Aβ was added to the cells in our study, they all experienced cell death, albeit to differing degrees depending on the amount of PrPc they were expressing. HEK293 cells which were overexpressing PrPc underwent significantly increased levels of cytotoxicity in response to Aβ in comparison to non-transfected samples, while HpL3-4 cells which lack PrPc expression were significantly more resistant to Aβ-induced cytotoxicity than N2a cells which endogenously express PrPc. The findings confirm that PrPc plays a significant role in mediating Aβ-induced cytotoxicity.
However, these cells responded to anti-LRP/LR treatment in varying degrees. In N2a cells and HEK293 cells overexpressing PrPc, treatment with the anti-LRP/LR antibody resulted in a significant rescuing from Aβ-induced cell death, while having no effect in HpL3-4 cells. This clearly shows that blockage of LRP/LR has the ability to rescue cells from the cytotoxic effects of Aβ, however, only in the presence of PrPc, suggesting an interesting mode of action for LRP/LR in AD. The blockage of LRP/LR with IgG1-iS18 would hinder the interaction between LRP/LR and PrPc thereby effectively preventing the internalization of PrPc. Since the LRP/LR-mediated internalization of Aβ was never examined in a PrPc free environment, it is plausible that the mechanism by which LRP/LR internalized Aβ might well have been PrPc dependent. This is highly likely since PrPc and Aβ and the complex between these two proteins has previously been reported to be internalized together [44]. The blockage of LRP/LR may thus be hindering the internalization of the PrPc-Aβ complex. Furthermore, examination of the binding sites for Aβ (aa 95 – 110) and LRP/LR (aa 53–93 as well as aa 129 – 179) on PrPc (Fig. 6) reveal that these proteins bind in very close proximity to one another. This may suggest that a ternary complex forms between these three proteins that is crucial for eliciting cytotoxic events. Blockade of LRP/LR would prevent this complex from forming thus negating the cytotoxicity of the Aβ and PrPc complex. The abovementioned possibilities open up many new interesting avenues for research that will help to further elucidate the role of LRP/LR in AD, while also substantiating the important role that LRP/LR plays in AD and highlighting how IgG1-iS18 may have potential therapeutic uses for the treatment of this devastating disease in the future.
Footnotes
ACKNOWLEDGMENTS
This work is based upon research supported by the National Research Foundation (NRF), the Republic of South Africa (RSA). The research from which this publication emanated was co-funded by the South African Medical Research Council (SAMRC). 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.
