N-acetyl L-cysteine and Growth Factors Impede Endoplasmic Reticulum Stress and Inflammatory Responses in Astrocytes to Amyloid-β in Serum-free Culture

Abstract

Background

Astrocytes play an integral role in Alzheimer’s disease (AD) pathology, where they may act as a double-edged sword. The existing serum-supplemented in vitro astrocyte culture models are not suitable to study certain stress response mechanisms that occur in AD.

Purpose

Here, we tried to develop a serum-free murine primary cortical astrocyte culture model to study endoplasmic reticulum (ER) stress and inflammation to investigate the effect of amyloid-beta (Aβ_1–42).

Methods

Astrocytes were cultured in a controlled serum-free environment to minimise interference from serum components. Serum-free astrocytes were exposed to oligomeric Aβ and subjected to imaging, immunocytochemistry, real-time PCR and western blot analysis.

Results

Using an established protocol, no significant activation of eIF2α, a key marker of ER stress, was observed under serum-free conditions, but with the removal of N-acetyl cysteine (NAC), ER stress response was enhanced after 24 hours of Aβ exposure. Subsequently, the Aβ-induced inflammatory response, assessed through TNF-α expression, which was minimal in the presence of growth factors, became pronounced when these factors were withdrawn. Concomitantly, a significant increase in astrocytes reactivity, assessed by GFAP expression upon 24 hours of Aβ exposure, was observed. Transcript analysis revealed a time-dependent shift in the expression of inflammatory molecules, with early time points showing an increase in anti-inflammatory markers, while late exposure promoting pro-inflammatory responses.

Conclusion

This study identifies that NAC and growth factors impede ER stress and inflammatory responses in astrocytes upon Aβ exposure in serum-free culture. These findings also highlight the potential of a serum-free culture system for studying ER stress and inflammation in astrocytes to understand the complex role of these cells in AD pathophysiology.

Introduction

Astrocytes play a vital role in maintaining homeostasis in the central nervous system (CNS) by supporting neurons, regulating blood flow, and managing neurotransmitter levels in the brain. ¹ In Alzheimer’s disease (AD), astrocytes become reactive, thus promoting neuroinflammation and reducing the ability to effectively clear amyloid-beta (Aβ), leading to increased plaque burden. Dysfunctional astrocytes further accelerate neurodegeneration by disrupting synaptic function and overall brain health.^2–4 The accumulation of misfolded proteins, such as Aβ, induces endoplasmic reticulum (ER) stress, triggering the unfolded protein response (UPR) in both neurons and glial cells. Prolonged ER stress hinders neuron function by interfering with protein quality control, resulting in synaptic dysfunction, ultimately leading to neuron death in AD. ⁵ In astrocytes, ER stress impedes Aβ clearance, which may be linked to the neuroinflammatory response, worsening AD pathology. ⁶

Although the blood-brain barrier (BBB) prevents direct access of serum components to brain cells, certain substances like proteins, hormones and growth factors can still affect the brain. For instance, hormones like insulin and cortisol can enter the brain under physiological conditions, potentially influencing brain activity, mood, etc. This is due to the selective permeability of the BBB, which allows specific molecules to cross via active transport or passive diffusion. Furthermore, entry of serum into the brain indicates a possible leakage of BBB under pathological conditions. Many current in vitro astrocyte culture models use medium supplemented with serum, which does not allow for the study of certain stress conditions in the brain during disease. The main goal of our work was to develop a serum-free astrocyte culture for examining ER stress in response to Aβ. ⁷

Inflammation or changes in the systemic circulation (such as cytokines or other signalling molecules in the blood) can change the permeability of the BBB and cause serum entry, triggering significant effects on the brain cells.^8–10 The serum-free culture system we developed in this study eliminated the variability introduced by serum components (like growth factors, hormones, and proteins), which can otherwise interfere with the correct interpretation of experimental results.^{11, 12}

MD (McCarthy-de Vellis) astrocyte culture containing serum may be useful to address certain biological questions, ¹⁰ but complicates the interpretation for pathological models, including AD. Serum-free primary astrocyte cultures are being developed, ¹¹ but the existing protocols are either very expensive (e.g., immunopanning) or the addition of several serum-free factors may not suitably allow for the examination of specific biological questions. These limitations highlight the need for a new, effective protocol that allows for a mechanistic analysis of how Aβ accumulation triggers ER stress in astrocytes. Serum-free cultures may be used to better study the direct impact of Aβ on astrocyte function without interference from serum components. In this study, we developed a serum-free murine primary cortical astrocyte culture model to study ER stress and inflammation to see the effect of Aβ on these processes.

Methods

Materials

Cell culture dishes and flasks were purchased from Nunc (USA). DMEM powder, DMEM (1X), FBS, Trypsin-EDTA, Neurobasal, Sodium pyruvate and L-glutamine were purchased from Thermo Fisher Scientific. BSA, Apotransferrin, Putrescine, Progesterone, Selenite, PDL, NAC, HB-EGF, FGF-2 and β-Actin antibody (conjugated with HRP) were procured from Sigma. We obtained eIF2α and Phospho-eIF2α antibodies from Cell Signalling Technology. XBP1 antibody was purchased from Affinity Biosciences. GFAP antibody was purchased from Novus Biologicals. TNF-α antibody was purchased from Abclonal. The secondary antibodies used for immunocytochemistry and western blot were purchased from Thermo Fisher Scientific and Cell Signalling Technology, respectively.

Primary Astrocyte Culture

Isolation of Brain from C57BL/6 Mouse Pups

Prior to brain dissection, 10 ml of 1X Hank’s Balanced Salt Solution (HBSS) was placed in a 100 mm petri dish, and 10 ml of 0.05% trypsin-BSA solution was prepared. 0.01% Poly D-Lysine solution was used to coat T-75 flasks as per requirement. Sterile dissection tools, 70% ethanol, ice, and a microscope were arranged for the dissection of mouse pups’ brains. Five to six mouse pups, postnatal day 0 to 1 (P0–P1), were obtained from the Institutional Animal House. The neck region of the pups was sprayed with 70% ethanol to avoid contamination. The mouse pups were decapitated in a single cut using sharp scissors, and the brains were collected into a sterile 10 cm dish. The cranium was carefully separated using forceps to expose the brain. Small bent forceps were used to lift the brain and disconnect it from the skull base. The brain was transferred to a sterile 10 cm petri dish containing ice-cold 1X HBSS, ensuring it was fully submerged. Once the meninges were fully removed, the brain tissue was chopped into 6–10 pieces using forceps and sharp blades to improve dissociation efficiency. Afterwards, 1X HBSS was replaced by 0.05% trypsin-BSA solution to the finely chopped cortical tissue, which was then incubated at 37°C for 15 minutes. Following incubation, the trypsin-BSA solution was carefully aspirated using a Pasteur pipette. The tissues were transferred to a 15 ml tube with complete astrocyte growth medium (Medium 1), containing DMEM supplemented with 10% fetal bovine serum (FBS) and streptomycin-penicillin (1X).

Preparation of Serum-free Primary Astrocyte Culture for Treatment

After enzymatic digestion, the tissue was mechanically dissociated into a single-cell suspension by gently triturating or pipetting the tissue to break it into smaller pieces and disperse the cells. The goal was to isolate the astrocytes from the surrounding cells and extracellular matrix while minimising cell damage. We had to triturate at least 20 times for achieving a single cell suspension, which was then poured through a mesh filter (or cell strainer), which allowed smaller individual cells to pass through while capturing larger cell clumps, debris or tissue fragments. This process created a homogenous single-cell suspension. We then centrifuged the cells at 750 rpm for 5 mins, followed by plating the cells into a PDL-coated T75 flask until 90% confluency in an incubator at 37°C with 5% CO₂. The media is changed every alternate day using Medium 1. When confluency is reached in the mixed cell culture, the flask is gently tapped on both sides several times to remove oligodendrocyte progenitor cells. ¹³ After culturing the cells in Medium 1 for 10 days, they were collected from the T-75 flask, followed by trypsinisation and centrifugation. The cells were then plated onto dishes and coverslips with Medium 1 for biochemical analysis. On the 12th day, that is, two days after subculture, astrocytes were switched from Medium 1 to serum-free Medium 2 containing 5 ng/ml heparin-binding EGF-like growth factor (HBEGF), 50% 1X DMEM (without glutamine), 50% Neurobasal medium, 1 mM L-glutamine, 100 µg/ml bovine serum albumin, 100 µg/ml apotransferrin, 16 µg/ml putrescine dihydrochloride, 60 ng/ml progesterone, 40 ng/ml sodium selenite, 5 µg/ml N-acetyl-L-cysteine (NAC), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin) for three days. ¹² Since cells were dying in Medium 2, later this medium was replaced with Medium 3, which had all components of Medium 2 except NAC and was supplemented with 5 ng/mL of FGF-2. After maintaining the cells in Medium 3 for three days, on 15th day astrocytes were transitioned to Medium 4 containing 50% 1X DMEM (without glutamine), 50% Neurobasal medium, 1mM L-glutamine, 100 µg/ml bovine serum albumin, 100 µg/ml apotransferrin, 16 µg/ml putrescine dihydrochloride, 60 ng/ml progesterone, 40 ng/ml sodium selenite, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin. Finally, on the 16th day, the cells were ready for treatment (Figure 1).

Figure 1.

Schematic Illustration of Serum-free Culture Procedure.

Oligomeric Aβ Preparation

The lyophilised Aβ_1–42 peptide (American Peptide, Sunnyvale, CA, USA) was first resuspended in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) (Sigma-Aldrich) to a concentration of 1 mM and then centrifuged under vacuum conditions in a speed vac (Eppendorf, Hamburg, Germany) until all the HFIP was evaporated. The resulting peptide pellet was dissolved in DMSO (Sigma-Aldrich) at a concentration of 5 mM and subjected to sonication in a 37°C water bath for 10 minutes. The solution was then diluted further with phosphate-buffered saline (PBS: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 2 mM, pH 7.2) and SDS (0.2%) to a final concentration of 400 µM and incubated at 37°C for 6–18 hours. Finally, PBS was added to achieve a final concentration of 100 µM, and the solution was incubated for an additional 18–24 hours at 37°C. 1.5 µM Aβ_1–42 was used to treat the mature astrocyte culture on the 16th day at different time points.

Preparation of Cell Lysate

All treated cells, along with the controls, were washed with PBS and collected by scraping in PBS. The cells were then harvested by centrifugation at 1200 rpm at 4°C for 5 minutes, followed by lysis using lysis buffer [10 mM Tris (pH 4), 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 0.2 mM Orthovanadate, Protease Inhibitors], and incubated on ice for 10 minutes. The cell lysates were then centrifuged at 14,000 rpm at 4°C for 15 minutes. The supernatant was collected, and protein concentration was determined using the Lowry method.

Western Blotting

Lysates containing equal amounts of protein from each condition were separated using SDS-PAGE. The protein bands were then transferred onto a PVDF membrane. After blocking the membrane with 5% BSA solution for 1.5 hours at room temperature, the membranes were incubated with primary antibodies: anti-mouse β-Actin conjugated with peroxidase (1:10000) for 1 hour at room temperature, and anti-rabbit phospho-eIF2α (1:1000), anti-rabbit total-eIF2α (1:1000), anti-mouse GFAP (1:2500), and anti-rabbit TNF-α (1:1000) antibodies overnight at 4°C. Following incubation, the membranes were washed three times with TBST [1.5 M NaCl, 1 M Tris (pH 7.5), 0.1% Tween20] and then incubated with HRP-conjugated secondary antibodies for one to two hours at room temperature. Protein bands were detected using the Invitrogen iBright 1500 Imaging System with ECL reagents, following three washes with TBST. Densitometric analysis of the bands was performed using the NIH-ImageJ software.

Immunocytochemistry

Control and treated cells cultured on glass cover slips were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. To permeabilise and block the cells, they were incubated with 3% goat serum and 0.3% Triton X-100 in PBS for one to two hours. The cells were then incubated overnight at 4°C with anti-mouse GFAP (1:500). The following day, the cells were washed with PBST (0.3% Triton X-100 in PBS). Next, the cells were incubated with species-specific secondary antibodies conjugated to Alexafluor 546/488 for one to two hours at room temperature. Nuclei were stained with Hoechst 33342 at 2 µg/ml in PBS for 30 minutes at room temperature. Images were captured using a LeicaCTR4000 fluorescence microscope with a 40X objective. The corrected total cell fluorescence (CTCF) was calculated using the formula: CTCF = Integrated density – (area of selected cell × mean fluorescence of background readings). Brightfield Microscopic Images were captured without fixation with 20X objective and then adjusted with Photoshop software.

RNA Isolation and Real Time PCR

Total RNA of each sample is isolated from cultured astrocytes by using RNAiso (Takara). From RNA cDNA has been prepared. With cDNA, quantitative PCR was performed using TB Green Premix Ex Taq (Tli RNase H Plus) in an Applied Biosystems 7500 Fast Real Time PCR System following the manufacturer’s specifications. Primers used for qPCR are given in the above table.

Actin:	Forward Primer	5’-GAAATCGTGCGTGACATCAAAG-3’
	Reverse Primer	5’-TGTAGTTTCATGGATGCCACAG-3’
Ptx3:	Forward Primer	5’-ATGGTTGCTGTGTAGGTGGG-3’
	Reverse Primer	5’-GACGACATTTCCCCGGATGT-3’
Sphk1:	Forward Primer	5’-GAAACCCCTGTGTAGCCTCC-3’
	Reverse Primer	5’-AGCAGGTTCATGGGTGACAG-3’
S100A10:	Forward Primer	5’-CAGGTTTGCAGGCGACAAAG-3’
	Reverse Primer	5’-CAGCCAGAGGGTCCTTTTGA-3’
C3:	Forward Primer	5’-AGCTTCAGGGTCCCAGCTAC-3’
	Reverse Primer	5’-GCTGGAATCTTGATGGAGACG-3’
Iigp1:	Forward Primer	5’-GCCACCAATCTTCCTGCTCT-3’
	Reverse Primer	5’-CAGCAGCAAATCCTTCCAGC-3’
Ggta1:	Forward Primer	5’-CCAGTCCCGAGAAGTTCACC-3’
	Reverse Primer	5’-CGTTCCTCCAAAAATGGCCG-3’

Statistical Analysis

Data are presented as mean ± SEM (standard error of the mean). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc analysis. Results were considered statistically significant at p < .05.

Result

Developing Murine Primary Cortical Astrocyte Culture in Absence of NAC and Growth Factors in a Serum-free Medium

To develop a serum-free murine primary cortical astrocyte culture for studying ER stress and inflammation in response to Aβ, we employed a protocol that has been described earlier. ¹² After maintaining the astrocytes in Medium 1 with serum for 10 days, we observed that the flask was nearly 90% confluent, with astrocytes being the predominant cell type, displaying a polygonal shape (Figure 2A). To remove microglia, the cells were subjected to orbital shaking at 100 rpm at 37°C for 8 hours. Once pure astrocytes were obtained, the cells were trypsinised for 5 minutes and then centrifuged at 750 rpm for 5 minutes before being subcultured in serum-free Medium 2 (Please see Figure 1 for composition). After 24 hours, the cells began to exhibit signs of deterioration (Figure 2B), although they still maintained their typical stellate shape. By 72 hours, the majority of the astrocytes had died, and only a small number of viable cells remained (Figure 2C). However, when the cells were replated with Medium 1, following trypsinisation and without any shaking, they regained their viability. Microscopic observation over the next two days revealed that, by the 12th day, the flat astrocyte monolayer appeared healthy (Figure 2D). On 16th day, after being cultured in Medium 3 without NAC for three days followed by one day in Medium 4 without NAC and growth factors (please see Methods section and Figure 1 for details), astrocytes with branched processes emerged (Figure 2E), mimicking astrocyte’s typical physiological morphology appropriate for our experimental study. ¹⁰

Figure 2.

Brightfield Microscopic Image of Astrocyte Culture (Scale Bar is 100 µm). (A) Microscopic Image of Subcultured Astrocytes on the 10th Day. (B) Image of Astrocytes after 24 Hours of Replate (Stellate Shape) in Medium 2. (C) Image of Astrocyte Death after 72 Hours of Subculture Maintained in Medium 2. (D) Image of Subcultured Astrocytes, Monolayer of Polygonal-shaped Astrocytes on the 12th Day in Medium 1. (E.) Image of Astrocytes with Branched Processes (White Arrow) on the 16th Day, After Maintaining Them in Medium 3 for Three Days, Followed by 1 Day in Medium 4.

Validation of ER Stress in Astrocytes in Response to Aβ

Next, we assessed the impact of Aβ on ER stress response, focusing on eIF2α, a key component of the UPR pathway. ¹⁴ Under conditions of significant ER stress, eIF2α undergoes phosphorylation, which serves as a critical marker of cellular stress. ¹⁵ After maintaining the astrocytes for 16 days in Medium 2, the cells were treated with 1.5 µM of oligomeric Aβ_1–42 (henceforth referred to as Aβ) at various time points, that is, 0, 3, 6 and 24 hours. Western blot analysis was performed to evaluate the phosphorylation of eIF2α. The results showed that irrespective of the duration of treatment, there was no significant change in the phosphorylation of eIF2α, indicating that Aβ treatment did not induce notable ER stress (Figure 3A–3C) in the astrocytes in Medium 2.

Figure 3.

Effect of Aβ on Components of UPR. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different Time Points after Treatment as Indicated in the Figure and Cell Lysates were Subjected to Western Blot Analysis. (A) Immunoblot of Temporal Changes in the Levels of p-eIF2α, eIF2α & β-Actin upon Aβ Treatment. (B, C) Graphical Representations of Fold Change - p-eIF2α/eIF2α (B), eIF2α (C). Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). ns p > .05. β-Actin was Used as a Loading Control.

NAC restores glutathione levels, which is one of the most powerful antioxidants. ¹⁶ Increased oxidative stress is commonly associated with the onset or exacerbation of ER stress. Studies have shown that NAC can affect ER stress biomarkers, such as eIF2α and sXBP1. Specifically, treatment with NAC has been shown to reduce ER stress.^{17, 18} Hence, we examined whether the removal of NAC from Medium 2 would allow us to study the effect of Aβ on serum-free astrocytes. On the 12th day, astrocytes were shifted to Medium 3 (without NAC), and as in the previous experiment, Aβ treatment was done at the same time points (0, 3, 6 and 24 hours) on day 16. Analysis for the phosphorylation of eIF2α revealed Aβ-induced activation of eIF2α at 24 hours (Figure 4A–4C) following Aβ treatment. The expression level of spliced X-box binding protein 1 (sXBP1), which is another ER stress downstream marker, significantly increases after 24 hours exposure (Figure 4D and 4E) to 1.5 µM Aβ. This indicates that prolonged exposure to Aβ (24 hours) leads to a more pronounced activation of the UPR pathway when the cells were maintained in Medium 3.

Figure 4.

Effect of Aβ on the Component of UPR When Cells are Maintained Without NAC Since the 12th Day. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different Time Points after Treatment as Indicated in the Figure and Cell Lysates were Subjected to Western Blot Analysis. (A) Immunoblot of Temporal Changes in Expression of p-eIF2α, eIF2α & β-Actin upon Aβ Treatment. (B, C) Graphical Representations of Fold Change - p-eIF2α/eIF2α (B), eIF2α (C). (D) Immunoblot of Temporal Changes in Expression of sXBP1 and β-Actin upon Aβ Treatment. (E) Graphical Representations of Fold Change of sXBP1. Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). ns p > .05, **p < .01, ***p < .001. β-Actin was Used as a Loading Control.

We next analysed the effect of Aβ on inflammation and ER stress in this astrocyte model maintained in Medium 3. We measured the protein expression of TNF-α, a key inflammatory cytokine. There was no significant change in TNF-α expression at any of the time points (0, 3, 6 and 24 hours) after Aβ treatment to primary astrocytes on the 16th day in Medium 3 (Figure 5A and 5B).

Figure 5.

Effect of Aβ on TNF-α. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different Time Points after Treatment as Indicated in the Figure and Cell Lysates were Subjected to Western Blot Analysis. (A) Immunoblot of Temporal Changes in Expression of TNF-α & β-Actin upon Aβ Treatment. (B) Graphical Representation of Fold Change of TNF-α. Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). ns p > .05. β-Actin was Used as a Loading Control.

The presence of specific growth factors can sometimes mask the effect of Aβ on astrocytes. ¹⁹ To study this further, we removed the growth factors which might interfere with inflammatory response, Hence, on the 16th day, we exposed the cells to Aβ at four different time points (0, 3, 6, and 24 hour) to evaluate the inflammatory response in astrocytes in absence of growth factors FGF-2 & HBEGF (Medium 4). Remarkably, we found that the expression of TNF-α was significantly elevated following 24 hours of Aβ treatment (Figure 6A and 6B).

Figure 6.

Effect of Aβ on TNF-α When FGF-2 & HBEGF Have Been Withdrawn from the Medium on the 15th Day. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different Time Points after Treatment as Indicated in the Figure and Cell Lysates were Subjected to Western Blot Analysis. (A) Immunoblot of Temporal Changes in Expression of TNF-α & β-Actin upon Aβ Treatment. (B) Graphical Representation of Fold Change of TNF-α. Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). ns p > .05, **p < .01. β-Actin was Used as a Loading Control.

Astrogliosis in Response to Aβ Treatment

Further, we examined how Medium 4 changes the reactivity of astrocytes in the presence of Aβ at the same time points as earlier to evaluate the morphological shift and changes in expression of GFAP, an astrocyte marker. Using western blot analysis, we found that GFAP was increased significantly after 24-hour exposure to Aβ compared to the 0, 3 and 6 hours time points (Figure 7A and 7B). Immunofluorescence analysis of GFAP expression revealed that upon 24 hours of Aβ exposure, the CTCF (corrected total cell fluorescence) of GFAP was significantly increased in astrocytes in Medium 4 (Figure 7C and 7D) versus 0, 3 and 6 hours treatment. Moreover, cortical astrocytes transformed into a more fibrous shape after 24 hours of Aβ treatment assessed by individual cell perimeter, which was significantly increased after 24 hours exposure of Aβ (Figure 7E).

Figure 7.

Astrocyte Reactivity Upon Aβ Treatment. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different time Points after Treatment as Indicated in the Figure and Cell Lysates were Subjected to Western Blot Analysis. Additionally, the Treated Astrocytes were Fixed for Immunocytochemistry at Particular Time Points. (A) Immunoblot of GFAP & β-Actin of Primary Astrocyte Treated with Oligomeric Aβ, (B) Graphical Analysis of Fold Change of GFAP, (C) Images of Primary Astrocyte Treated with Oligomeric Aβ. Immunocytochemistry was Done Using anti-GFAP (green) Antibody & DAPI (blue) for Nucleus (Scale bar is 100 µm), (D) Graphical Representation of Corrected Total Cell Fluorescence of GFAP and (E) Length of Astrocytes Perimeter Treated with Aβ. Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). *p < .05, **p < .01, ***p < .001. β-Actin was Used as a Loading Control.

Characterisation of Astrocyte-specific Inflammatory Molecules Upon Aβ Treatment

Finally, we characterised pure astrocytes in Medium 4 for astrocyte-specific inflammatory molecules. Accordingly, the Aβ-treated samples (at different time points: 0, 3, 6, and 24 hours) were collected to examine the gene expression levels of several inflammatory molecules, ⁹ and we found that the transcript levels of anti-inflammatory molecules like Ptx3, S100a10 and Sphk1, ²⁰ were increased (Figure 8D–8F) at 3 and 6 hours, whereas the pro-inflammatory molecules like C3, Iigp1 and Ggta1, ²¹ were increased at 24 hours of Aβ exposure (Figure 8A–8C). This suggests that during early time-points, astrocytes display a protective role evident from high levels of anti-inflammatory markers, which switches to a detrimental paradigm at 24-hour of Aβ exposure.

Figure 8.

Characteristics of Inflammatory Molecules in Serum-free Astrocytes Upon Aβ Treatment by RT-PCR. Astrocytes were Treated with 1.5 µM Aβ on the 16th Day of Culture, Cells were Harvested at Different Time Points after Treatment as Indicated in the Figure, and Expression of the Indicated Genes was Quantified by RT-PCR. Graphical Representation of Fold Change of A. C3, B. Iigp1, C. Ggta1, D. PTX3, E. Sphk1, F. S100A10. Statistical Analysis was Performed Using ANOVA. Data are Presented as Mean ± SEM (N = 3). *p < .05, **p < .01, ***p < .001. β-Actin was Used as a Loading Control in RT-PCR.

Discussion

ER stress and inflammatory pathways play an important role in AD pathogenesis.^{15, 22} How subtle ER stress leads to inflammatory responses in diseased conditions remains elusive. ER stress disrupts the proper folding and clearance of amyloid precursor protein (APP), resulting in increased production and aggregation of Aβ. ²³ The accumulation of Aβ further activates the UPR, which escalates ER dysfunction. ER stress also triggers tau hyperphosphorylation, contributing to the formation of neurofibrillary tangles. ²⁴ Chronic ER stress activates pro-apoptotic proteins, such as CHOP, via the PERK-eIF2α pathway, leading to synaptic loss and neuronal death. ²⁵ Additionally, ER stress amplifies inflammatory responses to activate astrocytes and microglia. ²⁶ The IRE-1α-TRAF2 complex facilitates the interaction between active IRE-1α and the NF-κB and MAPK signalling pathways. The production of inflammatory cytokines induced by ER stress in infected mice relies on the interplay between TRAF2, NOD1/2, and RIPK2. The PERK-JAK1 complex phosphorylates STAT3, revealing a novel pathway that links UPR to the regulation of neuroinflammation, exacerbating AD pathology. ²⁶ Studying the interplay between ER stress and inflammatory responses in astrocytes is critical for an integrative mechanistic understanding of these pathways in AD. Although there are well-established methods for culturing neurons to study ER stress responses in vitro, current literature lacks cell culture protocols dedicated to study ER stress responses in astrocytes. In this study, we aimed to study ER stress in primary cortical astrocytes in response to Aβ. We found no change in ER stress markers in response to Aβ in serum-free astrocyte cultures using a protocol reported earlier. ¹⁰ Upon investigation, we identified a compound, NAC, which appears to have an inhibitory effect on ER stress, explaining why there were no alterations in ER stress upon Aβ exposure. ¹⁸

NAC is a potent antioxidant that has been shown to have protective effects against various forms of cellular stress, including ER stress. ¹⁶ NAC functions primarily by replenishing intracellular levels of glutathione, a key antioxidant in the cell. By enhancing glutathione levels, NAC reduces oxidative stress, which is a significant contributor to ER stress. In the context of ER stress, NAC has been reported to alleviate cellular damage caused by the accumulation of misfolded proteins in the ER lumen, a key feature of the UPR. It can mitigate oxidative stress, promote protein folding, and restore normal cellular functions, thus potentially protecting cells from the harmful effects of prolonged ER stress. ¹⁷ FGF-2 effectively countered TNFα through MAPK activation and reduced proinflammatory cytokine levels in the hippocampus of rats.^{27, 28} EGF can mitigate inflammatory signals by modulating TLR2 and associated transcription factors such as p38, NFκB. Moreover, EGF treatment suppresses the expression of inflammatory markers in macrophages.^{29, 30} To explore this further, we excluded these components from the modified medium, which is devoid of NAC and on the 15th day (24 hours prior to Aβ treatment), the cells were cultured in modified medium without FGF-2 and HBEGF (referred to as Medium 4). FGF-2 has been shown to regulate inflammatory responses by reducing the production of pro-inflammatory cytokines, ²⁸ while HBEGF helps to reduce the secretion of these mediators. Furthermore, HBEGF can act as a protective factor under inflammatory conditions by promoting cell survival and preventing the overall inflammatory response. Astrocytes were maintained in Medium 4 on the 16th and 17th days without the inclusion of growth factors to eliminate their potential inhibitory effects on inflammation, ensuring that even subtle effects of Aβ exposure could be quantified, which further emphasised the impact of these growth factors in modulating the inflammatory response in astrocytes.^{28, 29} The use of the specific medium for culturing astrocytes in this study is critical for maintaining cell viability, promoting growth, and preserving the appropriate cellular responses, such as ER stress. The proposed medium provides the necessary conditions to sustain astrocytes in an optimal state for both maintenance and experimental treatment of Aβ, removing factors that might hinder interpreting results in studies of ER stress, inflammation and Aβ effects on astrocytes.

The significant increase in GFAP expression suggests that astrocytes undergo reactive astrogliosis in response to Aβ exposure. This is consistent with previous reports that have shown that astrocytes become reactive and upregulate GFAP expression in response to neurotoxic stimuli.^31–33 The evaluation of astrocytes by transcripts profile indicated a time-dependent shift in the expression of inflammatory molecules in astrocytes exposed to Aβ. Early time points (3 and 6 hours) showed increased transcript levels of anti-inflammatory molecules such as Ptx3, S100a10, and Sphk1, suggesting an initial protective or regulatory response. However, after 24 hours of Aβ exposure, pro-inflammatory molecules like C3, Iigp1, and Ggta1 were significantly upregulated, indicating a transition towards a pro-inflammatory state in astrocytes that drives neurodegeneration in AD. ⁹ When astrocytes first encounter Aβ, they upregulate anti-inflammatory genes to protect neurons and try to maintain homeostasis. This is possibly a defence mechanism through the release of anti-inflammatory proteins, including cytokines TIMP-1 and ICAM-1 from astrocytes.^{33, 34} However, persistent Aβ aggregation can activate the NLRP3 inflammasome as well as pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β),^35–37 triggering a chronic inflammatory response that shifts from anti-inflammatory to pro-inflammatory signalling, where neuroprotective molecules are reduced.^{20, 21, 38} These findings have important implications for understanding the dynamic role of astrocytes in AD. The early anti-inflammatory response could be an attempt by astrocytes to counteract Aβ-induced damage, but the prolonged exposure appears to push astrocytes toward a pro-inflammatory phenotype, which may contribute to neurodegeneration.^{20, 39, 40}

Conclusion

In conclusion, the serum-free culture conditions established in this study provide a controlled and reproducible model for studying astrocyte responses to Aβ. Our results demonstrate that astrocytes exhibit a time-dependent shift from anti-inflammatory to pro-inflammatory responses in response to Aβ oligomers, highlighting their complex role in neuroinflammation and neurodegeneration. Hence, this unique culture medium, which supports astrocyte health and function, is crucial for investigating ER stress and inflammation pathways together in AD.