Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi:10.21203/rs.3.rs-4694859/v1

Download PDF

Research Article

Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

https://doi.org/10.21203/rs.3.rs-4694859/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Unfolded protein response (UPR) signaling in cells stimulates UPR signaling in adjacent cells, facilitating disease progression by upregulating UPR target genes. However, whether this dissemination occurs between nerve cells and its molecular basis remains unclear. The supernatant of endoplasmic reticulum (ER) stress in rat astrocytes was prepared and treated with rat adrenal medulla pheochromocytoma cells to simulate the propagation of ER stress between nerve cells. The results showed that ER stress may propagate between rat nerve cells, ultimately leading to cell death. It was also found that the mediators mediating ER stress transmission have non-vesicular, oxidative-linked molecules with molecular weights > 100 kD. In conclusion, ER stress propagation might play a significant role in neuronal death following ER stress in central nervous system (CNS) diseases, suggesting novel therapeutic targets for these conditions.

astrocytes

endoplasmic reticulum stress

endoplasmic reticulum stress propagation

neurons

rats

mediators

The endoplasmic reticulum (ER) is one of the organelles of eukaryotic cells and is named after its lumen-like structure (Sun et al., 2024). The ER is mainly responsible for protein synthesis, lipid synthesis, Ca²⁺ storage, and signal transduction (Celik et al., 2023; Zhang et al., 2023). Intracellular and extracellular factors that interfere with any of the homeostatic functions of the ER can induce ER stress (Ghemrawi et al., 2020; Hetz et al., 2020). In response to ER stress, the unfolded protein response (UPR) is induced to overcome the stress and reduce protein synthesis through transcription and translation, increasing ER protein assembly ability, and reducing misfolded or unfolded proteins to overcome stress and recover protein balance and ER homeostasis (Ajoolabady et al., 2023; Li et al., 2023). Long-term ER stress induces chronic UPR activation, which alters body homeostasis even if low-level UPR stimulation protects the cell (Oakes & Papa, 2015; Han et al., 2021; Marciniak et al., 2022). ER stress may propagate between different cells (Hu et al., 2021; Li et al., 2023). Conditioned culture medium from ER-stressed tumor cells may cause ER stress in bone marrow cells, which will then release unknown molecules that help tumor cell growth (Mahadevan et al., 2011). In obesity, ER stress propagates between liver cells and disrupts systemic metabolism (Tirosh et al., 2021).

ER stress is directly related to the pathogenesis of central nervous system (CNS) diseases (Sims et al., 2022). A recent study discovered that strong ER stress markers, such as p-eIF-2α, ATF4, and CHOP, were still present in the cerebral cortex days after a traumatic brain injury (Sun et al., 2022). Another study using rats to observe neuropathic pain has suggested that the spinal dorsal horn has higher levels of ER stress markers, including BIP, p-PERK, p-IRE1α, and ATF6, accompanied by NF-κB phosphorylation (Jiao et al., 2024). However, can ER stress propagate among different types of nerve cells? Can ER stress signals produced by damaged nerve cells propagate to other types of nerve cells, promoting disease progression? In CNS diseases, ER stress signaling transmission may be the basic root cause of neuronal death; however, it's not yet known.

The current research has confirmed that thapsigargin (TG) can successfully induce ER stress in vitro (Je et al., 2023). Therefore, this work intends to explore whether ER stress signals can propagate between different nerve cell types and preliminary explore the possible mediators mediating their propagation. We hypothesized that ER stress may propagate between rat nerve cells, ultimately leading to cell death.

Cell culture

The PC12 cells were grown in a complete medium consisting of 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 U/mL streptomycin. These cells were incubated at 37°C with 5% CO₂ in a cell culture incubator provided by the Chinese Academy of Sciences Stem Cell Bank and CTX (Rat Brain Astrocytes, ASC) (iCell Bioscience Inc.). The cells were passaged when they attained 90% confluence. The growing medium was discarded to initiate passage, and 0.25% trypsin was introduced for digestion. The cells were then collected by pipetting and centrifuged. After removing the supernatant, the cells were resuspended in a fresh culture medium. The cells were evenly distributed into culture bottles and passaged every 3 days (Ni et al., 2022).

ASC ER stress supernatant preparation

The ASCs were treated with 0.5 µmol/L TG (Sigma, USA) for 12 h, and the medium was taken out. The cells were washed three times with serum-free DMEM (Gibco, USA), and then a complete medium was added to continue culturing for 12 h. The cells after 12 h of culture were collected. The culture medium was spun at 2000 × g at 4°C for 20 min to discard cell debris. The supernatant was collected as ASC supernatant (astrocyte conditioned medium, or ACM) and used for subsequent experiments.

Experimental grouping

ASC ER stress validation experiment

The ASC was divided into 4 groups. Untreated group (UT group): Complete medium incubated for 24 h; DMEM + Complete medium group: DMEM incubated for 12 h, complete medium incubated for 12 h; TG + Complete medium group: 0.5 µmol/L TG incubated for 12 h, and complete medium incubated for 12 h; TG group: 0.5 µmol/L TG incubated for 24 h.

ER stress propagation experiment

PC12 cells were divided into 4 groups. UT group: Complete medium incubated for 24 h; Untreated-ACM group, (UT-ACM group): Untreated ACM was incubated for 24 h; TG-ACM group: 0.5 µmol/L TG-treated ACM was incubated for 24 h; TG group: 0.5 µmol/L TG was incubated for 24 h.

ER stress propagation mediator factor-molecular weight size probe experiment

PC12 cells were divided into 8 groups. UT group: Complete medium incubated for 24 h; UT-ACM group: Untreated ACM incubated for 24 h; Untreated-ACM-Filtrates group (UT-ACM-F group): Untreated ACM filtrates incubated for 24 h; Untreated-ACM-Residue group (UT-ACM-R group): Untreated ACM residue incubated for 24 h; TG-ACM group: 0.5 µmol/L TG-treated ACM incubated for 24 h; TG-ACM-Filtrates group (TG-ACM-F group): 0.5 µmol/L TG-treated ACM filtrate incubated for 24 h; TG-ACM-Residue group (TG-ACM-R group): 0.5 µmol/L TG-treated ACM residue incubated for 24 h; TG group: 0.5 µmol/L TG incubated for 24 h.

ER stress propagation mediator factor-vesicle/non-vesicle nature exploration experiment

PC12 cells were divided into 8 groups. UT group: Complete medium incubated for 24 h; UT-ACM group: Untreated ACM incubated for 24 h; UT-ACM-(no) Vesicles group: Untreated de-vesicularized ACM incubated for 24 h; UT-ACM-Vesicles group: Untreated vesicularized ACM incubated for 24 h; TG-ACM group: 0.5 µmol/L TG-treated ACM incubated for 24 h; TG-ACM-(no) Vesicles group: 0.5 µmol/L TG-treated de-vesicularized ACM incubated for 24 h; TG-ACM-Vesicles group: 0.5 µmol/L TG-treated vesicularized ACM incubated for 24 h; TG group: 0.5 µmol/L TG incubated for 24 h.

ER stress propagation mediator factor-oxidative activity probing experiment

PC12 cells were divided into 5 groups. UT group: Complete medium incubated for 24 h; UT-ACM group: Untreated ACM incubated for 24 h; The TG-ACM group: 0.5 µmol/L TG-treated ACM incubated for 24 h; The TG-ACM + Vc group: 0.5 µmol/L TG-treated ACM and supplemented with 100 µmol/L Vc, then incubated for 24 h; The TG group: 0.5 µmol/L TG incubated for 24 h.

qRT-PCR

The RNA was collected using the RNA-easyTM extraction kit (Vazyme, China) following the manufacturer's instructions. Nucleic acid protein method (Eppendorf, Germany) was used to determine the quantity and integrity of the whole RNA. The A260/A280 ratio obtained for RNA was 1.8-2.0, with a concentration of approximately 300 mg/mL. The cDNA was produced using the Quantscript RT Kit (Vazyme, China). The cDNA was then mixed with the SuperReal PreMix Plus kit (SYBR Green, Vazyme, China) for qRT-PCR. The gene amplification primer sequences are indicated in Table 1.

Table 1

Primer sequences
Gene	Sequence (5′–3′)	Size (bp)
β-actin	F-GCTGTGCTATGTTGCCCTAGACTTC R-GGAACCGCTCATTGCCGATAGTG	122
GRP78	F-CGGAGGAGGAGGACAAGAAGGAG R-ATACGACGGTGTGATGCGGTTG	104
CHOP	F-TGGCATCACCTCCTGTCTGTCTC R-CCCTCTCCTTTGGTCTACCCTCAG	95
ATF4	F-CTGCTTGCTCTGTGGTAGATGTCTC R-CTCTGCTGCCTCTAATACGCCATG	107

Western blot

Cells were taken from the previous culture and rinsed twice with PBS. The tissue and cells were cleansed with ice-cold lysis buffer (100 µL RIPA + 1 µL protease inhibitor) (Beyotime, China). Total protein was calculated by nucleic acid protein assay (Eppendorf, Germany). The samples of proteins (100 µg total protein/lane) were weighed and run in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel for 2 h before transferring to polyvinylidene difluoride (PVDF) membrane (Sigma, USA). After blocking the membrane with 5% skim milk for 2 h at room temperature, primary antibodies were incubated overnight at 4°C (Table 2). The membrane was rinsed three times with Tris-buffered saline with Tween 20 (TBST) (Biosharp, China) and then treated with fluorescent secondary antibody (1:5000 dilution) for 1 h. The protein bands were identified by ECL substrate (Vazym, China), and the optical intensity of protein bands was determined by Lane1D.

Table 2

Complete antibody list for all immunofluorescence and western blot
Antibody	Immunogen comment	Manufacturer, Catalog, and RRID	Concentration
β-actin	Human β synthetic peptide immunity of residues near the amino terminus of actin	CST, Cat# 4970, Rabbit mAb, AB_2223172	WB 1:1000
GRP78	Synthetic peptide	Abcam, Cat# ab108615, Rabbit pAb, AB_10890641	WB 1:1000
p-eIF-2α	A synthesized peptide derived from human Phospho-EIF2S1 (S51)	Boster, Cat# BM3942, Rabbit mAb	WB 1:500
ATF-4	A synthesized peptide derived from human ATF4	Boster, Cat# BM5179, Rabbit mAb	WB 1:1000
CHOP	Synthetic peptide immunity of human CHOP sequences	CST, Cat# 2895, Mouse mAb, AB_2089254	WB 1:1000 IF 1:200

Immunofluorescence staining to detect cell PDI expression

The cells were planted in a six-well plate at a density of 5×10⁴/mL. Following treatment, the culture media was detached and washed twice with PBS. The cells were restored with a 4% paraformaldehyde solution for 30 min. After the relocation of the paraformaldehyde, the cells were rinsed three times in PBS for 5 min each. The cells were permeated with 0.1% Triton X-100 for 30 min. The cells were washed three times with PBS for five min each. A CyTM3-labeled secondary antibody (1:200) (Abcam, USA) was introduced and incubated at room temperature for 1 h. DAPI counterstaining was performed, accompanied by rinsing three times with PBS for 20 min each time. Eventually, the cells were placed under a fluorescence microscope (OLYMPUS, Japan), photos were taken, and observations were made.

Apoptosis probe staining to detect cell apoptosis

Cells were introduced on a six-well plate at a density of 5×10⁴/mL. After an intervention, the culture medium was detached and washed three times with PBS. Following that, 1 mL comprising Annexin V-FITC (5 µL) and PI (5 µL) was then transferred to each well (Vazyme, China). The cells were incubated in the dark at 37°C for 15 min and examined with a fluorescence microscope.

Determination of TG in the supernatant using a full-wavelength spectrophotometer

TG has a distinctive absorption peak at 230 nm, referring to the literature (Mahadevan et al., 2011). The samples were added into the cuvette to be tested sequentially and the absorption peak at 230 nm was measured. TG was added to DMEM and measured as a positive control.

ACM filtrate and filter residue preparation

After collecting the ACM, the supernatant was added to an ultrafiltration centrifuge tube containing a 100 K polyethersulfone membrane. The mixture was then centrifuged at 5000 × g for 40 min at 4°C. The ACM was divided into two parts. The lower layer is called ACM filtrate (ACM-F). An equal quantity of culture medium was transferred into the upper layer and diluted for later use. This upper layer is known as the ACM filter residue (ACM-R).

Measurement of the molecular weight of ACM filtrate and filter residue by Coomassie brilliant blue stainings

The ACM was separated from the filtrate residue and then was analyzed by using the SDS-PAGE. To separate the samples, the electrophoresis was performed first at 70 V for 30 min and then increased to 110 V for 1 h until the electrophoresis process was accomplished. Then the gel was stained with 2.5% Coomassie Brilliant Blue dye for 2 h on a shaker following electrophoresis. The gel was finally incubated overnight at ambient temperature in a dissolving solution containing acetic acid (Gao et al., 2019; Zhang et al., 2019).

ACM-vesicle Nanoparticle Size Analysis

Firstly, the ACM was isolated and centrifuged at 2,000×g for 5 minutes at 4°C to acquire the supernatant. Then the sample was spun at 12,000×g and 4°C for 15 minutes to discard cell debris and impurities prior to discarding the pellet. The whole obtained supernatant was ultracentrifuged at 100,000×g for 2 hours. After rinsing with PBS, a second ultracentrifugation was done under the same conditions. The supernatant was removed, and 200 µL of PBS was introduced to resuspend the pellet in the solution. 20 µL of the sample was administered to an equal volume of RIPA lysis buffer and lysed on ice for 1 h to determine the concentration. The vesicles were appropriately diluted and injected into the instrument sample chamber, analyzed, and their particle size was determined through nanoparticle tracking analysis software.

MTT assay to detect changes in cell viability

Cell viability was assessed using MTT (Macklin, China). PC12 cells were seeded in a 96-well plate at a density of 5×10⁴/mL. After treatment, the medium was replaced with an MTT mix (MTT: DMEM = 1:9), and the culture was incubated for 4 h. The MTT mix was then discarded, and 150 µL of DMSO solution was transferred to each well. The 96-well plate was wrapped with tin foil and shaken on an agitator for 10 min. Cell viability was assessed by measuring the optical density (D) value of each well at 490 nm with a microplate reader. Cell survival rate (%) is calculated as (D value of the experimental group – D value of the blank group)/ (D value of the control group – D value of the blank group) × 100%.

Determination of Vc content in supernatant using a full-wavelength spectrophotometer

The 0.01 g of Vc (Sigma, USA) was accurately weighed, diluted in distilled water, and introduced to a 100 mL volumetric flask. The volume was then adjusted to the mark, stirred thoroughly, and set aside. A pipette transferred 50 mL, 40 mL, 30 mL, 20 mL, 10 mL, and 0 mL of the Vc dilution to separate 100 mL volumetric flasks. To adjust the pH to 6, 1% hydrochloric acid and 0.1 mol/L NaOH were used. The solutions were then diluted to the mark using distilled water, shaken well, and the absorbance was measured at 243 nm to draw a standard curve. Finally, the pH of the sample was adjusted, and its absorbance was measured.

Data analysis

Data analysis was executed utilizing GraphPad Prism software (Version 8, GraphPad Software Inc., San Diego, CA, USA). The Shapiro-Wilk test (P > 0.05) was employed to assess the data's normality. An independent student t-test was employed to compare the differences between the two groups. When assuming variance homogeneity, a one-way analysis of variance (ANOVA) was applied to compare differences between three or more groups. Tukey's test was applied for post-hoc comparisons if the assumption of variance uniformity was met. If the assumption of variance uniformity was not met, the Brown-Forsythe test was applied to compare differences between three or more groups, followed by the Games-Howell test for post-hoc testing. Differences were assumed when P < 0.05. All quantitative data are indicated as mean ± standard deviation (mean ± SD).

TG induces endoplasmic reticulum stress in ASCs

As shown in Fig. 1, the qRT-PCR findings indicated that the GRP78, ATF4, and CHOP mRNA expression levels in the TG + Complete medium group significantly enhanced (all P < 0.01) compared to the UT group and DMEM + Complete medium group. Western blot findings indicated that the GRP78, ATF4, CHOP, and p-eIF-2α protein levels in the TG + complete medium group also significantly enhanced (all P < 0.01) than the UT group and DMEM + Complete medium group which confirmed the ER stress in ASCs.

Endoplasmic reticulum stress propagates between ASC and PC12 cells

It was observed under an ordinary light microscope that, compared with the UT group and UT-ACM group, the number of cells in the TG-ACM group and TG group was significantly reduced, the morphology was abnormal, and the cells were damaged. In addition, the qRT-PCR findings indicated that the GRP78, ATF4, and CHOP mRNA expression levels in the TG-ACM group significantly upregulated (all P < 0.01) than the UT group and UT-ACM group. Western blot findings revealed that the protein levels of GRP78, ATF4, CHOP, and p-eIF-2α in the TG-ACM group also significantly upregulated (all P < 0.01) than the UT group and UT-ACM group. The findings of PDI immunofluorescence staining confirmed that the PDI fluorescence intensity of cells in the TG-ACM group and TG group was significantly weakened, and even appeared as granular spots, than the UT group and UT-ACM group, indicating that the ER was severely damaged. Finally, PC12 cells were stained with apoptosis probes. The results showed that the TG-ACM group and TG group accounted for more Annexin V and PI co-stained cells than the UT group and UT-ACM group, indicating endoplasmic reticulum stress. In vitro dissemination can induce cell apoptosis, as shown in Fig. 2.

Mediators mediating endoplasmic reticulum stress propagation-Exploration on molecular weight

As indicated in Fig. 3, the results of the full-wavelength spectrophotometer method showed that compared with the TG + DMEM group, the absorption peaks in the supernatant of the UT-ACM group and TG-ACM group have basically disappeared, indicating that there is basically no TG in the TG-ACM residue. Furthermore, ultrafiltration centrifugation was used to successfully separate the components of the supernatant filtrate and filter residue. Coomassie brilliant blue staining experiments confirmed that the molecular weight of the filtrate was < 100 kD and the molecular weight of the filter residue was > 100 kD. The qRT-PCR results indicated that the GRP78, ATF4, and CHOP mRNA expression levels significantly enhanced in the TG-ACM group and TG-ACM-R group (all P < 0.01) than the UT group.

Study on the vesicular/non-vesicular properties of mediators mediating endoplasmic reticulum stress propagation

As shown in Fig. 4, the nanoparticle size analyzer detected the particle size of the supernatant vesicles, and the results showed that the particle sizes were all around 180 nm. The effects of supernatant vesicle/non-vesicle components on ER stress in PC12 cells were further examined. The qRT-PCR results indicated that GRP78 in the TG-ACM group and TG-ACM-(no) Vesicles group, ATF4, and CHOP mRNA expression levels significantly upregulated (all P < 0.01) than the UT group.

Study on oxidative activity as a mediator of endoplasmic reticulum stress propagation

As shown in Fig. 5, the ordinary light microscope observed that compared with the TG-ACM group and TG group, the number of cells in the TG-ACM + Vc group enhanced, and the morphology was restored. The findings of the MTT experiment observed that the cell viability of the TG-ACM group significantly decreased (P < 0.01) than the UT group, while the cell viability of the TG-ACM + Vc group increased by about 10% than the TG-ACM group (P < 0.01). In addition, immunoblot results indicated that the GRP78, ATF4, and p-eIF-2α protein levels also significantly increased in the TG-ACM group (all P < 0.01) than the UT group, and the GRP78, ATF4, and p-eIF-2α protein levels significantly decreased in the ACM group (P < 0.01 or P < 0.05) than the TG group. Furthermore, the results of PDI immunofluorescence staining confirmed that the PDI fluorescence intensity of cells in the TG-ACM + Vc group was enhanced, and the punctate and granular staining areas were reduced than the TG-ACM group and TG group. Spectrophotometer test results indicated that the Vc concentration in the TG-ACM + Vc group significantly decreased (P < 0.01) than the UT-ACM + Vc group.

Over the past decade, cellular damage secondary to endoplasmic reticulum stress has increasingly been identified as a central factor in the pathophysiology of various diseases (Wang et al., 2022; Guillén-Samander & De-Camilli, 2023; Li et al., 2023). Research shows that endoplasmic reticulum stress may propagate between different cells and aggravate disease progression. For example, ER stress can be propagated from cancer cells to myeloid cells to promote tumor progression or from cardiomyocytes to macrophages, promoting inflammatory infiltration (Hu et al., 2021; Mahadevan et al., 2011; Wei et al., 2019). However, the cause and mechanism of ER stress propagation between CNS cells remain unclear. Therefore, ER stress propagation between CNS cells has become the focus of this study.

ASCs, which are glial cells found in the CNS, have the largest volume and quantity among all regulating glial cells. They are one of the first to detect stress and transmit it to other cells (Frakes et al., 2020; Smith et al., 2020). In the current study, ASCs were administered with TG. The qRT-PCR and western blot findings confirmed that the expression levels of ER stress-related mRNA and protein in ASCs were significantly enhanced, indicating that they experienced endoplasmic reticulum stress. Furthermore, ASC supernatant was obtained and used to treat PC12 cells. We observed that the number of cells in the TG-ACM group was reduced, the morphology was changed, while the expression levels of ER stress-related mRNA and protein were significantly upregulated than the control group. In addition, cell apoptosis was enhanced. This confirmed that ER stress may propagate from ASCs to PC12 cells, leading to cell death, which may be the main cause of neuronal death in CNS diseases.

It is currently unknown which mediator elements facilitate the propagation of ER stress between ASC and PC12 cells. A recent study reported that the macromolecules components mediate the development of ER stress (Sprenkle et al., 2019). In the present study, the supernatant was separated to obtain the filter residue. Experimental results indicated that supernatant residues with a molecular weight > 100 kD may induce ER stress in PC12 cells. Moreover, recent studies have confirmed that extracellular vesicles containing DNA, RNA, proteins, and lipids can be taken up by recipient cells and contribute to disease progression. They could be implicated in mediating the propagation of ER stress (Kisielewska et al., 2024; Fevrier et al., 2004; Yamagata & Freire, 2021; Wu et al., 2021). We used ultracentrifugation to separate vesicles and non-vesicle components in the supernatant. Interestingly, we found that the expression of ER stress-related mRNA was significantly upregulated in the TG-ACM-(no) Vesicles group than the control group, but the TG-ACM-Vesicles group did not induce endoplasmic reticulum stress in PC12 cells. This indicates that the mediators facilitating the propagation of endoplasmic reticulum stress are non-vesicle components.

Studies have shown that cells under sustained stress release oxidative components into the supernatant, which further inhibits their own growth (Jiang et al., 2020; Rodvoldet al., 2017). Our study found that adding Vc to the supernatant partially blocks the role of the supernatant in propagating ER stress. Spectrophotometer detection results showed that Vc is significantly consumed in the supernatant of the TG-ACM + Vc group. Therefore, we speculate that Vc may interact with oxidative substances in the supernatant to block the propagation of ER stress.

This study found that ER stress can propagate from ASC to PC12 cells. The mediators that facilitate the propagation of endoplasmic reticulum stress have non-vesicular, oxidative-linked molecules with molecular weights > 100 kD. However, the specific medium factor remains to be further explored.

Conflict of Interest

All authors recommended ‘No’ competing conflict of interest.

Author Contribution

Yating Ling: Conceptualization, Methodology, Data Collection, Resources, Writing-Original Draf; Muhammad Abid Hayat: Conceptualization, Data Analysis, Supervision, Writing-Original, Review, and Editing; Xiaorui Lv: Software, Methodology; Dongdong Niu: Writing-Review and Editing; Yu Zeng: Writing-Review and Editing; Yun Qiu: Writing-Review and Editing; Bo Chen: Writing-Review and Editing; Jiabo Hu: Conceptualization, Methodology, Resources, Funding acquisition, Supervision, Writing-Review, and Editing. All authors read and approved the final manuscript.

Founding

This study was supported by the National Natural Science Foundation of China (Grant No. 81571221), the Science and Technology Cooperation Foundation of Health Biomed (Grant No. 20200605), and the Qing Lan Project of Jiangsu Province.

Date availability statement

The data are contained within the manuscript.

Ajoolabady A, Kaplowitz N, Lebeaupin C, Kroemer G, Kaufman RJ, Malhi H, Ren J (2023) Endoplasmic reticulum stress in liver diseases. Hepatology 77 (2):619-639. doi:10.1002/hep.32562
Celik C, Lee SYT, Yap WS, Thibault G (2023) Endoplasmic reticulum stress and lipids in health and diseases. Prog Lipid Res 89:101198. doi:10.1016/j.plipres.2022.101198
Fevrier B, Vilette D, Archer F, Loew D, Faigle W, Vidal M, Laude H, Raposo G (2004) Cells release prions in association with exosomes. Proc Natl Acad Sci U S A 101 (26):9683-9688. doi:10.1073/pnas.0308413101
Frakes AE, Metcalf MG, Tronnes SU, Bar-Ziv R, Durieux J, Gildea HK, Kandahari N, Monshietehadi S, Dillin A (2020) Four glial cells regulate ER stress resistance and longevity via neuropeptide signaling in C. elegans. Science 367 (6476):436-440. doi:10.1126/science.aaz6896
Gao J, Zhang L, Wei Y, Chen T, Ji X, Ye K, Yu J, Tang B, Sun X, Hu J (2019) Human hair keratins promote the regeneration of peripheral nerves in a rat sciatic nerve crush model. J Mater Sci Mater Med 30 (7):82. doi:10.1007/s10856-019-6283-1
Ghemrawi R, Khair M (2020) Endoplasmic Reticulum Stress and Unfolded Protein Response in Neurodegenerative Diseases. Int J Mol Sci 21 (17). doi:10.3390/ijms21176127
Guillen-Samander A, De Camilli P (2023) Endoplasmic Reticulum Membrane Contact Sites, Lipid Transport, and Neurodegeneration. Cold Spring Harb Perspect Biol 15 (4). doi:10.1101/cshperspect.a041257
Han Y, Yuan M, Guo YS, Shen XY, Gao ZK, Bi X (2021) Mechanism of Endoplasmic Reticulum Stress in Cerebral Ischemia. Front Cell Neurosci 15:704334. doi:10.3389/fncel.2021.704334
Hetz C, Zhang K, Kaufman RJ (2020) Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol 21 (8):421-438. doi:10.1038/s41580-020-0250-z
Hu X, Duan T, Xiong Y, He Z, Wei W, Cao Z (2021) Intercellular transmission of chronic ER stress: a new mechanism of metabolic diseases. Acta Biochim Biophys Sin (Shanghai) 53 (8):1109-1111. doi:10.1093/abbs/gmab081
Je S, Lee Y, Yamaoka Y (2023) Effect of Common ER Stress-Inducing Drugs on the Growth and Lipid Phenotypes of Chlamydomonas and Arabidopsis. Plant Cell Physiol 64 (4):392-404. doi:10.1093/pcp/pcac154
Jiang Z, Zhang G, Huang L, Yuan Y, Wu C, Li Y (2020) Transmissible Endoplasmic Reticulum Stress: A Novel Perspective on Tumor Immunity. Front Cell Dev Biol 8:846. doi:10.3389/fcell.2020.00846
Jiao B, Zhang W, Zhang C, Zhang K, Cao X, Yu S, Zhang X (2024) Protein tyrosine phosphatase 1B contributes to neuropathic pain by aggravating NF-kappaB and glial cells activation-mediated neuroinflammation via promoting endoplasmic reticulum stress. CNS Neurosci Ther 30 (2):e14609. doi:10.1111/cns.14609
Kisielewska M, Rakoczy K, Skowron I, Gorczynska J, Kacer J, Bochenska A, Choromanska A (2024) Utilizing Extracellular Vesicles for Eliminating 'Unwanted Molecules': Harnessing Nature's Structures in Modern Therapeutic Strategies. Molecules 29 (5). doi:10.3390/molecules29050948
Li T, Zhao H, Guo G, Xia S, Wang L (2023a) VMP1 affects endoplasmic reticulum stress sensitivity via differential modulation of the three unfolded protein response arms. Cell Rep 42 (3):112209. doi:10.1016/j.celrep.2023.112209
Li X, Zhong Y, Yue R, Xie J, Zhang Y, Lin Y, Li H, Xu Y, Zheng D (2023b) Inhibition of MiR-106b-5p mediated by exosomes mitigates acute kidney injury by modulating transmissible endoplasmic reticulum stress and M1 macrophage polarization. J Cell Mol Med 27 (19):2876-2889. doi:10.1111/jcmm.17848
Li Y, Li M, Feng S, Xu Q, Zhang X, Xiong X, Gu L (2024) Ferroptosis and endoplasmic reticulum stress in ischemic stroke. Neural Regen Res 19 (3):611-618. doi:10.4103/1673-5374.380870
Mahadevan NR, Rodvold J, Sepulveda H, Rossi S, Drew AF, Zanetti M (2011) Transmission of endoplasmic reticulum stress and pro-inflammation from tumor cells to myeloid cells. Proc Natl Acad Sci U S A 108 (16):6561-6566. doi:10.1073/pnas.1008942108
Marciniak SJ, Chambers JE, Ron D (2022) Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov 21 (2):115-140. doi:10.1038/s41573-021-00320-3
Ni W, Zhou J, Ling Y, Lu X, Niu D, Zeng Y, Qiu Y, Si Y, Wang J, Zhang W, Wang Z, Hu J (2022) Neural stem cell secretome exerts a protective effect on damaged neuron mitochondria in Parkinson's disease model. Brain Res 1790:147978. doi:10.1016/j.brainres.2022.147978
Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol 10:173-194. doi:10.1146/annurev-pathol-012513-104649
Rodvold JJ, Chiu KT, Hiramatsu N, Nussbacher JK, Galimberti V, Mahadevan NR, Willert K, Lin JH, Zanetti M (2017) Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells. Sci Signal 10 (482). doi:10.1126/scisignal.aah7177
Sims SG, Cisney RN, Lipscomb MM, Meares GP (2022) The role of endoplasmic reticulum stress in astrocytes. Glia 70 (1):5-19. doi:10.1002/glia.24082
Smith HL, Freeman OJ, Butcher AJ, Holmqvist S, Humoud I, Schatzl T, Hughes DT, Verity NC, Swinden DP, Hayes J, de Weerd L, Rowitch DH, Franklin RJM, Mallucci GR (2020) Astrocyte Unfolded Protein Response Induces a Specific Reactivity State that Causes Non-Cell-Autonomous Neuronal Degeneration. Neuron 105 (5):855-866 e855. doi:10.1016/j.neuron.2019.12.014
Sprenkle NT, Lahiri A, Simpkins JW, Meares GP (2019) Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system. J Neurochem 148 (4):516-530. doi:10.1111/jnc.14642
Sun G, Zhao Z, Lang J, Sun B, Zhao Q (2022) Nrf2 loss of function exacerbates endoplasmic reticulum stress-induced apoptosis in TBI mice. Neurosci Lett 770:136400. doi:10.1016/j.neulet.2021.136400
Sun S, Zhao G, Jia M, Jiang Q, Li S, Wang H, Li W, Wang Y, Bian X, Zhao YG, Huang X, Yang G, Cai H, Pastor-Pareja JC, Ge L, Zhang C, Hu J (2024) Stay in touch with the endoplasmic reticulum. Sci China Life Sci 67 (2):230-257. doi:10.1007/s11427-023-2443-9
Tirosh A, Tuncman G, Calay ES, Rathaus M, Ron I, Tirosh A, Yalcin A, Lee YG, Livne R, Ron S, Minsky N, Arruda AP, Hotamisligil GS (2021) Intercellular Transmission of Hepatic ER Stress in Obesity Disrupts Systemic Metabolism. Cell Metab 33 (8):1716. doi:10.1016/j.cmet.2021.07.005
Wang L, Liu Y, Zhang X, Ye Y, Xiong X, Zhang S, Gu L, Jian Z, Wang H (2022) Endoplasmic Reticulum Stress and the Unfolded Protein Response in Cerebral Ischemia/Reperfusion Injury. Front Cell Neurosci 16:864426. doi:10.3389/fncel.2022.864426
Wei C, Yang X, Liu N, Geng J, Tai Y, Sun Z, Mei G, Zhou P, Peng Y, Wang C, Zhang X, Zhang P, Geng Y, Wang Y, Zhang X, Liu X, Zhang Y, Wu F, He X, Zhong H (2019) Tumor Microenvironment Regulation by the Endoplasmic Reticulum Stress Transmission Mediator Golgi Protein 73 in Mice. Hepatology 70 (3):851-870. doi:10.1002/hep.30549
Wu Q, Zhang H, Sun S, Wang L, Sun S (2021) Extracellular vesicles and immunogenic stress in cancer. Cell Death Dis 12 (10):894. doi:10.1038/s41419-021-04171-z
Yamagata AS, Freire PP (2021) Are cachexia-associated tumors transmitTERS of ER stress? Biochem Soc Trans 49 (4):1841-1853. doi:10.1042/BST20210496
Zhang L, Gao J, Chen T, Chen X, Ji X, Ye K, Yu J, Tang B, Wei Y, Xu H, Hu J (2019) Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70. Stem Cells Int 2019:6452684. doi:10.1155/2019/6452684
Zhang SX, Wang JJ, Starr CR, Lee EJ, Park KS, Zhylkibayev A, Medina A, Lin JH, Gorbatyuk M (2024) The endoplasmic reticulum: Homeostasis and crosstalk in retinal health and disease. Prog Retin Eye Res 98:101231. doi:10.1016/j.preteyeres.2023.101231

No competing interests reported.

Download PDF

Editor assigned by journal
24 Aug, 2024
Submission checks completed at journal
16 Aug, 2024
First submitted to journal
05 Jul, 2024

You are reading this latest preprint version

Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture

ASC ER stress supernatant preparation

Experimental grouping

ASC ER stress validation experiment

ER stress propagation experiment

ER stress propagation mediator factor-molecular weight size probe experiment

ER stress propagation mediator factor-vesicle/non-vesicle nature exploration experiment

ER stress propagation mediator factor-oxidative activity probing experiment

qRT-PCR

Western blot

Immunofluorescence staining to detect cell PDI expression

Apoptosis probe staining to detect cell apoptosis

Determination of TG in the supernatant using a full-wavelength spectrophotometer

ACM filtrate and filter residue preparation

ACM-vesicle Nanoparticle Size Analysis

MTT assay to detect changes in cell viability

Determination of Vc content in supernatant using a full-wavelength spectrophotometer

Data analysis

Results

TG induces endoplasmic reticulum stress in ASCs

Endoplasmic reticulum stress propagates between ASC and PC12 cells

Mediators mediating endoplasmic reticulum stress propagation-Exploration on molecular weight

Study on the vesicular/non-vesicular properties of mediators mediating endoplasmic reticulum stress propagation

Study on oxidative activity as a mediator of endoplasmic reticulum stress propagation

Discussion

Declarations

Conflict of Interest

Author Contribution

Date availability statement

References

Additional Declarations

Supplementary Files

Status:

Version 1