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Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway

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Abstract

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer’s disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP’s induction of Cdk5 activity and potential involvement in neurodegeneration.

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Acknowledgements

The authors thank all the members of the Center for Immunology and Inflammation for their support on this study.

Funding

This work was supported by the National Institutes of Health grants R01 AA028947 (PW, PM) and R35 GM118337 (PW).

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Authors and Affiliations

  1. Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, 350 Community Dr, Manhasset, NY, 11030, USA

    Archna Sharma, Ezgi Sari, Yongchan Lee, Shivani Patel, Max Brenner & Ping Wang

  2. Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Manhasset, NY, 11030, USA

    Archna Sharma, Max Brenner, Philippe Marambaud & Ping Wang

  3. Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Manhasset, NY, 11030, USA

    Max Brenner & Ping Wang

  4. The Litwin-Zucker Center for Alzheimer’s Disease Research, The Feinstein Institutes for Medical Research, Manhasset, NY, 11030, USA

    Philippe Marambaud

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Contributions

AS conceived and designed the experiments and wrote the manuscript; ES and AS conducted all the experiments, acquired, and analyzed the data; SP performed some immunoblots and calpain activity assays, YC performed confocal and some immunofluorescent imaging; PM provided reagents and critical input in some experimental designs; PW, MB and PM critically reviewed the manuscript, and PW supervised the research. All authors read and approved the final manuscript.

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Correspondence to Ping Wang.

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12035_2023_3273_Fig11_ESM.png

Supplementary Fig. 1. eCIRP Transiently Increases Neuronal Calpain 1 not Calpain 2. a N2a cells were treated with vehicle or 2.5 µg/ml eCIRP for 16 h. RNA was isolated from N2a cells and subjected to qPCR. Relative CT values for calpain 1 (Cal 1) (n = 7/group) and calpain 2 (Cal 2) (n = 9/group) mRNA in vehicle treated control N2a cells. Data are means ± SEM compared by Student’s t-test. * p < 0.05 vs. Cal 1. b N2a cells were treated with vehicle or eCIRP at indicated doses for 48 h. Total cell lysates were prepared and subjected to WB. Representative WB images for calpain 1 and actin and bar graphs (n = 4–6/group) from the densitometric analysis of blots are shown. The dotted lines on the WB image reflect different samples from the corresponding groups shown in the bar graph below. Data are means ± SEM compared by ANOVA/Tukey analysis and no significant differences were found. c N2a cells were treated with vehicle or eCIRP at indicated doses for 16 h. Total cell lysates were prepared and subjected to WB. Representative WB images for calpain 2 and actin and bar graphs (n = 6/group) from the densitometric analysis of blots are shown. The dotted lines on the WB image reflect different samples from the corresponding groups shown in the bar graph below. Data are means ± SEM compared by ANOVA/Tukey analysis and differences were not significant (ns) (PNG 179 kb)

High resolution image (TIF 993 kb)

12035_2023_3273_Fig12_ESM.png

Supplementary Fig. 2. eCIRP Induces Intracellular Ca2+ in HT22 Cells in a Dose-Dependent Manner. HT22 cells (n = 12/group) were stimulated with indicated doses of eCIRP or no eCIRP as control in a clear 96 well-black plate. After 3 h the medium is removed, the cells were loaded with Fluo-4 AM dye. a Fluorescence measurement for intracellular Ca2+ levels performed at 485/535 nm. Fluo-4 fluorescence units for control cells was set as 100%. Data are means ± SEM and compared by ANOVA/Tukey analysis. * p < 0.05 vs. no eCIRP and # p < 0.05 vs. eCIRP. b Representative confocal images of the Fluo-4 dye loaded HT22 cells under untreated control, and eCIRP treatment are shown. Scale bar, 100 µm. c Bar graphs from the fluorescence intensity analysis of the images with relative fluorescence units for all HT22 cells in the field are shown. Data are means ± SEM and compared by ANOVA/SNK analysis. * p < 0.05 vs. no eCIRP and # p < 0.05 vs. 0.1 µg/ml eCIRP (PNG 389 kb)

High resolution image (TIF 1916 kb)

12035_2023_3273_Fig13_ESM.png

Supplementary Fig. 3. eCIRP Does not Affect Extracellular Ca2+ Influx. HT22 cells were treated with vehicle or 2.5 μg/ml eCIRP for 3 h. After 3 h the medium is removed, the cells were washed and loaded with Fluo-4 AM dye followed with Ca2+ depletion (0 mM CaCl2) and Ca2+ added back (1.4 mM CaCl2). Fluorescence measurements for intracellular Ca2+ levels were performed at 485/535 nm and data shown as a kinetic graph of relative fluorescence units (RFU) (n = 4/group) expressed as means ± SEM over time for control cells (open black circles with black lines for SEM) and eCIRP treated cells (filled red circles with red lines for SEM). No significant differences were found when compared by ANOVA/Tukey analysis (PNG 178 kb)

High resolution image (TIF 945 kb)

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Sharma, A., Sari, E., Lee, Y. et al. Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway. Mol Neurobiol 60, 3311–3328 (2023). https://doi.org/10.1007/s12035-023-03273-3

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