Restoration of Sarco/Endoplasmic Reticulum Ca2+-ATPase Activity Functions as a Pivotal Therapeutic Target of Anti-Glutamate-Induced Excitotoxicity to Attenuate Endoplasmic Reticulum Ca2+ Depletion

Abstract

Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2b) is a membrane-embedded P-type ATPase pump that manages the translocation of calcium ions (Ca²⁺) from cytosol into the lumen of the endoplasmic reticulum (ER) calcium stores. It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca²⁺ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca²⁺ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca²⁺ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca²⁺ leakage attenuated Ca²⁺ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca²⁺-depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS.

Keywords: CDN1163; SERCA2b; calcium depletion; endoplasmic reticulum stress; excitotoxicity; mitochondria.

FIGURE 1

SERCA2 mutants exacerbate hypo-glutamate-induced ERS and cell death in HT22 cells. (A,B): MTT assay, CCK-8 assay (A), and LDH release assay (B) show decreased cellular activity in a glutamate concentration-dependent manner in native HT22 cells. (C,D): CCK-8 assay (C) and LDH assay (D) show SERCA2 mutants exacerbated hypo-glutamate-induced cell death. *p < 0.05 compared with respective 0 mM glutamate stimulation. ^# p > 0.05 compared with respective 0 mM glutamate stimulation. ^& p < 0.05 compared with WT in 1 mM glutamate stimulation, respectively. (E,F): Representative images (E) and summary data (F) show increased cell apoptosis of SRCA2 mutants under hypo-glutamate stimulation by TUNEL staining. *p < 0.05 compared with WT group, respectively. Scale bar, 200 µm. (G,H): Increased expression of ERS marker of indicated proteins (G) and mRNAs (H) under hypo-glutamate stimulation by Western blotting, real-time qPCR, respectively. *p < 0.05 compared with WT group.

FIGURE 2

Declined pump activity and cytoplasmic Ca²⁺ clearance efficiency in SERCA2 mutants. (A) Representative [Ca²⁺]_cyto response of SERCA2 mutants. The addition of glutamate (G), caffeine (C), EGTA (E) is indicated with black arrows. Scale bar, 200 s. Horizontal and vertical red lines represent the level of the [Ca²⁺]_cyto clearance efficiency and [Ca²⁺]_ER storage, respectively. (B) Quantification of [Ca²⁺]_ER storage in response to caffeine. *p < 0.05 compared with WT group, ^# p > 0.05 compared with WT group, respectively. (C) Quantification of [Ca²⁺]_cyto clearance efficiency in response to glutamate. *p < 0.05 compared with WT group, respectively. (D,E): Quantification of SERCA2 activity of mutants when transfected alone (D) or co-transfected (E), SERCA2 mutants were inactive and even inhibited endogenous SERCA2 activity. (D): *p < 0.05 compared with CON group, ^# p > 0.05 compared with CON groups respectively. (E): *p < 0.05 compared with WT group.

FIGURE 3

LOF of SERCA2 is caused by insoluble and low-expression of mutants protein. A- B: Intracellular distribution and expression of SERCA2 in mutant cells. Anti-Flag antibody was used to detect the expression of WT and mutant proteins by immunostaining (A) and Western Blotting (B). L: lactacystin, T/U: Cells were treated with an additional 1% Triton-X100 and ultrasonic fragmentation. Scale bar, 5 µm. (C): Expression level of SERCA2 mRNA by qPCR. ^# p > 0.05 compared with WT group, respectively. (D): Quantification of SERCA2 protein level with or without lactacystin and T/U treatment. *p < 0.05 compared with L (-) treatment cells in each groups, ^# p > 0.05 compared with L (-) treatment cells in each groups, respectively.

FIGURE 4

SERCA2 mutants increase mitochondrial-ER contacts leading to mitochondrial Ca²⁺ overload and dysfunction under hypo-glutamate stimulation. (A): Representative electron micrographs of mitochondria-ER contact SERCA2 mutants. M: mitochondria; red arrows indicate ER. The scale bar is 1 µm (left line), 500 nm (right line). (B) Quantification of the mitochondrial surface percentage in close apposition to the ER. *p < 0.05 compared with WT group. (C): Increasing of the calcium channel protein (IP3R and Mfn2) from specifically isolated MAM fractions in SERCA2 mutants by Western Blotting. (D): Representative normalized fluorescence of mitochondria Ca²⁺ by Rhod-2AM. Scale bar, 200 s. (G): glutamate. (E,F): Decreased mitochondrial membrane potential in SERCA2 mutants. Representative immunostaining (E) and quantification (F) of TMRM fluorescence. *p < 0.05 compared with no-glutamate treatment. ^# p > 0.05 compared with no-glutamate treatment. (G): Increased level of ROS in SRECA2 mutants by DCFH immunostaining. *p < 0.05 compared with WT group.

FIGURE 5

CDN1163 inhibits hypo-glutamate-induced excitotoxicity by reducing SERCA2 mutant-mediated ER Ca²⁺ depletion and stress. (A): Representative normalized fluorescence of cytoplasmic Ca²⁺ by Furo-4 AM. CDN1163 enhanced cytoplasmic Ca²⁺ clearance of SERCA2 mutants. The addition of glutamate (G), caffeine (C), EGTA (E) is indicated with black arrows. Scale bar, 200 s. (B): CDN1163 rescues the cell viability of SERCA2 mutants under hypo-glutamate loading. *p < 0.05 compared with no-CDN1163 treatment, ^# p > 0.05 compared with no-CDN1163 treatment. (C): Low protein level of ERS marker in SERCA2 mutants with CDN1163 treatment by Western blotting.

FIGURE 6

Inhibition of GOF-type pump leakage reduces ER Ca²⁺ depletion and attenuates glutamate-induced excitotoxicity. (A–C): Thapsigargin inhibits pump leakage of G860S mutant. Representative normalized fluorescence of cytoplasmic Ca²⁺ by Furo-4 AM (A). The addition of glutamate (G), caffeine (C), and EGTA (E) is indicated with black arrows. (B): Quantification of the [Ca²⁺]_cyto clearance efficiency in response to glutamate. T: Thapsigargin. (C): Quantification of the [Ca²⁺]_ER storage in response to caffeine. (D): Thapsigargin rescues the cell viability of the G860S mutant under glutamate loading. (B–D)*p < 0.05 compared with no-thapsigargin treatment, ^# p > 0.05 compared with no-thapsigargin treatment.

FIGURE 7

Schematic model depicting the ER Ca²⁺ depletion in response to glutamate-induced excitotoxicity. SERCA2b mutants increase susceptibility to glutamate-induced excitotoxicity by exacerbating ER Ca²⁺ depletion. ER Ca²⁺ depletion leads to increased levels of the [Ca²⁺]_cyto and ER-mitochondria contact, which in turn leads to abnormal activation of many Ca²⁺-dependent pathways, such as ERS, oxidative stress, apoptosis, autophagy, and ultimately promotes cell death. CDN1163 enhances SERCA2b activity and inhibits ER Ca²⁺ depletion, maintaining intracellular Ca²⁺ homeostasis. The affected dysfunctional mitochondria, lysosomes, and ER are highlighted in red.