Korean J Physiol Pharmacol.  2016 Jul;20(4):387-397. 10.4196/kjpp.2016.20.4.387.

Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis

• Kim SI
• Lee HJ
• Kim SS
• Kwon YS
• Chun W

• KMID: 2371056
• DOI: http://doi.org/10.4196/kjpp.2016.20.4.387

Abstract

Neurofi brillary tangles (NFTs) of microtubule-associated protein tau are a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of AD. However, the exact role of ER stress in tau pathology has not yet been clearly elucidated. In present study, the possible relationship between tau pathology and ER stress was examined in terms of sorcin, which is a calcium binding protein and plays an important role in calcium homeostasis. Our previous yeast two hybrid study showed that sorcin is a novel tau interacting protein. Caspase-3-cleaved tau (T4C3) showed significantly increased tau-sorcin interaction compared to wild type tau (T4). Thapsigargin-induced ER stress and co-expression of constitutively active GSK3β (GSK3β-S9A) also exhibited significantly increased tau-sorcin interactions. T4C3-expressing cells showed potentiated thapsigargin-induced apoptosis and disruption of intracellular calcium homeostasis compared to T4-expressing cells. Overexpression of sorcin signifi cantly attenuated thapsigargin-induced apoptosis and disruption of calcium homeostasis. In contrary, siRNA-mediated knock-down of sorcin showed significantly increased thapsigargin-induced apoptosis and disruption of calcium homeostasis. These data strongly suggest that sequestration of sorcin by aberrant forms of tau compromises the function of sorcin, such as calcium homeostasis and cellular resistance by ER stress, which may consequently result in the contribution to the progression of AD.

Keyword

Alzheimer's disease; Endoplasmic reticulum stress; Sorcin; Tau; Thapsigargin

MeSH Terms

Alzheimer Disease
Apoptosis
Calcium*
Carrier Proteins
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Homeostasis*
Pathology
Thapsigargin
Yeasts
Calcium
Carrier Proteins
Thapsigargin

Figure

• 

  Fig. 1 Tau interacts with sorcin.Representative images (A) and quantitative analysis (B) of immunoprecipitation of tau and sorcin. Intracellular localization of tau and sorcin (C). HEK 293 and SH-SY5Y cells were transfected with T4, T4C3, or vector. After 48 hr of transfection, cells were harvested. Cell lysates were immunoprecipitated with tau or sorcin antibody, and detected with Western blotting with respective antibodies. Vector was used for transfection control and agarose beads control in immunoprecipitation data to conform binding of agarose beads to tau or sorcin. Data were obtained from independent experiments (n=3) and expressed as mean±SD. *p<0.05 indicates significant differences between the indicated groups. To examine the intracellular localization of tau and sorcin, T4 or T4C3 transfected HEK 293 cells were stained with sorcin (red) or tau (green) antibody. Nuclei were visualized by Hoechst staining (blue). Scale bar 10 µm. Vec stands for vector (pcDNA3.1-) and IP for immunoprecipitation.

• 

  Fig. 2 Fibrillogenic tau species preferentially associates with sorcin and increases ER stress induced by thapsigartin compared to wild type tau.Immunoprecipitation of tau and sorcin (A). Quantitative analyses of Tau-sorcin interaction (B) and bip expression (C). HEK 293 cells were co-transfected T4 or T4C3 with GSK3β-S9A or vector, respectively. After 48 hr of transfection, cells were treated 2.5 µM thapsigartin for 24 hr to induce ER stress. Cell lysates were immunoprecipitated with tau or sorcin antibody, and performed Western blotting. BiP was used as an ER stress maker. Data were obtained from independent experiments (n=3) and expressed as mean±SD. *p<0.05 and **p<0.01 indicate significant differences between the indicated groups. Vec stands for vector (pcDNA3.1-), cont for control, Tg for thapsigargin, and IP for immunoprecipitation.

• 

  Fig. 3 Expression of caspase-3 cleaved tau and knock-down of sorcin increase thapsigartin-induced cytotoxicity but over-expression of sorcin decreases thapsigartin-induced cytotoxicity.HEK293 cells were transiently transfected with T4 (A, B) or T4C3 (C, D) constructs in the absence or presence of sorcin overexpression or sorcin siRNA. After 48 hrs of transfection, cells were treated 2.5 µM thapsigartin. Cell viability was measured with MTT assay (A, C) and statistical analyses of cell viability were determined at 48 hr after thapsigartin treatment (B, D). Data were obtained from independent experiments (n=3) and expressed as mean±SD. *p<0.05 and **p<0.01 indicate significant differences between the indicated groups. Vec stands for vector (pcDNA3.1-), Sor for sorcin, Cont for control, and Tg for thapsigargin.

• 

  Fig. 4 Overexpression of sorcin significantly attenuates thapsigartin-induced apoptosis in T4C3 cells.Representative immunoblots (A) of examined proteins and quantitative analyses of cleaved PARP (B) and cleaved caspase-12 (C). HEK293 cells were transiently transfected T4 or T4C3 constructs and then co-transfected with sorcin or vector, respectively. After 48 hrs of transfection, cells were treated 2.5 µM thapsigartin for 24 hr. Western blotting was carried out with cleaved PARP and cleaved caspase-12 antibodies to determine role of sorcin on thapsigartin-induced apoptosis. Data were obtained from independent experiments (n=3) and expressed as mean±SD. *p<0.05 and **p<0.01 indicate significant differences between the indicated groups. Vec stands for vector (pcDNA3.1-), Sor for sorcin, Cont for control, and Tg for thapsigargin.

• 

  Fig. 5 Knock-down of sorcin significantly accelerates thapsigartin-induced apoptosis in T4 cells.Representative immunoblots (A) and quantitative analyses of cleaved PARP (B) and cleaved caspase-12 (C). HEK293 cells were transiently transfected T4 or T4C3 constructs and then co-transfected with sorcin siRNA or control siRNA, respectively. After 48 hr of transfection, cells were treated with 2.5 µM thapsigartin for 24 hr. Western blotting was carried out with cleaved PARP and cleaved caspase-12 antibodies to determine role of sorcin on thapsigartin-induced apoptosis. Data were obtained from independent experiments (n=3) and expressed as mean±SD. **p<0.01 indicates significant differences between the indicated groups. Vec stands for vector Sor for sorcin, Cont for control, and Tg for thapsigargin.

• 

  Fig. 6 Expression of caspse-3-cleaved tau and silencing of sorcin potentiate the thapsigargin-induced perturbation of intracellular calcium homeostasis.Representative image (A) and quantitative analysis (B) of intracellular calcium levels. Cultured cells on coverslips were co-transfected with indicated constructs. After 48 hr transfection, cells were incubated with 2 µM Flou-4AM for 30 min to stain intracellular calcium. Data were obtained from randomly selected 30 cells from three independent experiments (n=3) and expressed as mean±SD. *p<0.05 and **p<0.01 indicate significant differences between the indicated groups. Vec stands for vector (pcDNA3.1-) and Sor for sorcin.

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