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PMCA4 (ATP2B4) mutation in familial spastic paraplegia causes delay in intracellular calcium extrusion.

Ho PW, Pang SY, Li M, Tse ZH, Kung MH, Sham PC, Ho SL - Brain Behav (2015)

Bottom Line: More than 50 disease loci have been described with different modes of inheritance.Further to this finding, here we describe the functional effect of this mutation.Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology, Department of Medicine, University of Hong Kong Hong Kong, China ; Research Centre of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong Hong Kong, China.

ABSTRACT

Background: Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation.

Methods: As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4.

Results: Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin.

Conclusion: Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP.

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Related in: MedlinePlus

(A) Western blot showing stable overexpression of either wild-type (WT) or R268Q mutant ATP2B4 protein at similar level in SH-SY5Y cells. (B) ATP2B4 mRNA levels in WT and mutant stably overexpressing cells were similar as shown by quantitative real-time PCR. ** represents statistical significance at p<0.01 as compared to vector. ns: Not statistically significant.
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fig01: (A) Western blot showing stable overexpression of either wild-type (WT) or R268Q mutant ATP2B4 protein at similar level in SH-SY5Y cells. (B) ATP2B4 mRNA levels in WT and mutant stably overexpressing cells were similar as shown by quantitative real-time PCR. ** represents statistical significance at p<0.01 as compared to vector. ns: Not statistically significant.

Mentions: To assess the cellular functional impact of the p.R268Q mutation on PMCA4, we overexpressed either wild-type or mutant PMCA4 protein in human SH-SY5Y cells and assessed changes in intracellular calcium transient. Overexpression of either wild-type or mutant PMCA4 was confirmed by western blotting using antibodies against PMCA4 and β-actin (Fig.1A), and real-time RT-PCR (Fig.1B). To assess the efficiency of intracellular calcium extrusion, addition of KCl was used to induce membrane depolarization and calcium transient. KCl treatment caused immediate increase in [Ca2+]i in all test groups, as indicated by a rapid increase in OD340/380 ratio under confocal microscopy (Fig.2A). Cells overexpressing either wild-type or mutant PMCA4 had lower maximum [Ca2+]i than empty-vector control (P < 0.01) (Vector controls: 1051.87 ± 54.9 μmol/L; PMCA4(WT): 769.0 ± 16.1 μmol/L; PMCA4(R268Q): 843.9 ± 20.6 μmol/L) (Fig.2B), suggesting that overexpression of either wild-type or mutant PMCA4 both increased calcium extrusion activity. However, R268Q mutant cells demonstrated significantly higher maximum calcium surge compared to wild-type (all P < 0.0001) (Fig.2A). After the initial influx, OD340/380 ratio was gradually restored back towards basal level by plasma membrane calcium transporters. There was no statistical difference in time-dependent decay (t1/2) of calcium surge between cells overexpressing WT (t1/2 = 23.06) and mutant PMCA4 (t1/2 = 22.26), (95% confidence intervals: WT = 22.36–23.80; R268Q mutant = 21.51–23.07; P = 0.1502) based on extra sum-of-squares F test in one phase decay analysis. To further elucidate the contribution of PMCA4 to the calcium transient, we performed additional experiments to measure the steady state [Ca2+]i after incubation with SERCA inhibitor, thapsigargin (TG; 500 nmol/L) (Fig.3A). The steady-state [Ca2+]i in cells overexpressing mutant PMCA4 after exposure to TG was significantly higher than cells overexpressing WT PMCA4 (WT: 732.3 ± 13.0 μmol/L vs. R268Q: 785.4 ± 16.1 μmol/L; P < 0.05; n > 92) (Fig.3B).


PMCA4 (ATP2B4) mutation in familial spastic paraplegia causes delay in intracellular calcium extrusion.

Ho PW, Pang SY, Li M, Tse ZH, Kung MH, Sham PC, Ho SL - Brain Behav (2015)

(A) Western blot showing stable overexpression of either wild-type (WT) or R268Q mutant ATP2B4 protein at similar level in SH-SY5Y cells. (B) ATP2B4 mRNA levels in WT and mutant stably overexpressing cells were similar as shown by quantitative real-time PCR. ** represents statistical significance at p<0.01 as compared to vector. ns: Not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356846&req=5

fig01: (A) Western blot showing stable overexpression of either wild-type (WT) or R268Q mutant ATP2B4 protein at similar level in SH-SY5Y cells. (B) ATP2B4 mRNA levels in WT and mutant stably overexpressing cells were similar as shown by quantitative real-time PCR. ** represents statistical significance at p<0.01 as compared to vector. ns: Not statistically significant.
Mentions: To assess the cellular functional impact of the p.R268Q mutation on PMCA4, we overexpressed either wild-type or mutant PMCA4 protein in human SH-SY5Y cells and assessed changes in intracellular calcium transient. Overexpression of either wild-type or mutant PMCA4 was confirmed by western blotting using antibodies against PMCA4 and β-actin (Fig.1A), and real-time RT-PCR (Fig.1B). To assess the efficiency of intracellular calcium extrusion, addition of KCl was used to induce membrane depolarization and calcium transient. KCl treatment caused immediate increase in [Ca2+]i in all test groups, as indicated by a rapid increase in OD340/380 ratio under confocal microscopy (Fig.2A). Cells overexpressing either wild-type or mutant PMCA4 had lower maximum [Ca2+]i than empty-vector control (P < 0.01) (Vector controls: 1051.87 ± 54.9 μmol/L; PMCA4(WT): 769.0 ± 16.1 μmol/L; PMCA4(R268Q): 843.9 ± 20.6 μmol/L) (Fig.2B), suggesting that overexpression of either wild-type or mutant PMCA4 both increased calcium extrusion activity. However, R268Q mutant cells demonstrated significantly higher maximum calcium surge compared to wild-type (all P < 0.0001) (Fig.2A). After the initial influx, OD340/380 ratio was gradually restored back towards basal level by plasma membrane calcium transporters. There was no statistical difference in time-dependent decay (t1/2) of calcium surge between cells overexpressing WT (t1/2 = 23.06) and mutant PMCA4 (t1/2 = 22.26), (95% confidence intervals: WT = 22.36–23.80; R268Q mutant = 21.51–23.07; P = 0.1502) based on extra sum-of-squares F test in one phase decay analysis. To further elucidate the contribution of PMCA4 to the calcium transient, we performed additional experiments to measure the steady state [Ca2+]i after incubation with SERCA inhibitor, thapsigargin (TG; 500 nmol/L) (Fig.3A). The steady-state [Ca2+]i in cells overexpressing mutant PMCA4 after exposure to TG was significantly higher than cells overexpressing WT PMCA4 (WT: 732.3 ± 13.0 μmol/L vs. R268Q: 785.4 ± 16.1 μmol/L; P < 0.05; n > 92) (Fig.3B).

Bottom Line: More than 50 disease loci have been described with different modes of inheritance.Further to this finding, here we describe the functional effect of this mutation.Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology, Department of Medicine, University of Hong Kong Hong Kong, China ; Research Centre of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong Hong Kong, China.

ABSTRACT

Background: Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation.

Methods: As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4.

Results: Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin.

Conclusion: Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP.

Show MeSH
Related in: MedlinePlus