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Published on: November 7, 2012
PMCA4 (ATP2B4) mutation in familial spastic paraplegia causes delay in intracellular calcium extrusion
Philip Wing-Lok Ho1, Shirley Yin-Yu Pang2, Miaoxin Li3
1Division 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.
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.
Insights
A novel mutation in the plasma membrane calcium ATPase (PMCA4) gene impairs calcium regulation in neuronal cells. This calcium dysregulation may contribute to the development of familial spastic paraplegia (FSP).
Area of Science:
- Neuroscience
- Genetics
- Cell Biology
Background:
- Familial spastic paraplegia (FSP) encompasses diverse inherited disorders causing progressive lower limb spasticity and weakness.
- Over 50 genetic loci are linked to FSP, with various inheritance patterns.
- A specific missense mutation (p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene was identified in a Chinese family with autosomal dominant FSP.
Purpose of the Study:
- To investigate the functional consequences of the p.R268Q mutation in the PMCA4 gene.
- To determine the role of PMCA4 dysfunction in neuronal calcium homeostasis.
- To explore the potential link between PMCA4 mutations and the pathogenesis of FSP.
Main Methods:
- Utilized fura-2 fluorescent dye and confocal microscopy to visualize cytosolic calcium levels in human SH-SY5Y neuroblastoma cells.
- Compared calcium transients and decay in cells overexpressing wild-type PMCA4, R268Q mutant PMCA4, or a vector control.
- Assessed the impact of PMCA4 variants on calcium handling following KCl-induced depolarization and SERCA inhibition by thapsigargin.
Main Results:
- Overexpression of both wild-type and mutant PMCA4 reduced calcium surge post-depolarization compared to controls.
- Cells expressing the R268Q mutant PMCA4 exhibited a significantly higher calcium surge than those with wild-type PMCA4.
- Mutant PMCA4-expressing cells showed persistently elevated steady-state cytosolic calcium levels after SERCA inhibition.
Conclusions:
- The p.R268Q mutation in PMCA4 leads to altered calcium homeostasis in human neuronal cells.
- Dysregulation of calcium signaling pathways is implicated in the pathogenesis of FSP.
- This study highlights the critical role of PMCA4 in neuronal calcium regulation and FSP.
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