Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients

Abstract

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca²⁺ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca²⁺ activity ([Ca²⁺]_i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca²⁺]_i following Ca²⁺ re-addition after Ca²⁺-store depletion with sarcoendoplasmatic Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

Neurons, Erythrocytes and Beyond –The Diverse Functions of Chorein

“…Similar observations were made in fibroblasts [37]. Fibroblasts of ChAc patients express less ORAI1 protein than fibroblasts of healthy controls [37].…”

“…3). ORAI1 and STIM1 transcript levels and protein abundance, SOCE and cell survival are all increased by a 24 hours treatment with the antidepressant lithium [12, 37, 75], effects reversed by pharmacological inhibition of serum & glucocorticoid inducible kinase SGK1 or of ORAI1 [12]. Thus, chorein deficiency impairs the SGK1-dependent expression of ORAI1 and STIM1 thus leading to blunted SOCE and enhanced cell death, effects all reversed by lithium [12].…”

“…Fibroblasts of ChAc patients express less ORAI1 protein than fibroblasts of healthy controls [37]. Accordingly, SOCE is decreased and apoptosis enhanced in ChAc fibroblasts as compared to control fibroblasts [37].…”

“…Accordingly, SOCE is decreased and apoptosis enhanced in ChAc fibroblasts as compared to control fibroblasts [37]. Lithium again increases SOCE and pharmacological inhibition of ORAI1 decreases SOCE [37]. Lithium decreases suicidal death of ChAc fibroblasts, an effect abrogated by pharmacological ORAI1 inhibition [37].…”

“…Chorein participates in the regulation of diverse functions [33, 34], including cytoskeletal architecture [33-35], exocytosis [12, 34, 36] and cell survival [12, 37]. …”