Effect of Lithium treatment on SOCE components in Chorea Acanthocytosis

Chorea Acanthocytosis (ChAc) is an autosomal recessive neurodegenerative disease characterized by limb chorea, dystonia, epilepsy and acanthocytosis. The patients have a reduced life expectancy of only around 60 years. This disorder is caused by a loss-of-function mutation of the VPS13A (vacuolar protein sorting-associated protein A) gene, which is the encoding gene of chorein. Normally, chorein stimulates phosphoinositide 3-kinase (PI3K), which is involved in the regulation of Ca2+ influx. Oscillations of intracellular Ca2+ concentration regulate, among other cell components, proliferation, differentiation and apoptosis. Emptying of intracellular Ca2+ stores activates the Store-operated Ca2+ entry (SOCE), which leads to a conformational change and opening of the channel through the interaction between the Ca2+ concentration sensor protein, the Stromal Interacting Molecule 1 (STIM1), and the pore-forming channel, Orai1. The Ca2+ channel is known also as Ca2+ release-activated channel (CRAC). SOCE is increased by lithium via stimulation of serum & glucocorticoid-inducible kinase (SGK1), which is responsible in cells for stimulating the expression of Orai1 and STIM1 as well regulating the transcription factor: Nuclear Factor 'kappa-light-chain-enhancer' of activated B-cells (NFкB). Lithium is used in the treatment of bipolar disorders and can cross the blood-brain barrier (BBB). In this study, fibroblasts and neurons of ChAc patients have been shown to have decreased SOCE and an increased rate of apoptosis. Fibroblasts were isolated from six ChAc patients and six healthy donors. In addition, fibroblasts were obtained from three additional patients and healthy donors and induced pluripotent stem cells (iPSCs) were generated in order to differentiate them into neurons. Western blotting and calcium imaging showed a significant reduction in the amount of Orai1 protein and SOCE, respectively, in fibroblasts and iPSC-differentiated neurons of ChAc patients compared to healthy donors. RT-PCR showed a significant decrease in the mRNA levels of Orai1 and STIM1 in iPSC-differentiated neurons of ChAc patients. Apoptosis was detected by annexin-V / propidium iodide staining via flow cytometry and was in fibroblasts and iPSC-differentiated neurons of ChAc patients significantly higher than in those of healthy donors. In this study, it could be shown that the treatment of fibroblasts and neurons of ChAc patients with lithium positively influenced SOCE, an effect significantly reduced by the Orai1 blocker, 2-aminoethoxy diphenyl borate (2-APB). Lithium induced a significant decrease in apoptosis, an effect again abrogated by 2-APB. The mRNA and protein expression of Orai1 and STIM1 were increased by lithium, an effect reversed by inhibition of SGK1 and NFкB. In conclusion, the apoptotic effect of chorein deficiency in fibroblasts and neurons of ChAc patients is in part due to the decreased expression of Orai1, STIM1 and SOCE. Treatment with lithium could reverse this effect, via the SGK1/NFкB signaling pathway, which shows that lithium could be a new treatment for Chorea Acanthocytosis

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition