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

Abstract

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.

STIM2 Mediates Excessive Store-Operated Calcium Entry in Patient-Specific iPSC-Derived Neurons Modeling a Juvenile Form of Huntington's Disease

“…We showed that STIM2 is upregulated in HD76 neurons and the pre-incubation of HD76 neurons with EVP4593 resulted in lowering the STIM2 level (Figures 4A,B). This result correlates well with the published data, demonstrating the attenuation of both lithium-induced and normal STIM2 expression by application of another NF-κB antagonist wogonin (Sukkar et al, 2018). Then we hypothesized that STIM2 drives the high activity of SOC channels in HD.…”

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes

“…and FGF23 total RNA was extracted according to manufacturer's instructions with TriFast (Peqlab, Erlangen, Germany) [60][61][62][63][64] . DNAse digestion was performed to avoid DNA contamination and was followed by reverse transcription using random hexamers (Promega, Manheim, Germany) and GoScript ™ reverse transcription system (Promega, Manheim, Germany).…”

“…Western blotting. Protein abundance of NFAT5, ORAI1, and GAPDH was determined by Western blotting [60][61][62][63][64] . After isolation of platelets from control donors and patients with impaired kidney function, platelets were centrifuged for 5 minutes at 600 g and 4 °C.…”

“…Calcium measurements in megakaryocytes. To determine cytosolic Ca 2+ concentration ([Ca 2+ ] i ), Fura-2/ AM fluorescence was utilized [60][61][62][63][64][65] . Cells were preincubated for 15-30 minutes with Fura-2/AM (2 µM, Invitrogen, Goettingen, Germany) at 37 °C and excited alternatively at 340 nm and 380 nm in an inverted phase-contrast www.nature.com/scientificreports www.nature.com/scientificreports/ microscope (Axiovert 100, Zeiss, Oberkochen, Germany) through an objective (Fluor 40×/1.30 oil).…”

The Mystery of EVP4593: Perspectives of the Quinazoline-Derived Compound in the Treatment of Huntington’s Disease and Other Human Pathologies

“…On the other hand, it is extremely important that EVP4593 reduces excessive STIM2 levels to control values since it has been reported that downregulation of STIM2 can be dangerous because STIM2-dependent stability of mushroom spines was shown to be a mechanism of hippocampal synaptic loss in a mice model of Alzheimer’s disease [ 46 ]. Curiously, despite the expression of the STIM2 encoding gene, it does not depend on NF-κB; another blocker of NF-κB signaling, wogonin, can also reduce STIM2 levels [ 47 ]. Wogonin also inhibits the mTOR pathway and can have a similar effect as rapamycin on the STIM protein level [ 48 ].…”