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

Vladimir A Vigont,Sergey N Illarioshkin,Lilia D Shuvalova,Sergey A Klyushnikov,Elena V Kaznacheyeva,Anton Yu Skopin,Olga S Lebedeva,Konstantin O Gusev,Ekaterina A Khomyakova,Lyubov N Glushankova,Olga A Zubkova,Dmitriy A Grekhnev,Egor A Volovikov,Alexandra N Bogomazova,Maria A Lagarkova

doi:10.3389/fcell.2021.625231

Abstract

Huntington's disease (HD) is a severe autosomal-dominant neurodegenerative disorder caused by a mutation within a gene, encoding huntingtin protein. Here we have used the induced pluripotent stem cell technology to produce patient-specific terminally differentiated GABA-ergic medium spiny neurons modeling a juvenile form of HD (HD76). We have shown that calcium signaling is dramatically disturbed in HD76 neurons, specifically demonstrating higher levels of store-operated and voltage-gated calcium uptakes. However, comparing the HD76 neurons with the previously described low-repeat HD models, we have demonstrated that the severity of calcium signaling alterations does not depend on the length of the polyglutamine tract of the mutant huntingtin. Here we have also observed greater expression of huntingtin and an activator of store-operated calcium channels STIM2 in HD76 neurons. Since shRNA-mediated suppression of STIM2 decreased store-operated calcium uptake, we have speculated that high expression of STIM2 underlies the excessive entry through store-operated calcium channels in HD pathology. Moreover, a previously described potential anti-HD drug EVP4593 has been found to attenuate high levels of both huntingtin and STIM2 that may contribute to its neuroprotective effect. Our results are fully supportive in favor of the crucial role of calcium signaling deregulation in the HD pathogenesis and indicate that the cornerstone of excessive calcium uptake in HD-specific neurons is a calcium sensor and store-operated calcium channels activator STIM2, which should become a molecular target for medical treatment and novel neuroprotective drug development.

Highlights

Since neurodegenerative disorders are one of the most acute and socially significant problems facing modern medicine, adequate models for these diseases are highly demanded
To differentiate induced pluripotent stem cells (iPSCs) into GABA-ergic medium spiny neurons (MSNs), we used a protocol based on double inhibition of the SMAD cascade, followed by purmorphamine treatment directing cells into lateral ganglionic eminence progenitors (LGE) using purmorphamine, and further maturation of neurons with neurotrophic factors BDNF and GDNF
Immunocytochemical analysis showed that up to 100% of the cells were stained for neuronal marker MAP2 and up to 80% of MAP2-positive cells were stained for DARPP-32, a known GABA MSNs specific marker (Figure 1)

Summary

Introduction

Since neurodegenerative disorders are one of the most acute and socially significant problems facing modern medicine, adequate models for these diseases are highly demanded. Numerous potential drugs have demonstrated a pronounced specific effect on the pathological functioning of calcium signaling (Chen et al, 2011; Wu et al, 2011, 2016; Weber et al, 2019). The mutant huntingtin deregulates calcium signaling by many ways including interactions with mitochondria membranes (Panov et al, 2002; Choo et al, 2004) and calcium-binding proteins (Bao et al, 1996), impact on NMDA receptor trafficking (Fan et al, 2007), changes in the expression of genes responsible for calcium homeostasis (Luthi-Carter et al, 2002; Czeredys et al, 2013; Nekrasov et al, 2016) modulation of voltage-gated calcium channels activity (Silva et al, 2017; Chen et al, 2018). Mutant huntingtin was shown to interact with and potentiate the receptor for inositol-1,4,5-trisphosphate (InsP3R) thereby promoting calcium leakage from endoplasmic reticulum (ER) to cytosol (Tang et al, 2003, 2005)

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