Huntington’s disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms. Here, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.

Huntingtons disease (HD) is a severe inherited neurodegenerative disorder characterized by motor dysfunction, cognitive decline, and mental impairment. At the molecular level, HD is caused by a mutation in the first exon of the gene encoding the huntingtin protein. The mutation results in an expanded polyglutamine tract at the N-terminus of the huntingtin protein, causing the neurodegenerative pathology. Calcium dyshomeostasis is believed to be one of the main causes of the disease, which underlies the great interest in the problem among experts in molecular physiology. Recent studies have focused on the development of animal and insect HD models, as well as patient-specific induced pluripotent stem cells (HD-iPSCs), to simulate the diseases progression. Despite a sesquicentennial history of HD studies, the issues of diagnosis and manifestation of the disease have remained topical. The present review addresses these issues.

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntington’s disease (HD) pathological phenotype. In view of recent data suggesting that improper regulation of store-operated calcium (SOC) channels is involved in neurodegenerative processes, we investigated influence of expression of the mutant huntingtin N-terminal fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells (Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from mice. The results show that SOCE in these cells is enhanced upon lentiviral expression of the Htt138Q-1exon. Moreover we demonstrated that RNAi-mediated knockdown of TRPC1, Orai1 or STIM1 proteins leads to dramatic reduction of abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus we concluded that abnormal SOCE in these cells is maintained by both TRPC1- and Orai1-containing channels and required STIM1 for its activation. Furthermore, EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila screening system has proved to be capable of reducing SOCE to the normal level in MSNs expressing the Htt138Q-1exon.

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.

The endoplasmic reticulum calcium sensors STIM1 and STIM2 are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in the norm and pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. It is in only a few studies that STIM2-induced CRAC currents have been recorded. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also TRPC channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, while STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch Imin channels regulation between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca2+ influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels: Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.

Neurodegenerative pathologies are among the most serious and socially significant problems of modern medicine, along with cardiovascular and oncological diseases. Several attempts have been made to prevent neuronal death using novel drugs targeted to the cell calcium signaling machinery, but the lack of adequate models for screening markedly impairs the development of relevant drugs. A potential breakthrough in this field is offered by the models of hereditary neurodegenerative pathologies based on endogenous expression of mutant proteins in neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). Here, we study specific features of store-operated calcium entry (SOCE) using an iPSCs-based model of Huntington’s disease (HD) and analyze the pharmacological effects of a specific drug targeted to the calcium channels. We show that SOCE in gamma aminobutyric acid-ergic striatal medium spiny neurons (GABA MSNs) was mediated by currents through at least two different channel groups, I_CRAC and I_SOC. Both of these groups were upregulated in HD neurons compared with the wild-type neurons. Thapsigargin-induced intracellular calcium store depletion in GABA MSNs resulted in predominant activation of either I_CRAC or I_SOC. The potential anti-HD drug EVP4593, which was previously shown to have neuroprotective activity in different HD models, affected both I_CRAC and I_SOC.