Vladimir Vigont

Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment

Huntingtons disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons

BackgroundHuntington’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.ResultsHere, 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.ConclusionsOur 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.

Pharmacological protein targets in polyglutamine diseases: Mutant polypeptides and their interactors

Polyglutamine diseases are a group of pathologies affecting different parts of the brain and causing dysfunction and atrophy of certain neural cell populations. These diseases stem from mutations in various cellular genes that result in the synthesis of proteins with extended polyglutamine tracts. In particular, this concerns huntingtin, ataxins, and androgen receptor. These mutant proteins can form oligomers, aggregates, and, finally, aggresomes with distinct functions and different degrees of cytotoxicity. In this review, we analyze the effects of different forms of polyQ proteins on other proteins and their functions, which are considered as targets for therapeutic intervention.

Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntingtons 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.

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

Store-operated calcium entry into SK-N-SH human neuroblastoma cells modeling huntington’s disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of polyglutamine at the N-terminus of the huntingtin protein. Striatal medium spiny neurons (MSN) are the primary targets of HD pathology. In our study, a cellular model of HD was based on the human neuroblastoma cells SK-N-SH transfected with plasmid for expression of the mutant huntingtin protein Htt138Q. Expression of Htt138Q increased store-dependent calcium entry into SK-N-SH cells. EVP4593 reversibly blocked the abnormal store-dependent response, probably generated by the channels incorporating TRPC1 ( transient receptor potential canonical 1) subunit.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca2+ channels in non-excitable cells has been studied. Receptor- and store-operated Ca2+ influxes have been measured using the fluorescent method of detection of cytosolic Ca2+ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca2+ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels Imax and non-selective channels Ins in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance Imin channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.

Patient-Specific iPSC-Based Models of Huntington’s Disease as a Tool to Study Store-Operated Calcium Entry Drug Targeting

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, ICRAC and ISOC. 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 ICRAC or ISOC. The potential anti-HD drug EVP4593, which was previously shown to have neuroprotective activity in different HD models, affected both ICRAC and ISOC.

Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

Huntington’s disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca2+ release from the endoplasmic reticulum (ER) induces Ca2+ influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca2+ depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IP3R1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IP3R1 was detected while the ionomycin-sensitive ER Ca2+ pool decreased. 6-Bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (C20H22BrClN2), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP3 sponge also restored the elevation of SOCE and increased the release of Ca2+ from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IP3R1 activity.