An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD.

Dithiadiazole derivative 3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxide - Novel modulator of store-operated calcium entry.

Pathological calcium homeostasis accompanies the development of a large number of different diseases, therefore, the search for new modulators of calcium signaling remains highly actual. Last decades store-operated calcium channels have been repeatedly postulated as a therapeutic target, so the compounds acting on them can be considered promising drug prototypes. Here, we tested several derivatives of 1,2,3,4-dithiadiazole, 1,3-thiazine, pyrazolopyrimidine and thiohydrazides for the ability to affect the thapsigargin-induced calcium response.

Store-Operated Calcium Entry in Mouse Cardiomyocytes.

The fluorescent dye fura-2 AM was employed to record activation of Ca entry in response to a decrease in Ca concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca currents with an amplitude of 0.46±0.

Molecular Pathogenesis in Huntington's Disease.

Huntington's disease (HD) is a severe autosomal dominant neurodegenerative disorder characterized by a combination of motor, cognitive, and psychiatric symptoms, atrophy of the basal ganglia and the cerebral cortex, and inevitably progressive course resulting in death 5-20 years after manifestation of its symptoms. HD is caused by expansion of CAG repeats in the HTT gene, which leads to pathological elongation of the polyglutamine tract within the respective protein - huntingtin. In this review, we present a modern view on molecular biology of HD as a representative of the group of polyglutamine diseases, with an emphasis on conformational changes of mutant huntingtin, disturbances in its cellular processing, and proteolytic stress in degenerating neurons.

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 Ca release from the endoplasmic reticulum (ER) induces Ca influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT.

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).

Homer 1a Induces Calcium Channel Activation, but Does Not Change Their Properties in A431 Cells.

Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells.

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.

Huntington's Disease: Calcium Dyshomeostasis and Pathology Models.

Huntington's 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.

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

Background: 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.

Results: 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.

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 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.

STIM1 Protein Activates Store-Operated Calcium Channels in Cellular Model of Huntington's Disease.

We have shown that the expression of full-length mutated huntingtin in human neuroblastoma cells (SK-N-SH) leads to an abnormal increase in calcium entry through store-operated channels. In this paper, the expression of the N-terminal fragment of mutated huntingtin (Htt138Q-1exon) is shown to be enough to provide an actual model for Huntington's disease. We have shown that Htt138Q-1exon expression causes increased store-operated calcium entry, which is mediated by at least two types of channels in SK-N-SH cells with different reversal potentials.

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.

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.

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

Huntington's 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.