Pharmacological inhibition of the LIF/LIFR autocrine loop reveals vulnerability of ovarian cancer cells to ferroptosis.

Of all gynecologic cancers, epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts, 90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa, we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover, treatment with LIFR inhibitor, EC359 significantly reduced OCa cell viability and cell survival with an IC50 ranging from 5-50 nM. Furthermore, EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro, ex vivo and in vivo models including cell-based xenografts, patient-derived explants, organoids, and xenograft tumors, we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally, EC359 therapy considerably improved tumor immunogenicity by robust CD45+ leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-, B-, and other immune cells. Collectively, our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa.

Metformin-induced activation of Ca2+ signaling prevents immune infiltration/pathology in Sjogren's syndrome-prone mouse models.

Immune cell infiltration and glandular dysfunction are the hallmarks of autoimmune diseases such as primary Sjogren's syndrome (pSS), however, the mechanism(s) is unknown. Our data show that metformin-treatment induces Ca2+ signaling that restores saliva secretion and prevents immune cell infiltration in the salivary glands of IL14α-transgenic mice (IL14α), which is a model for pSS. Mechanistically, we show that loss of Ca2+ signaling is a major contributing factor, which is restored by metformin treatment, in IL14α mice. Furthermore, the loss of Ca2+ signaling leads to ER stress in salivary glands. Finally, restoration of metformin-induced Ca2+ signaling inhibited the release of alarmins and prevented the activation of ER stress that was essential for immune cell infiltration. These results suggest that loss of metformin-mediated activation of Ca2+ signaling prevents ER stress, which inhibited the release of alarmins that induces immune cell infiltration leading to salivary gland dysfunction observed in pSS.

Assessment of dental student education and preparedness in periodontics in two different models: Discipline-based and general practice-based learning.

PURPOSE: To compare dental students' preparedness in clinical periodontal education following their participation in a discipline-based predoctoral periodontics clinic with dental students that received a general practice-based periodontal education. METHODS: 114 third-year dental students and 112 fourth-year dental students at the University of Texas Health Science Center in San Antonio, were emailed an online survey consisting of questions on their proficiency and confidence levels in diagnosing periodontal diseases and conditions, treatment planning, performing non-surgical periodontal treatment, identifying the need for referral, and their perceived impediments on their clinical periodontics education. RESULTS: Approximately 97% of third-year dental students that participated in discipline-based predoctoral periodontics agree that they are overall confident in providing excellent periodontal care to their patients, and 91% of these students feel that their participation in the predoctoral periodontics clinic has enhanced their knowledge in periodontics as well as their confidence level when providing periodontal patient care. While 95% of fourth-year dental students agree that they feel confident in providing excellent periodontal care to their patients, only 83% felt that level of confidence during their third-year in dental school, and 77% believe that given the option to participate in the pre-doctoral periodontics clinic, their periodontal education would have been strengthened. CONCLUSIONS: Our results show that the integration of a discipline based predoctoral periodontics clinic has benefited the dental students and boosted their knowledge and confidence in diagnosing and treating periodontal patients. This model can be improved by addressing space and time limitations.

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. METHODS: Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed to identify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. RESULTS: We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. CONCLUSIONS: These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Ablation of TRPC3 disrupts Ca2+ signaling in salivary ductal cells and promotes sialolithiasis.

Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca2+) and salivary stone formation, sialolithiasis; however, the process and the mechanism leading to Ca2+ overload during sialolithiasis is not well understood. Here, we show that TRPC3 null (-/-) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3-/-) and patient (sialolithiasis) salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes (BMP2/6, Runx2) in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory (IL6, NLRP3), fibrotic (FN1, TGFß1) and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3-/- leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland stone formation.

Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.

The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-µM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, ß-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2-/- mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.

Inhibition of Ca2+ entry by capsazepine analog CIDD-99 prevents oral squamous carcinoma cell proliferation.

Oral cancer patients have a poor prognosis, with approximately 66% of patients surviving 5-years after diagnosis. Treatments for oral cancer are limited and have many adverse side effects; thus, further studies are needed to develop drugs that are more efficacious. To achieve this objective, we developed CIDD-99, which produces cytotoxic effects in multiple oral squamous cell carcinoma (OSCC) cell lines. While we demonstrated that CIDD-99 induces ER stress and apoptosis in OSCC, the mechanism was unclear. Investigation of the Bcl-family of proteins showed that OSCC cells treated with CIDD-99 undergo downregulation of Bcl-XL and Bcl-2 anti-apoptotic proteins and upregulation of Bax (pro-apoptotic). Importantly, OSCC cells treated with CIDD-99 displayed decreased calcium signaling in a dose and time-dependent manner, suggesting that blockage of calcium signaling is the key mechanism that induces cell death in OSCC. Indeed, CIDD-99 anti-proliferative effects were reversed by the addition of exogenous calcium. Moreover, electrophysiological properties further established that calcium entry was via the non-selective TRPC1 channel and prolonged CIDD-99 incubation inhibited STIM1 expression. CIDD-99 inhibition of calcium signaling also led to ER stress and inhibited mitochondrial complexes II and V in vitro. Taken together, these findings suggest that inhibition of TRPC mediates induction of ER stress and mitochondrial dysfunction as a part of the cellular response to CIDD-99 in OSCC.

Corrigendum: Lipidomic Analysis of TRPC1 Ca2+-Permeable Channel-Knock Out Mouse Demonstrates a Vital Role in Placental Tissue Sphingolipid and Triacylglycerol Homeostasis Under Maternal High-Fat Diet.

[This corrects the article DOI: 10.3389/fendo.2022.854269.].

Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo.

BACKGROUND: Current treatments for salivary gland (SG) hypofunction are palliative and do not address the underlying cause or progression of the disease. SG-derived stem cells have the potential to treat SG hypofunction, but their isolation is challenging, especially when the tissue has been damaged by disease or irradiation for head and neck cancer. In the current study, we test the hypothesis that multipotent bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model are capable of trans-differentiating to the SG epithelial cell lineage when induced by a native SG-specific extracellular matrix (SG-ECM) and thus may be a viable substitute for repairing damaged SGs. METHODS: Rat BM-MSCs were treated with homogenates of decellularized rat SG-ECM for one hour in cell suspension and then cultured in tissue culture plates for 7 days in growth media. By day 7, the cultures contained cell aggregates and a cell monolayer. The cell aggregates were hand-selected under a dissecting microscope, transferred to a new tissue culture dish, and cultured for an additional 7 days in epithelial cell differentiation media. Cell aggregates and cells isolated from the monolayer were evaluated for expression of SG progenitor and epithelial cell specific markers, cell morphology and ultrastructure, and ability to form SG-like organoids in vivo. RESULTS: The results showed that this approach was very effective and guided the trans-differentiation of a subpopulation of CD133-positive BM-MSCs to the SG epithelial cell lineage. These cells expressed amylase, tight junction proteins (Cldn 3 and 10), and markers for SG acinar (Aqp5 and Mist 1) and ductal (Krt 14) cells at both the transcript and protein levels, produced intracellular secretory granules which were morphologically identical to those found in submandibular gland, and formed SG-like organoids when implanted in the renal capsule in vivo. CONCLUSIONS: The results of this study suggest the feasibility of using autologous BM-MSCs as an abundant source of stem cells for treating SG hypofunction and restoring the production of saliva in these patients.

Lipidomic Analysis of TRPC1 Ca2+-Permeable Channel-Knock Out Mouse Demonstrates a Vital Role in Placental Tissue Sphingolipid and Triacylglycerol Homeostasis Under Maternal High-Fat Diet.

The transient receptor potential canonical channel 1 (TRPC1) is a ubiquitous Ca2+-permeable integral membrane protein present in most tissues, including adipose and placenta, and functionally regulates energetic homeostasis. We demonstrated that elimination of TRPC1 in a mouse model increased body adiposity and limited adipose accumulation under a high fat diet (HFD) even under conditions of exercise. Additionally, intracellular Ca2+ regulates membrane lipid content via the activation of the protein kinase C pathway, which may impact placental membrane lipid content and structure. Based upon this we investigated the effect of HFD and TRPC1 elimination on neutral lipids (triacylglycerol and cholesteryl ester), membrane lipids (phosphatidylcholine and phosphatidylethanolamine), and other multifunctional lipid species (unesterified cholesterol, sphingomyelins, ceramides). The concentration of unesterified cholesterol and sphingomyelin increased with gestational age (E12.5 to E 18.5.) indicating possible increases in plasma membrane fluidity. Diet-dependent increases ceramide concentration at E12.5 suggest a pro-inflammatory role for HFD in early gestation. TRPC1-dependent decreases in cholesterol ester concentration with concomitant increases in long-chain polyunsaturated fatty acid -containing triacylglycerols indicate a disruption of neutral lipid homeostasis that may be tied to Ca2+ regulation. These results align with changes in lipid content observed in studies of preeclamptic human placenta.

SARS-CoV-2 infection enhances mitochondrial PTP complex activity to perturb cardiac energetics.

SARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.

Resolving macrophage polarization through distinct Ca2+ entry channel that maintains intracellular signaling and mitochondrial bioenergetics.

Transformation of naive macrophages into classically (M1) or alternatively (M2) activated macrophages regulates the inflammatory response. Here, we identified that distinct Ca2+ entry channels determine the IFNγ-induced M1 or IL-4-induced M2 transition. Naive or M2 macrophages exhibit a robust Ca2+ entry that was dependent on Orai1 channels, whereas the M1 phenotype showed a non-selective TRPC1 current. Blockade of Ca2+ entry suppresses pNF-κB/pJNK/STAT1 or STAT6 signaling events and consequently lowers cytokine production that is essential for M1 or M2 functions. Of importance, LPS stimulation shifted M2 cells from Orai1 toward TRPC1-mediated Ca2+ entry and TRPC1-/- mice exhibited transcriptional changes that suppress pro-inflammatory cytokines. In contrast, Orai1-/- macrophages showed a decrease in anti-inflammatory cytokines and exhibited a suppression of mitochondrial oxygen consumption rate and inhibited mitochondrial shape transition specifically in the M2 cells. Finally, alterations in TRPC1 or Orai1 expression determine macrophage polarization suggesting a distinct role of Ca2+ channels in modulating macrophage transformation.

Increasing cytosolic Ca2+ levels restore cell proliferation and stem cell potency in aged MSCs.

Aging is an inescapable complex physiological but extendable process, and all cells, including stem cells, are altered over time. Diverse mechanism(s) could modulate stem cell number, their proliferation rate, and promote tissue repair during aging that leads to longevity. However, the factors that could restore aging stem cell potency and would lead to healthy aging are not fully identified. Here we show that maintaining cytosolic Ca2+ levels was essential for modulating stem cells function in aged mesenchymal stem cells (MSCs). Increasing external Ca2+ induced spindle shape stem cell morphology and maintained stem cell surface marker expression in aged bone marrow-derived MSCs. Similarly, stem cell survival and proliferation of aged MSCs was dependent on cytosolic Ca2+ levels. Importantly, Ca2+ entry potentiated cell cycle progression, and stem cell potential was increased in cells incubated with higher external Ca2+. Moreover, blocking Ca2+ entry using SKF 96365, decreased stem cell survival and its proliferation but, treatment with 2-APB did not significantly affected cell proliferation, rather only modulated cell viability. Evaluation of Ca2+ entry channels, showed that TRPC1/Orai1/Orai3 and their regulator STIM1 was essential for MSCs proliferation/viability as gene silencing of Orai1/Orai3/TRPC1/STIM1 significantly inhibited stem cell viability. Finally, MSCs isolated from aged mice that were subjected to higher Ca2+ levels, were able to rescue age-induced loss of MSCs function. Together these results suggest that Ca2+ entry is essential for preventing the loss of aged stem cell function and supplementing Ca2+ not only restored their proliferative potential but, allowed them to develop into younger stem cell lineages that could be critical for regenerative medicine.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration.

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Calcium Signaling Regulates Autophagy and Apoptosis.

Calcium (Ca2+) functions as a second messenger that is critical in regulating fundamental physiological functions such as cell growth/development, cell survival, neuronal development and/or the maintenance of cellular functions. The coordination among various proteins/pumps/Ca2+ channels and Ca2+ storage in various organelles is critical in maintaining cytosolic Ca2+ levels that provide the spatial resolution needed for cellular homeostasis. An important regulatory aspect of Ca2+ homeostasis is a store operated Ca2+ entry (SOCE) mechanism that is activated by the depletion of Ca2+ from internal ER stores and has gained much attention for influencing functions in both excitable and non-excitable cells. Ca2+ has been shown to regulate opposing functions such as autophagy, that promote cell survival; on the other hand, Ca2+ also regulates programmed cell death processes such as apoptosis. The functional significance of the TRP/Orai channels has been elaborately studied; however, information on how they can modulate opposing functions and modulate function in excitable and non-excitable cells is limited. Importantly, perturbations in SOCE have been implicated in a spectrum of pathological neurodegenerative conditions. The critical role of autophagy machinery in the pathogenesis of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, would presumably unveil avenues for plausible therapeutic interventions for these diseases. We thus review the role of SOCE-regulated Ca2+ signaling in modulating these diverse functions in stem cell, immune regulation and neuromodulation.

Helminth derived factors inhibit neutrophil extracellular trap formation and inflammation in bacterial peritonitis.

Despite their protective antimicrobial function, neutrophil extracellular traps (NETs) have been implicated in propagation of inflammatory responses in several disease conditions including sepsis. Highly diffusible exogenous ROS produced under such inflammatory conditions, can induce exuberant NETs, thus making inhibition of NETs desirable in inflammatory diseases. Here we report that helminth parasite excretory/secretory factors termed as parasitic ligands (PL) inhibit ROS-induced NETs by blocking the activation of nonselective calcium permeable channel Transient Receptor Potential Melastatin 2 (TRPM2). Therapeutic implication of PL mediated blockage of NET formation was tested in preclinical model of septic peritonitis, where PL treatment regulated neutrophil cell death modalities including NET formation and mitigated neutrophil mediated inflammatory response. This translated into improved survival and reduced systemic and local bacterial load in infected mice. Overall, our results posit PL as an important biological regulator of neutrophil functions with implications to a variety of inflammatory diseases including peritonitis.

Chloride channel accessory 1 integrates chloride channel activity and mTORC1 in aging-related kidney injury.

The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging-related kidney injury, we performed RNA-Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H2 S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H2 S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl- current via the Ca++ -dependent Cl- channel TMEM16A (anoctamin-1) by patch-clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence-associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1-TMEM16A-Cl- current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H2 S.

Calcium channels and their role in regenerative medicine.

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types. Based on their plasticity potential, they are divided into totipotent (morula stage cells), pluripotent (embryonic stem cells), multipotent (hematopoietic stem cells, multipotent adult progenitor stem cells, and mesenchymal stem cells [MSCs]), and unipotent (progenitor cells that differentiate into a single lineage) cells. Though bone marrow is the primary source of multipotent stem cells in adults, other tissues such as adipose tissues, placenta, amniotic fluid, umbilical cord blood, periodontal ligament, and dental pulp also harbor stem cells that can be used for regenerative therapy. In addition, induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells, and thus could be another source for regenerative medicine. Several diseases including neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, virus infection (also coronavirus disease 2019) have limited success with conventional medicine, and stem cell transplantation is assumed to be the best therapy to treat these disorders. Importantly, MSCs, are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair. Moreover, MSCs have the potential to migrate towards the damaged area, which is regulated by various factors and signaling processes. Recent studies have shown that extracellular calcium (Ca2+) promotes the proliferation of MSCs, and thus can assist in transplantation therapy. Ca2+ signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors, Ca2+ channels/pumps/exchangers, Ca2+ buffers, and Ca2+ sensors, which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity, which will be discussed in this review.

Sigma1 Receptor Inhibits TRPC1-Mediated Ca2+ Entry That Promotes Dopaminergic Cell Death.

Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated Ca2+ entry channel in dopaminergic cells, and loss of TRPC1 function leads to neurodegeneration; however, its regulation is not fully identified. Here we provide evidence that the sigma 1 receptor contributes to the loss of dopaminergic cells by blocking TRPC1-mediated Ca2+ entry. Importantly, downregulation of sigma 1 receptor expression significantly decreased neurotoxin-induced loss of dopaminergic cells as measured by MTT assays and caspase activity was also inhibited. Importantly, sigma 1 receptor inhibited TRPC1-mediated Ca2+ entry and silencing of sigma 1 receptor significantly restored store-dependent Ca2+ influx. Although co-immunoprecipitation failed to show an interaction between the TRPC1 and sigma 1 receptor, store depletion promoted a decrease in the sigma 1 receptor-STIM1 association. Neurotoxin-induced loss of Ca2+ entry was significantly restored in cells that had decreased sigma 1 receptor expression. Furthermore, TRPC1 or STIM1 silencing inhibited store-mediated Ca2+ entry, which was further increased upon the downregulation of the sigma 1 receptor expression. TRPC1 silencing prevented the increased neuroprotection and caspase activity observed upon the downregulation of sigma 1 receptor. Finally, sigma 1 receptor activation also significantly decreased TRPC1-mediated Ca2+ entry and lead to an increase in neurodegeneration. In contrast, addition of sigma 1 receptor antagonist prevented neurotoxin-induced neurodegeneration and facilitated TRPC1-mediated Ca2+ influx. Together these results suggest that the sigma 1 receptor is involved in the inhibition of TRPC1- mediated Ca2+ entry, which leads to the degeneration in the dopaminergic cells, and prevention of sigma 1 receptor function could protect neuronal cell death as observed in Parkinson's disease.