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.

Direct Outreach in Bars and Clubs to Enroll Cigarette Smokers in Mobile Cessation Services: Exploratory Study.

BACKGROUND: Cigarette smoking and alcohol use are well known to be concomitant behaviors, but there is a lack of studies related to recruitment of smokers for mobile cessation services at places where alcohol is consumed, such as bars and clubs. Adapting recruitment strategies to expand the reach of cessation programs to where tobacco users are located may help decrease the health-equity gap in tobacco control by improving reach and enrollment of underserved smokers residing in low-income and rural areas who are not reached by traditional cessation services. OBJECTIVE: The purpose of this exploratory study was to assess the feasibility of direct outreach in bars, clubs, and restaurants to recruit smokers to Quitxt, our mobile smoking cessation service. Quitxt is delivered through SMS text messaging or Facebook Messenger. METHODS: We collaborated with an advertising agency to conduct in-person recruitment of young adult smokers aged 18-29 years, focusing on urban and rural Spanish-speaking Latino participants, as well as English-speaking rural White and African American participants. Street team members were recruited and trained in a 4-hour session, including a brief introduction to the public health impacts of cigarette smoking and the aims of the project. The street teams made direct, face-to-face contact with smokers in and near smoking areas at 25 bars, clubs, and other venues frequented by young smokers in urban San Antonio and nearby rural areas. RESULTS: The 3923 interactions by the street teams produced 335 (8.5%) program enrollments. Most participants were English speakers with a mean age of 29.2 (SD 10.6) years and smoked a mean of 8.5 (SD 6.2) cigarettes per day. Among users who responded to questions on gender and ethnicity, 66% (70/106) were women and 56% (60/107) were Hispanic/Latino. Among users ready to make a quit attempt, 22% (17/77) reported 1 tobacco-free day and 16% (10/62) reported maintaining cessation to achieve 1 week without smoking. The response rate to later follow-up questions was low. CONCLUSIONS: Direct outreach in bars and clubs is a useful method for connecting young adult cigarette smokers with mobile cessation services. However, further research is needed to learn more about how mobile services can influence long-term smoking cessation among those recruited through direct outreach, as well as to test the use of incentives in obtaining more useful response rates.

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.

Using Tweetchats to Build Community Awareness and Advocacy around Alzheimer's Disease for Latinos.

U.S. Latinos are 1.5 times more likely to develop Alzheimer's disease (AD) than non-Latino Whites. To raise awareness of and action around this rising public health issue, Salud America!, a national Latino health advocacy network, organized three #SaludTues tweetchats on Twitter between 2018 and 2020. For the three Alzheimer's tweetchats ─Aug. 14, 2018, June 6, 2019, and Oct. 6, 2020─Salud America! partnered with global groups that advocate for AD solutions in Latino and other communities. We analyzed the three tweetchats' #Saludtues hashtag usage, participant demographics, and other metrics using Symplur analytics software. For the first tweetchat in 2018, there were 579 tweets with a total of 3.89 million impressions; the second tweetchat in 2019 had a bigger impact with 704 tweets with 5.72 million impressions; the third tweetchat had the biggest impact with 932 tweets and 6.62 million impressions. Most tweetchat participants were from states with large Latino populations, and most tweets indicated positive sentiment related to increasing awareness of solutions to AD issues among Latinos. The three Alzheimer's-focused #SaludTues tweetchats particularly served as unique testing grounds for the fast dissemination and increasingly exposed many people to the issue of AD and the need to advocate for the Latino community.

Use of #SaludTues Tweetchats for the Dissemination of Culturally Relevant Information on Latino Health Equity: Exploratory Case Study.

BACKGROUND: Latinx people comprise 18% of the US adult population and a large share of youth and continue to experience inequities that perpetuate health disparities. To engage Latinx people in advocacy for health equity based on this population's heavy share of smartphone, social media, and Twitter users, Salud America! launched the #SaludTues Tweetchat series. In this paper, we explore the use of #SaludTues to promote advocacy for Latinx health equity. OBJECTIVE: This study aims to understand how #SaludTues Tweetchats are used to promote dissemination of culturally relevant information on social determinants of health, to determine whether tweetchats serve to drive web traffic to the Salud America! website, and to understand who participates in #SaludTues Tweetchats and what we can learn about the participants. We also aim to share our own experiences and present a step-by-step guide of how tweetchats are planned, developed, promoted, and executed. METHODS: We explored tweetchat data collected between 2014 and 2018 using Symplur and Google Analytics to identify groups of stakeholders and web traffic. Network analysis and mapping tools were also used to derive insights from this series of chats. RESULTS: We conducted 187 chats with 24,609 reported users, 177,466 tweets, and more than 1.87 billion impressions using the hashtag #SaludTues during this span, demonstrating effective dissemination of and exposure to culturally relevant information. Traffic to the Salud America! website was higher on Tuesdays than any other day of the week, suggesting that #SaludTues Tweetchats acted effectively as a website traffic-driving tool. Most participants came from advocacy organizations (165/1000, 16.5%) and other health care-related organizations (162/1000, 16.2%), whereas others were unknown users (147/1000, 14.7%) and individual users outside of the health care sector (117/1000, 11.7%). The majority of participants were located in Texas, California, New York, and Florida, all states with high Latinx populations. CONCLUSIONS: Carefully planned, culturally relevant tweetchats such as #SaludTues can be a powerful tool for public health practitioners and advocates to engage audiences on Twitter around health issues, advocacy, and policy solutions for Latino health equity. Further information is needed to determine the effect that #SaludTues Tweetchats have on self- and collective efficacy for advocacy in the area of Latino health equity.

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.

Magnesium-Induced Cell Survival Is Dependent on TRPM7 Expression and Function.

Mg2+ homeostasis is essential for cell survival and the loss of this regulation has been associated with many neurodegenerative diseases, including loss of dopaminergic neurons. Although the neurotoxin-mediated loss of dopaminergic neurons in Parkinson disease models is extensively studied, the ion channel(s) that regulate Mg2+ homeostasis and thus could prevent neuronal cell death is not yet identified. Here, we show that TRPM7 (transient receptor potential melastatin 7) is involved in regulating Mg2+ homeostasis in dopaminergic cells. Importantly, transient loss of TRPM7 decreased intracellular Mg2+ levels and decreased dopaminergic cells/neurons survival. We provide further evidence that both increases in extracellular Mg2+ or transiently increasing TRPM7 levels protected dopaminergic SH-SY5Y cells against neurotoxin-mediated cell death. Neurotoxin treatment significantly decreased TRPM7 levels in both SH-SY5Y cells and the substantia nigra pars compacta region of mice, along with a decrease in Mg2+ influx. Moreover, Mg2+ supplementation showed a concentration-dependent decrease in caspase-3 activity, an increase in cell survival, restored mitochondrial membrane potential, and increase TRPM7 levels in neurotoxin-treated cells. In contrast, transient silencing of TRPM7 inhibited the positive effect of Mg2+ supplementation in protecting against neurotoxins. Whereas, TRPM7 overexpression not only maintained Mg2+ homeostasis but also inhibited caspase 3 activity that induced cell survival. Overall, these results suggest a significant role of TRPM7 channels in Mg2+ homeostasis and the survival of neurotoxin-induced loss of dopaminergic cells.

Ca2+ entry via TRPC1 is essential for cellular differentiation and modulates secretion via the SNARE complex.

Properties of adipocytes, including differentiation and adipokine secretion, are crucial factors in obesity-associated metabolic syndrome. Here, we provide evidence that Ca2+ influx in primary adipocytes, especially upon Ca2+ store depletion, plays an important role in adipocyte differentiation, functionality and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was found to be dependent on TRPC1-STIM1, and blocking Ca2+ entry with SKF96365 or using TRPC1-/- knockdown adipocytes inhibited adipocyte differentiation. Additionally, TRPC1-/- mice have decreased organ weight, but increased adipose deposition and reduced serum adiponectin and leptin concentrations, without affecting total adipokine expression. Mechanistically, TRPC1-mediated Ca2+ entry regulated SNARE complex formation, and agonist-mediated secretion of adipokine-loaded vesicles was inhibited in TRPC1-/- adipose. These results suggest an unequivocal role of TRPC1 in adipocyte differentiation and adiponectin secretion, and that loss of TRPC1 disturbs metabolic homeostasis.This article has an associated First Person interview with the first author of the paper.

TRPC1 expression and function inhibit ER stress and cell death in salivary gland cells.

Disturbances in endoplasmic reticulum (ER) Ca2+ homeostasis have been associated with many diseases including loss of salivary glands. Although significant progress has been accomplished which led to the increase in our understanding of the cellular responses to ER stress, the factors/ion channels that could inhibit ER stress are not yet identified. Here we show that TRPC1 (transient receptor potential canonical 1) is involved in regulating Ca2+ homeostasis and loss of TRPC1 decreased ER Ca2+ levels, inhibited the unfolded protein response (UPR), that induced loss of salivary gland cells. We provide further evidence that ER stress inducing agents (Tunicamycin and Brefeldin A) disrupts Ca2+ homeostasis by directly inhibiting TRPC1-mediated Ca2+ entry, which led to ER stress in salivary gland cells. Moreover, induction of ER stress lead to an increase in CHOP expression, which decreased TRPC1 expression and subsequently attenuated autophagy along with increased apoptosis. Importantly, TRPC1-/- mice showed increased ER stress, increased immune cell infiltration, loss of Ca2+ homeostasis, decreased saliva secretion, and decreased salivary gland survival. Finally, restoration of TRPC1 not only maintained Ca2+ homeostasis, but inhibited ER stress that induced cell survival. Overall these results suggest a significant role of TRPC1 Ca2+ channels in ER stress and homeostatic function/survival of salivary gland cells.

M1 Macrophage Polarization Is Dependent on TRPC1-Mediated Calcium Entry.

Macrophage plasticity is essential for innate immunity, but in-depth signaling mechanism(s) regulating their functional phenotypes are ill-defined. Here we report that interferon (IFN) γ priming of naive macrophages induces store-mediated Ca2+ entry and inhibition of Ca2+ entry impairs polarization to M1 inflammatory phenotype. In vitro and in vivo functional analyses revealed ORAI1 to be a primary contributor to basal Ca2+ influx in macrophages, whereas IFNγ-induced Ca2+ influx was mediated by TRPC1. Deficiency of TRPC1 displayed abrogated IFNγ-induced M1 inflammatory mediators in macrophages. In a preclinical model of peritonitis by Klebsiella pneumoniae infection, macrophages showed increased Ca2+ influx, which was TRPC1 dependent. Macrophages from infected TRPC1-/- mice showed inhibited expression of M1-associated signature molecules. Furthermore, in human patients with systemic inflammatory response syndrome, the level of TRPC1 expression in circulating macrophages directly correlated with M1 inflammatory mediators. Overall, TRPC1-mediated Ca2+ influx is essential for the induction/shaping of macrophage polarization to M1 inflammatory phenotype.

Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H2O2) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H2O2. This TRPM2-dependent H2O2-mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2-/- mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2-/- mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

TGFß-induced epithelial-to-mesenchymal transition in prostate cancer cells is mediated via TRPM7 expression.

Growth factors, such as the transforming growth factor beta (TGFß), play an important role in promoting metastasis of prostate cancer, thus understanding how TGFß could induce prostate cancer cell migration may enable us to develop targeted strategies for treatment of advanced metastatic prostate cancer. To more clearly define the mechanism(s) involved in prostate cancer cell migration, we undertook a series of studies utilizing non-malignant prostate epithelial cells RWPE1 and prostate cancer DU145 and PC3 cells. Our studies show that increased cell migration was observed in prostate cancer cells, which was mediated through epithelial-to-mesenchymal transition (EMT). Importantly, addition of Mg2+ , but not Ca2+ , increased cell migration. Furthermore, TRPM7 expression, which functions as an Mg2+ influx channel, was also increased in prostate cancer cells. Inhibition of TRPM7 currents by 2-APB, significantly blocked cell migration in both DU145 and PC3 cells. Addition of growth factor TGFß showed a further increase in cell migration, which was again blocked by the addition of 2-APB. Importantly, TGFß addition also significantly increased TRPM7 expression and function, and silencing of TRPM7 negated TGFß-induced cell migration along with a decrease in EMT markers showing loss of cell adhesion. Furthermore, resveratrol, which decreases prostate cancer cell migration, inhibited TRPM7 expression and function including TGFß-induced cell migration and activation of TRPM7 function. Together, these results suggest that Mg2+ influx via TRPM7 promotes cell migration by inducing EMT in prostate cancer cells and resveratrol negatively modulates TRPM7 function thereby inhibiting prostate cancer metastasis.

TRPM2 Promotes Neurotoxin MPP+/MPTP-Induced Cell Death.

In neurons, Ca2+ is essential for a variety of physiological processes that regulate gene transcription to neuronal growth and their survival. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ions (MPP+) are potent neurotoxins that selectively destroys the dopaminergic (DA) neurons and mimics Parkinson's disease (PD) like symptoms, but the mechanism as how MPP+/MPTP effects DA neuron survival is not well-understood. In the present study, we found that MPP+ treatment increased the level of reactive oxygen species (ROS) that activates and upregulates the expression and function of melastatin-like transient receptor potential (TRPM) subfamily member, melastatin-like transient receptor potential channel 2 (TRPM2). Correspondingly, TRPM2 expression was also increased in substantia nigra of MPTP-induced PD mouse model and PD patients. ROS-mediated activation of TRPM2 resulted in an increased intracellular Ca2+, which in turn promoted cell death in SH-SY5Y cells. Intracellular Ca2+ overload caused by MPP+-induced ROS also affected calpain activity, followed by increased caspase 3 activities and activation of downstream apoptotic pathway. On the other hand, quenching of H2O2 by antioxidants, resveratrol (RSV), or N-acetylcysteine (NAC) effectively blocked TRPM2-mediated Ca2+ influx, decreased intracellular Ca2+ overload, and increased cell survival. Importantly, pharmacological inhibition of TRPM2 or knockdown of TRPM2 using siRNA, but not control siRNA, showed an increased protection by preventing MPP+-induced Ca2+ increase and inhibited apoptosis. Taken together, we show here a novel role for TRPM2 expression and function in MPP+-induced dopaminergic neuronal cell death.

Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ions (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1. Prolonged neurotoxin treatment attenuated the binding of NF-κB to the TRPC1 promoter, which resulted in a decrease in TRPC1 expression, thereby attenuating autophagy and activating cell death. Restoration of TRPC1 expression rescued the effects of the dopaminergic neurotoxins in neuroblastoma cells by increasing Ca2+ entry, restoring NF-κB activity, and promoting autophagy. Overall, these results suggest that dopaminergic neurotoxins initially decreased Ca2+ entry, which inhibited the binding of NF-κB to the TRPC1 promoter, thereby inhibiting TRPC1 expression and resulting in cell death by preventing autophagy.-Sukumaran, P., Sun, Y., Antonson, N., Singh, B. B. Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

The TRPC1 Ca2+-permeable channel inhibits exercise-induced protection against high-fat diet-induced obesity and type II diabetes.

The transient receptor potential canonical channel-1 (TRPC1) is a Ca2+-permeable channel found in key metabolic organs and tissues, including the hypothalamus, adipose tissue, and skeletal muscle. Loss of TRPC1 may alter the regulation of cellular energy metabolism resulting in insulin resistance thereby leading to diabetes. Exercise reduces insulin resistance, but it is not known whether TRPC1 is involved in exercise-induced insulin sensitivity. The role of TRPC1 in adiposity and obesity-associated metabolic diseases has not yet been determined. Our results show that TRPC1 functions as a major Ca2+ entry channel in adipocytes. We have also shown that fat mass and fasting glucose concentrations were lower in TRPC1 KO mice that were fed a high-fat (HF) (45% fat) diet and exercised as compared with WT mice fed a HF diet and exercised. Adipocyte numbers were decreased in both subcutaneous and visceral adipose tissue of TRPC1 KO mice fed a HF diet and exercised. Finally, autophagy markers were decreased and apoptosis markers increased in TRPC1 KO mice fed a HF diet and exercised. Overall, these findings suggest that TRPC1 plays an important role in the regulation of adiposity via autophagy and apoptosis and that TRPC1 inhibits the positive effect of exercise on type II diabetes risk under a HF diet-induced obesity environment.

TRPC Channels and Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder, which involves degeneration of dopaminergic neurons that are present in the substantia nigra pars compacta (SNpc) region. Many factors have been identified that could lead to Parkinson's disease; however, almost all of them are directly or indirectly dependent on Ca2+ signaling. Importantly, though disturbances in Ca2+ homeostasis have been implicated in Parkinson's disease and other neuronal diseases, the identity of the calcium channel remains elusive. Members of the transient receptor potential canonical (TRPC) channel family have been identified as a new class of Ca2+ channels, and it could be anticipated that these channels could play important roles in neurodegenerative diseases, especially in PD. Thus, in this chapter we have entirely focused on TRPC channels and elucidated its role in PD.

Inhibition of L-Type Ca2+ Channels by TRPC1-STIM1 Complex Is Essential for the Protection of Dopaminergic Neurons.

Loss of dopaminergic (DA) neurons leads to Parkinson's disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca2+ channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1-/-, mice. Similarly, TRPC1-/- substantia nigra neurons showed increased L-type Ca2+ currents, decreased stimulation-dependent STIM1-Cav1.3 interaction, and decreased DA neurons. L-type Ca2+ currents and the open channel probability of Cav1.3 channels were also reduced upon TRPC1 activation, whereas increased Cav1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Cav1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, which was prevented by inhibiting L-type Ca2+ channels. Neurotoxins that mimic Parkinson's disease increased Cav1.3 function, decreased TRPC1 expression, inhibited Tg-mediated STIM1-Cav1.3 interaction, and induced caspase activation. Importantly, restoration of TRPC1 expression not only inhibited Cav1.3 function but increased cell survival. Together, we provide evidence that TRPC1 suppresses Cav1.3 activity by providing an STIM1-based scaffold, which is essential for DA neuron survival.SIGNIFICANCE STATEMENT Ca2+ entry serves critical cellular functions in virtually every cell type, and appropriate regulation of Ca2+ in neurons is essential for proper function. In Parkinson's disease, DA neurons are specifically degenerated, but the mechanism is not known. Unlike other neurons, DA neurons depend on Cav1.3 channels for their rhythmic activity. Our studies show that, in normal conditions, the pacemaking activity in DA neurons is inhibited by the TRPC1-STIM1 complex. Neurotoxins that mimic Parkinson's disease target TRPC1 expression, which leads to an abnormal increase in Cav1.3 activity, thereby causing degeneration of DA neurons. These findings link TRPC1 to Cav1.3 regulation and provide important indications about how disrupting Ca2+ balance could have a direct implication in the treatment of Parkinson's patients.

TRPC1-STIM1 activation modulates transforming growth factor ß-induced epithelial-to-mesenchymal transition.

Activation of Epithelial-to-Mesenchymal Transition (EMT) is important for tumor metastasis. Although growth factors such as TGFß and EGF have been shown to induce EMT in breast epithelial cells, the mechanism resulting in migration is not well understood. Herein, we provide evidence that Ca2+ entry into the cell, especially upon store-depletion, plays an important role in TGFß-induced EMT by promoting cellular migration and potentially leading to metastasis. The increased migration by TGFß in non-cancerous cells was due to the loss of E-cadherin along with a subsequent increase in N-cadherin levels. Importantly, TGFß-treatment increases store-mediated Ca2+ entry, which was essential for the activation of calpain leading to the loss of E-cadherin and MMP activation. Inhibition of Ca2+ entry by using Ca2+ channel blocker SKF-96365, significantly decreased Ca2+ entry, decreased TGFß-induced calpain activation, and suppressed the loss of E-cadherin along with inhibiting cell migration. Furthermore, TRPC1 function as an endogenous Ca2+ entry channel and silencing of either TRPC1 or its activator, STIM1, significantly decreased TGFß induced Ca2+ entry, inhibited TGFß-mediated calpain activation and cell migration. In contrast, overexpression of TRPC1 showed increased Ca2+ entry and promoted TGFß-mediated cell migration. Moreover, increased TRPC1 expression was observed in ductal carcinoma cells. Together these results suggest that disrupting Ca2+ influx via TRPC1/STIM1 mechanism reduces calpain activity, which could restore intercellular junction proteins thereby inhibiting EMT induced motility.

Functional role of TRP channels in modulating ER stress and Autophagy.

Intracellular calcium (Ca(2+)) levels play a vital role in regulating cellular fate. The coordination and interrelation among the cellular organelles, mainly the intracellular Ca(2+) stores in endoplasmic reticulum (ER), are crucial in maintaining cytosolic Ca(2+) levels and in general cellular homeostasis. Moreover, maintaining Ca(2+) homeostasis is essential for regulating diverse and sometimes opposing processes such as cell survival and cell death in disease conditions such as, neurodegeneration, cancer and aging. Ca(2+) is able to regulate opposing functions by either regulating the cellular "self-eating" phenomenon of autophagy to promote cell survival or by regulating the programmed cell death process of apoptosis. Autophagy is also important for cell survival especially after induction of ER stress and association between ER stress and autophagy may have relevance to numerous diseases. Moreover, a multitude of evidence is emerging that the functional regulation of TRP channels, their unique localization, and their interaction with other Ca(2+)-sensing elements define these diverse regulatory pathways. It is this unique function which allows individual TRP channels to contribute differently in the regulation of cell fate and, in turn, determines the precise effect of modulating Ca(2+) signaling via the particular channel. Thus, in this review we have focused on the aspects of TRP channel localization and function (Ca(2+) signaling) that affects the ER stress and autophagic process.

Resveratrol activates autophagic cell death in prostate cancer cells via downregulation of STIM1 and the mTOR pathway.

Resveratrol (RSV), a natural polyphenol, has been suggested to induce cell cycle arrest and activate apoptosis-mediated cell death in several cancer cells, including prostate cancer. However, several molecular mechanisms have been proposed on its chemopreventive action, the precise mechanisms by which RSV exerts its anti-proliferative effect in androgen-independent prostate cancer cells remain questionable. In the present study, we show that RSV activates autophagic cell death in PC3 and DU145 cells, which was dependent on stromal interaction molecule 1 (STIM1) expression. RSV treatment decreases STIM1 expression in a time-dependent manner and attenuates STIM1 association with TRPC1 and Orai1. Furthermore, RSV treatment also decreases ER calcium storage and store operated calcium entry (SOCE), which induces endoplasmic reticulum (ER) stress, thereby, activating AMPK and inhibiting the AKT/mTOR pathway. Similarly, inhibition of SOCE by SKF-96365 decreases the survival and proliferation of PC3 and DU145 cells and inhibits AKT/mTOR pathway and induces autophagic cell death. Importantly, SOCE inhibition and subsequent autophagic cell death caused by RSV was reversed by STIM1 overexpression. STIM1 overexpression restored SOCE, prevents the loss of mTOR phosphorylation and decreased the expression of CHOP and LC3A in PC3 cells. Taken together, for the first time, our results revealed that RSV induces autophagy-mediated cell death in PC3 and DU145 cells through regulation of SOCE mechanisms, including downregulating STIM1 expression and trigger ER stress by depleting ER calcium pool.