Two-pore channels and disease

Two-pore channels (TPCs) are Ca2+-permeable endo-lysosomal ion channels subject to multi-modal regulation. They mediate their physiological effects through releasing Ca2+ from acidic organelles in response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified

The cardiomyocyte "redox rheostat": Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death.

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death

Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx.

Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca(2+) release but their role in Ca(2+) signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca(2+) signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca(2+) release and Ca(2+) entry. Ca(2+) release required lysosomal and ER Ca(2+) stores suggesting that TRPMLs, like other endo-lysosomal Ca(2+) channels, are capable of 'chatter' with ER Ca(2+) channels. Our data identify new modalities for TRPML1 action

An Endosomal NAADP-Sensitive Two-Pore Ca(2+) Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling.

Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca(2+) derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca(2+)-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca(2+) fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca(2+) stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca(2+)-dependent hubs for signaling

IL-17A increases TNF-α-induced COX-2 protein stability and augments PGE<inf>2</inf> secretion from airway smooth muscle cells: Impact on β<inf>2</inf>-adrenergic receptor desensitization

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Background IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. Methods and Results We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. Conclusion As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation

Daily activity during stability and exacerbation of chronic obstructive pulmonary disease

BACKGROUND: During most COPD exacerbations, patients continue to live in the community but there is little information on changes in activity during exacerbations due to the difficulties of obtaining recent, prospective baseline data. METHODS: Patients recorded on daily diary cards any worsening in respiratory symptoms, peak expiratory flow (PEF) and the number of steps taken per day measured with a Yamax Digi-walker pedometer. Exacerbations were defined by increased respiratory symptoms and the number of exacerbations experienced in the 12 months preceding the recording of daily step count used to divide patients into frequent (> = 2/year) or infrequent exacerbators. RESULTS: The 73 COPD patients (88% male) had a mean (+/-SD) age 71(+/-8) years and FEV1 53(+/-16)% predicted. They recorded pedometer data on a median 198 days (IQR 134-353). At exacerbation onset, symptom count rose by 1.9(+/-1.3) and PEF fell by 7(+/-13) l/min. Mean daily step count fell from 4154(+/-2586) steps/day during a preceding baseline week to 3673(+/-2258) step/day during the initial 7 days of exacerbation (p = 0.045). Patients with larger falls in activity at exacerbation took longer to recover to stable level (rho = -0.56; p < 0.001). Recovery in daily step count was faster (median 3.5 days) than for exacerbation symptoms (median 11 days; p < 0.001). Recovery in step count was also faster in untreated compared to treated exacerbation (p = 0.030).Daily step count fell faster over time in the 40 frequent exacerbators, by 708 steps/year, compared to 338 steps/year in 33 infrequent exacerbators (p = 0.002). CONCLUSIONS: COPD exacerbations reduced physical activity and frequent exacerbations accelerate decline in activity over time

The lysosomotrope, GPN, mobilises Ca2+ from acidic organelles

Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. GPN is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. But whether these signals are due to a primary action on lysosomes is unclear in light of recent evidence showing GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN-action. GPN blocked Ca2+ responses evoked by the novel NAADP-like agonist, TPC2-A1-N. GPN-evoked Ca2+ signals were thus better correlated with associated pH changes in the lysosome compared to the cytosol and coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles

Voting is Healthcare: Talking Politics With Patients Can Improve Health Outcomes

Introduction: Many healthcare policy-related issues were hot topics during this unprecedented election year. Healthcare workers took it upon themselves to play a major role in the election by introducing voting as a topic of discussion within the patient encounter. At Wayne State University School of Medicine (WSUSOM), the Voting is Healthcare (VIH) Taskforce was created in order for students, residents and faculty to assist patients with voter registration. The Taskforce’s major partner was VotER, a non-profit, non-partisan organization dedicated to helping patients register to vote through a user-friendly and easily-accessible online platform. Methods: The VIH Taskforce distributed badge-backers with QR codes that patients could scan with their phones to learn more information regarding voter registration, polling location, and absentee voting. Codes were utilized in in-patient settings, clinics, and through tele-health encounters. The VIH Taskforce spearheaded a training session for the WSUSOM community where VotER representatives and local community partners across Michigan spoke. Results: Per VotER records, the VIH Taskforce and WSUSOM community helped 84 patients vote. Of those patients, 34 were previously unregistered and 50 cast absentee ballots. Conclusions: In order to increase voter turnout, the VIH Taskforce opened up the patient encounter to include the topic of political advocacy. This is an unprecedented accomplishment, but it is a necessary addition to the social history portion of the visit. Future studies aim to make the link between civic engagement and positive health outcomes explicit

Post-operative Day 1 versus Day 0 follow-up for Uncomplicated Cataract Surgeries: A comparison of post-operative outcomes and managements

Purpose: To compare the postoperative outcomes and management of uncomplicated cataract surgery (CEIOL) patients seen on post-operative day zero (POD 0) versus post-operative day one (POD 1). Methods: A retrospective chart review of 533 patients who had CEIOL at the Kresge Eye Institute from December 2017 to September 2019 was performed. Visual acuity (VA) and intraocular pressure (IOP) were collected from the pre-operative visit, and the first and second post-operative day visits. In addition, changes in management were recorded from the first post-operative day visit. Patients were excluded if they had a complex cataract procedure, had combined glaucoma filtering surgery, or did not complete two follow up visits within 14 days of surgery. Results: The pre-operative demographic data between patients seen on POD 0 (n=119) versus POD1 (N=414) were equally distributed. By unpaired t-test, the average VA of patients seen on POD 1 was significantly better than those seen on POD 0 (P0.50). Conclusion: There was no significant difference in management between POD 0 and POD 1 patients having undergone uncomplicated cataract surgeries. Therefore, surgeons can safely consider POD 0 or POD 1 evaluations for uncomplicated cataract surgeries and improve healthcare cost efficiency for CEIOL