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Abstract Background Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA2-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes. Methods/Results Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA2-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression. Conclusion These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

Objective: Until now, the clinically relevant improvement for the Xerostomia Inventory (XI) has not been defined. Therefore, our aim was to determine the Minimally Important Difference (MID) of the XI for improvement in dry-mouth symptoms in SjD patients., Method: The study recruited 34 SjD patients who underwent sialendoscopy of major salivary glands and 15 SjD patients in a nonintervention control group. XI scores were assessed at several time points. The MID was determined from the mean difference in XI scores between the groups with and without improvement., Results: In the control group, no significant XI score changes were seen. In the sialendoscopy group, a clinically relevant XI score change of four scale points was identified after 1 week. For a prolonged duration (≥16 weeks), a minimum reduction of seven scale points in the XI score was required to indicate clinically relevant improvement., Conclusion: In SjD patients, a minimum change of four points in the XI score indicates a clinically relevant improvement for evaluating short-term effects. For prolonged effects, a clinically relevant improvement requires a MID of seven points. The determination of the MID in XI could assist in future studies that evaluate changes in xerostomia., (© 2023 The Authors. Oral Diseases published by Wiley Periodicals LLC.)

Background and Aims: Vascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype., Methods: We used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y 2 receptor (P2Y 2 R) gene to evaluate resulting phenotypic alterations., Results: HASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y 2 R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin., Conclusions: Our data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y 2 R by basally released ATP is required for maintenance of the differentiated VSMC phenotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role of the P2Y 2 purinergic receptor (P2Y 2 R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y 2 Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y 2 R being highly expressed. Using syngeneic tumor grafts in wild-type mice, we observed that MOC2 tumors expressing P2Y 2 R were larger than P2Y 2 R -/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b + F4/80 + macrophages and CD3 +  cells, which were revealed to be CD3 + CD4 + IFNγ + T cells, compared to P2Y 2 R -/- tumors. These results were mirrored when utilizing P2Y 2 R -/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y 2 Rs. Results suggest that targeted suppression of the P2Y 2 R in HNSCC cells in vivo, rather than systemic P2Y 2 R antagonism, may be a more effective treatment strategy for HNSCCs., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Salivary gland damage and hypofunction result from various disorders, including autoimmune Sjögren's disease (SjD) and IgG4-related disease (IgG4-RD), as well as a side effect of radiotherapy for treating head and neck cancers. There are no therapeutic strategies to prevent the loss of salivary gland function in these disorders nor facilitate functional salivary gland regeneration. However, ongoing aquaporin-1 gene therapy trials to restore saliva flow show promise. To identify and develop novel therapeutic targets, we must better understand the cell-specific signaling processes involved in salivary gland regeneration. Transforming growth factor-β (TGF-β) signaling is essential to tissue fibrosis, a major endpoint in salivary gland degeneration, which develops in the salivary glands of patients with SjD, IgG4-RD, and radiation-induced damage. Though the deposition and remodeling of extracellular matrix proteins are essential to repair salivary gland damage, pathological fibrosis results in tissue hardening and chronic salivary gland dysfunction orchestrated by multiple cell types, including fibroblasts, myofibroblasts, endothelial cells, stromal cells, and lymphocytes, macrophages, and other immune cell populations. This review is focused on the role of TGF-β signaling in the development of salivary gland fibrosis and the potential for targeting TGF-β as a novel therapeutic approach to regenerate functional salivary glands. The studies presented highlight the divergent roles of TGF-β signaling in salivary gland development and dysfunction and illuminate specific cell populations in damaged or diseased salivary glands that mediate the effects of TGF-β. Overall, these studies strongly support the premise that blocking TGF-β signaling holds promise for the regeneration of functional salivary glands., (© 2024 The Authors.)

In sialendoscopy, ducts are dilated and the salivary glands are irrigated with saline. Contrast-enhanced ultrasound sialendoscopy (CEUSS), using microbubbles, may facilitate the monitoring of irrigation solution penetration in the ductal system and parenchyma. It is imperative to test CEUSS for its safety and feasibility in Sjögren's syndrome (SS) patients. CEUSS was performed on 10 SS patients. The primary outcomes were safety, determined by the occurrence of (serious) adverse events ((S)AEs), and feasibility. The secondary outcomes were unstimulated and stimulated whole saliva (UWS and SWS) flow rates, xerostomia inventory (XI), clinical oral dryness score, pain, EULAR Sjögren's syndrome patient reported index (ESSPRI), and gland topographical alterations. CEUSS was technically feasible in all patients. Neither SAEs nor systemic reactions related to the procedure were observed. The main AEs were postoperative pain (two patients) and swelling (two patients). Eight weeks after CEUSS, the median UWS and SWS flow had increased significantly from 0.10 to 0.22 mL/min ( p = 0.028) and 0.41 to 0.61 mL/min ( p = 0.047), respectively. Sixteen weeks after CEUSS, the mean XI was reduced from 45.2 to 34.2 ( p = 0.02). We conclude that CEUSS is a safe and feasible treatment for SS patients. It has the potential to increase salivary secretion and reduce xerostomia, but this needs further investigation.

G protein-coupled receptors are the target of more than 30% of all FDA-approved drug therapies. Though the purinergic P2 receptors have been an attractive target for therapeutic intervention with successes such as the P2Y 12 receptor antagonist, clopidogrel, P2Y 2 receptor (P2Y 2 R) antagonism remains relatively unexplored as a therapeutic strategy. Due to a lack of selective antagonists to modify P2Y 2 R activity, studies using primarily genetic manipulation have revealed roles for P2Y 2 R in a multitude of diseases. These include inflammatory and autoimmune diseases, fibrotic diseases, renal diseases, cancer, and pathogenic infections. With the advent of AR-C118925, a selective and potent P2Y 2 R antagonist that became commercially available only a few years ago, new opportunities exist to gain a more robust understanding of P2Y 2 R function and assess therapeutic effects of P2Y 2 R antagonism. This review discusses the characteristics of P2Y 2 R that make it unique among P2 receptors, namely its involvement in five distinct signaling pathways including canonical Gα q protein signaling. We also discuss the effects of other P2Y 2 R antagonists and the pivotal development of AR-C118925. The remainder of this review concerns the mounting evidence implicating P2Y 2 Rs in disease pathogenesis, focusing on those studies that have evaluated AR-C118925 in pre-clinical disease models., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The P2Y receptors are responsible for the regulation of various physiological processes including neurotransmission and inflammatory responses. These receptors are also considered as novel potential therapeutic targets for prevention and treatment of thrombosis, neurological disorders, pain, cardiac diseases and cancer. Previously, number of P2Y receptor antagonists has been investigated but they are less potent and non-selective with poor solubility profile. Herein, we present the synthesis of new class of benzimidazole derived sulfonylureas ( 1a-y ) as potent antagonists of P2Y receptors, with the specific aim to explore selective antagonists of P2Y1 receptors. The efficacy and selectivity of the synthesized derivatives 1 ) against four P2Y receptors i.e., t-P2Y1, h-P2Y2, h-P2Y4, and r-P2Y6Rs was carried out by calcium mobilization assay. The results revealed that except 1b, 1d, 1l, 1m, 1o, 1u, 1v, 1w, and 1y , rest of the synthesized derivatives exhibited moderate to excellent inhibitory potential against P2Y1 receptors. Among the potent antagonists, derivative 1h depicted the maximum inhibition of P2Y1 receptor in calcium signalling assay, with an IC 50 value of 0.19 ± 0.04 µM. The potential of inhibition was validated by computational investigations where bonding and non-bonding interactions between ligand and targeted receptor further strengthen the study. The best identified derivative 1h revealed the same binding mechanism as that of already reported selective antagonist of P2Y1 receptor i.e (1-(2- (2-tert-butyl-phenoxy) pyridin-3-yl)-3-4-(trifluoromethoxy) phenylurea but the newly synthesized derivative exhibited better solubility profile. Hence, this derivative can be used as lead candidate for the synthesis of more potential antagonist with much better solubility profile and medicinal importance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bano, Hussain, Langer, Weisman and Iqbal.)

Background: Sjögren syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and diminished secretory function of the salivary glands. Dexamethasone (DEX) resolves dry mouth and lymphocytic infiltration; however, this treatment is difficult to maintain because of multiple adverse effects (eg, osteoporosis and skin thinning); likewise, aspirin-triggered resolvin D1 (AT-RvD1) increases saliva secretion but cannot eliminate lymphocytic infiltration. Previous studies showed that a combination of low-dose DEX with AT-RvD1 before disease onset prevents SS-like features in a mouse model; however, this is not clinically practical because there are no reliable indicators of SS before disease onset. Therefore, the authors applied the combined treatment at disease onset to show its efficacy and comparative lack of adverse effects, so that it may reasonably be maintained over a patient's lifetime., Methods: NOD/ShiLtJ mice were treated with ethanol (vehicle control), high-dose DEX alone, AT-RvD1 alone, or a combination of low-dose DEX with AT-RvD1 at disease onset for 8 weeks. Then saliva flow rates were measured, and submandibular glands were harvested for histologic analyses., Results: A combined treatment of low-dose DEX with AT-RvD1 significantly decreased mast cell degranulation and lymphocytic infiltration, increased saliva secretion, and restored apical aquaporin-5 expression in submandibular glands of NOD/ShiLtJ mice., Conclusions: Low-dose DEX combined with AT-RvD1 reduces the severity of SS-like manifestation and prevents the development of advanced and potentially irreversible damage, all in a form that can reasonably be administered indefinitely without the need to cease treatment because of secondary effects.

Tuft cells are bottle-shaped, microvilli-projecting chemosensory cells located in the lining of a variety of epithelial tissues and, following their identification approximately 60 years ago, have been linked to immune system function in a variety of epithelia. Until recently, Tuft cells had not been convincingly demonstrated to be present in salivary glands with their detection by transmission electron microscopy only shown in a handful of earlier studies using rat salivary glands, and no follow-up work has been conducted to verify their presence in salivary glands of other species. Here, we demonstrate that Tuft cells are present in the submandibular glands of various species (i.e., mouse, pig and human) using transmission electron microscopy and confocal immunofluorescent analysis for the POU class 2 homeobox 3 (POU2F3), which is considered to be a master regulator of Tuft cell identity.

Purpose: To develop a mouse model of human dry eye disease (DED) for investigation of sex differences in autoimmune-associated dry eye pathology., Methods: Ocular surface disease was assessed by quantifying corneal epithelial damage with lissamine green stain in the NOD.H-2h4,IFNγ-/-,CD28-/- (NOD.H-2h4 DKO) mouse model of Sjögren's syndrome (SS). Lacrimal gland function was assessed by tear volume quantification with phenol red thread and lacrimal gland inflammation (i.e., dacryoadenitis) was assessed by quantification of immune cell foci, flow cytometric analysis of immune cell composition, and expression of proinflammatory markers., Results: The NOD.H-2h4 DKO mouse model of SS exhibits greater age-dependent increases in corneal damage than in NOD.H-2h4 parental mice and demonstrates an earlier disease onset in females compared to males. The severity of ocular surface disease correlates with loss of goblet cell density, increased conjunctivitis, and dacryoadenitis that is more pronounced in NOD.H-2h4 DKO than NOD.H-2h4 mice. B cells dominate lacrimal infiltrates in 16-week-old NOD.H-2h4 and NOD.H-2h4 DKO mice, but T helper cells and macrophages are also present. Lacrimal gland expression of proinflammatory genes, including the P2X7 and P2Y2 purinergic receptors, is greater in NOD.H-2h4 DKO than NOD.H-2h4 mice and correlates with dacryoadenitis., Conclusions: Our results demonstrate for the first time that autoimmune dry eye disease occurs in both sexes of NOD.H-2h4 DKO and NOD.H-2h4 mice, with earlier onset in female NOD.H-2h4 DKO mice when compared to males of the same strain. This study demonstrates that both NOD.H-2h4 and NOD.H-2h4 DKO mice are novel models that closely resemble SS-related and sex-dependent DED.

Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E 2 (PGE 2 ) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE 2 secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R -/- mice compared to wild-type mice. However, the role of PGE 2 signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE 2 action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE 2 production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE 2 induces salivary gland dysfunction, primary parotid gland cells treated with PGE 2 have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The in vivo effects of blocking PGE 2 production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE 2 signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gilman, Camden, Woods, Weisman and Limesand.)

Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A 1 , A 2A , A 2B and A 3 for extracellular adenosine, P2X1-7 receptors that are ATP-gated ion channels and G protein-coupled P2Y 1,2,4,6,11,12,13 and 14 receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Sjögren's syndrome (SS) is a chronic autoimmune exocrinopathy characterized by lymphocytic infiltration of the salivary and lacrimal glands and decreased saliva and tear production. Previous studies indicate that the G protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R) is upregulated in numerous models of salivary gland inflammation (i.e., sialadenitis), where it has been implicated as a key mediator of chronic inflammation. Here, we evaluate both systemic and localized P2Y 2 R antagonism as a means to resolve sialadenitis in the NOD.H-2 h4 ,IFNγ -/- ,CD28 -/- (NOD.H-2 h4 DKO) mouse model of SS., Design: Female 4.5 month old NOD.H-2 h4 DKO mice received daily intraperitoneal injections for 10 days of the selective P2Y 2 R antagonist, AR-C118925, or vehicle-only control. Single-dose localized intraglandular antagonist delivery into the Wharton's duct was also evaluated. Carbachol-induced saliva was measured and then submandibular glands (SMGs) were isolated and either fixed and paraffin-embedded for H&E staining, homogenized for RNA isolation or dissociated for flow cytometry analysis., Results: Intraperitoneal injection, but not localized intraglandular administration, of AR-C118925 significantly enhanced carbachol-induced salivation and reduced lymphocytic foci and immune cell markers in SMGs of 5 month old NOD.H-2 h4 DKO mice, compared to vehicle-injected control mice. We found that B cells represent the primary immune cell population in inflamed SMGs of NOD.H-2 h4 DKO mice that express elevated levels of P2Y 2 R compared to C57BL/6 control mice. We further demonstrate a role for P2Y 2 Rs in mediating B cell migration and the release of IgM., Conclusion: Our findings suggest that the P2Y 2 R represents a novel therapeutic target for the treatment of Sjögren's syndrome., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CLL is cofounder of Shift Pharmaceuticals but that has not influenced the work reported in this paper., (© 2021 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Objectives: To assess functional expression of the P2Y 2 nucleotide receptor (P2Y 2 R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC., Materials and Methods: In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y 2 R contributions to extracellular nucleotide-induced changes in intracellular free Ca 2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca 2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y 2 Rs using CRISPR technology or pharmacological inhibition with P2Y 2 R-selective antagonist AR-C118925 defined P2Y 2 R contributions to in vivo tumor growth., Results: P2Y 2 R agonists UTP and ATP increased intracellular Ca 2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y 2 R knockout. P2Y 2 R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y 2 R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume., Conclusion: P2Y 2 Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y 2 R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y 2 R represents a novel therapeutic target in HNSCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration., (Copyright © 2020 Khalafalla, Woods, Jasmer, Forti, Camden, Jensen, Limesand, Galtung and Weisman.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by three major histopathological markers: amyloid-β (Aβ) plaques, neurofibrillary tangles and gliosis in the central nervous system (CNS). It is now accepted that neuroinflammatory events in the CNS play a crucial role in the development of AD. This review focuses on neuroinflammatory signaling mediated by purinergic receptors (P1 adenosine receptors, P2X ATP-gated ion channels and G protein-coupled P2Y nucleotide receptors) and how therapeutic modulation of purinergic signaling influences disease progression in AD patients and animal models of AD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R -/- ) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R -/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R -/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E 2 (PGE 2 ) release from primary parotid cells of wild-type but not P2X7R -/- mice. To attempt to uncover the mechanism underlying differential PGE 2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE 2 release in P2X7R -/- mice. Irradiated P2X7R -/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies., Competing Interests: The authors declare no conflict of interest.

Objective: Interleukin-14α-transgenic (IL-14αTG) mice develop an autoimmune exocrinopathy with characteristics similar to Sjögren's syndrome, including sialadenitis and hyposalivation. The P2Y 2 receptor (P2Y 2 R) for extracellular ATP and UTP is upregulated during salivary gland inflammation (i.e., sialadenitis) where it regulates numerous inflammatory responses. This study investigated the role of P2Y 2 Rs in autoimmune sialadenitis in the IL-14αTG mouse model of Sjögren's syndrome., Materials and Methods: IL-14αTG mice were bred with P2Y 2 R -/- mice to generate IL-14αTG × P2Y 2 R -/- mice. P2Y 2 R expression, lymphocytic focus scores, B- and T-cell accumulation, and lymphotoxin-α expression were evaluated in the submandibular glands (SMG) along with carbachol-stimulated saliva secretion in IL-14αTG, IL-14αTG × P2Y 2 R -/- , and C57BL/6 control mice at 9 and 12 months of age., Results: Genetic ablation of P2Y 2 Rs in IL-14αTG mice significantly reduced B and T lymphocyte infiltration of SMGs. However, reduced sialadenitis did not restore saliva secretion in IL-14αTG × P2Y 2 R -/- mice. Decreased sialadenitis in IL-14αTG × P2Y 2 R -/- mice correlated with decreased lymphotoxin-α levels, a critical proinflammatory cytokine associated with autoimmune pathology in IL-14αTG mice., Conclusions: The results of this study suggest that P2Y 2 Rs contribute to the development of salivary gland inflammation in IL-14αTG mice and may also contribute to autoimmune sialadenitis in humans., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we sought to understand the requirement for CD40 signaling in the programmed cell death-1 (PD-1) checkpoint and CD28 costimulatory pathways important for maintenance of peripheral tolerance. Blocking either pathway can result in loss of self-tolerance and development of autoimmunity. We found that primary Sjögren's syndrome (pSS) and autoimmune thyroid diseases (ATDs) that develop spontaneously in CD28-deficient IFN-γ -/- NOD.H-2h4 (CD28 -/- ) mice required CD40 signaling. Specifically, blockade of CD40L with the anti-CD40L mAb, MR1, inhibited autoantibody production and inflammation in thyroid and salivary gland target tissues. Unexpectedly, however, ATD and pSS in PD-1-deficient IFN-γ -/- NOD.H-2h4 (PD-1 -/- ) mice developed independently of CD40/CD40L interactions. Treatment with MR1 had no effect and even exacerbated disease development in pSS and ATD, respectively. Most interesting, anti-thyroglobulin and pSS-associated autoantibodies were increased following anti-CD40L treatment, even though MR1 effectively inhibited the spontaneous splenic germinal centers that form in PD-1-deficient mice. Importantly, blockade of the PD-1 pathway by administration of anti-PD-1 mAb in CD28 -/- mice recapitulated the PD-1 -/- phenotype, significantly impacting the ability of MR1 to suppress ATD and pSS in these mice. These results indicate that there can be different pathways and requirements to autoimmune pathogenesis depending on the availability of specific checkpoint and costimulatory receptors, and an intact PD-1 pathway is apparently required for inhibition of autoimmunity by anti-CD40L.

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R -/- ). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na + and/or K + flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N -acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28 -/- , IFNγ -/- , NOD.H-2 h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection., (Copyright © 2017 American Society for Microbiology.)

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y 2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y 2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y 2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y 2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y 2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y 2 RGE mutant receptors. Consistent with the role of P2Y 2 receptors in vasodilation, receptor knockdown and overexpression of P2Y 2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y 2 receptor in shear stress-induced cytoskeletal alterations in HUVECs., Competing Interests: The authors declare that there are no conflicts of interest regarding the material discussed in the manuscript.

Sixty to seventy percent of IFN-γ(-/-) NOD.H-2h4 mice given sodium iodide (NaI)-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell (TEC) hyperplasia and proliferation (H/P). TEC H/P develops much earlier in CD28(-/-) mice and nearly 100% (both sexes) have severe TEC H/P at 4 mo of age. Without NaI supplementation, 50% of 5- to 6-mo-old CD28(-/-)IFN-γ(-/-) mice develop severe TEC H/P, and 2-3 wk of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid, and normalization of serum thyroxine levels does not reduce TEC H/P. Activated CD4(+) T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for, development of severe TEC H/P, as CD40(-/-)IFN-γ(-/-)CD28(-/-) mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ(-/-)CD28(-/-) mice is a result of reduced regulatory T cell (Treg) numbers, as CD28(-/-) mice have significantly fewer Tregs, and transfer of CD28(+) Tregs inhibits TEC H/P. Essentially all female IFN-γ(-/-)CD28(-/-) NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 mo of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 mo of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background and Aims: The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs., Methods: Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R-/- mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A)., Results: P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R-/- VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R-/- VSMCs versus cells transfected with the mutant P2Y2R., Conclusions: Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A., (Published by Elsevier Ireland Ltd.)

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-β plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7a(Nes) mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7a(Nes) mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7a(Nes) mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients., (Copyright © 2015 the American Physiological Society.)

ATP7A is a copper-transporting P-type ATPase that is essential for cellular copper homeostasis. Loss-of-function mutations in the ATP7A gene result in Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia and failure to thrive, due to systemic copper deficiency. Most recently, rare missense mutations in ATP7A that do not impact systemic copper homeostasis have been shown to cause X-linked spinal muscular atrophy type 3 (SMAX3), a distal hereditary motor neuropathy. An understanding of the mechanistic and pathophysiological basis of SMAX3 is currently lacking, in part because the disease-causing mutations have been shown to confer both loss- and gain-of-function properties to ATP7A, and because there is currently no animal model of the disease. In this study, the Atp7a gene was specifically deleted in the motor neurons of mice, resulting in a degenerative phenotype consistent with the clinical features in affected patients with SMAX3, including the progressive deterioration of gait, age-dependent muscle atrophy, denervation of neuromuscular junctions and a loss of motor neuron cell bodies. Taken together, these data reveal autonomous requirements for ATP7A that reveal essential roles for copper in the maintenance and function of the motor neuron, and suggest that SMAX3 is caused by a loss of ATP7A function that specifically impacts the spinal motor neuron., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Transforming growth factor-β (TGF-β) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-β may contribute to salivary gland fibrosis, the expression of TGF-β signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-β1 and TGF-β3 were significantly upregulated in the SMG, as were TGF-β receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-β signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-β receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-β signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-β signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-β signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-β signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution.

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta-amyloid peptides (Aβ) and neuronal death in brain regions involved in learning and memory. Although Aβ is neurotoxic, the primary mechanisms by which Aβ affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aβ have revealed that Aβ has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aβ and contributes to AD pathology. In recent years, Aβ has been shown to affect ATP release from brain and blood cells and alter the expression of G protein-coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aβ production and elimination, neuroinflammation, neuronal function and cerebral blood flow., (© 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is a progressive, age-dependent neurodegenerative disorder affecting specific brain regions that control memory and cognitive functions. Epidemiological studies suggest that exercise and dietary antioxidants are beneficial in reducing AD risk. To date, botanical flavonoids are consistently associated with the prevention of age-related diseases. The present study investigated the effects of 4 months of wheel-running exercise, initiated at 2-months of age, in conjunction with the effects of the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) administered orally in the drinking water (50 mg/kg daily) on: (1) behavioral measures: learning and memory performance in the Barnes maze, nest building, open-field, anxiety in the light-dark box; and (2) soluble amyloid-β (Aβ) levels in the cortex and hippocampus in TgCRND8 (Tg) mice. Untreated Tg mice showed hyperactivity, relatively poor nest building behaviors, and deficits in spatial learning in the Barnes maze. Both EGCG and voluntary exercise, separately and in combination, were able to attenuate nest building and Barnes maze performance deficits. Additionally, these interventions lowered soluble Aβ1-42 levels in the cortex and hippocampus. These results, together with epidemiological and clinical studies in humans, suggest that dietary polyphenols and exercise may have beneficial effects on brain health and slow the progression of AD.

Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the G q protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y 2 R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y 2 R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y 2 R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y 2 R uses Src and VEGFR-2 to mediate association of the P2Y 2 R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5β1 integrin and the Rho GTPase Cdc42., (Copyright © 2014 the American Physiological Society.)

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ-CH2 (1), ATP-α-thio-β,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 μM inhibited thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 μM. Analogues 1-3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1-3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.

Neuroinflammation is a prominent feature in Alzheimer's disease (AD) and activation of the brain's innate immune system, particularly microglia, has been postulated to both retard and accelerate AD progression. Recent studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is an important regulator of innate immunity by assisting in the recruitment of monocytes to injured tissue, neutrophils to bacterial infections and eosinophils to allergen-infected lungs. In this study, we investigated the role of the P2Y2R in progression of an AD-like phenotype in the TgCRND8 mouse model that expresses Swedish and Indiana mutations in amyloid precursor protein (APP). Our results indicate that P2Y 2 R expression is upregulated in TgCRND8 mouse brain within 10 weeks of age and then decreases after 25 weeks of age, as compared to littermate controls expressing low levels of the P2Y 2 R. TgCRND8 mice with homozygous P2Y 2 R deletion survive less than 5 weeks, whereas mice with heterozygous P2Y 2 R deletion survive for 12 weeks, a time point when TgCRND8 mice are fully viable. Heterozygous P2Y 2 R deletion in TgCRND8 mice increased β-amyloid (Aβ) plaque load and soluble Aβ1-42 levels in the cerebral cortex and hippocampus, decreased the expression of the microglial marker CD11b in these brain regions and caused neurological deficits within 10 weeks of age, as compared to age-matched TgCRND8 mice. These findings suggest that the P2Y2R is important for the recruitment and activation of microglial cells in the TgCRND8 mouse brain and that the P2Y2R may regulate neuroprotective mechanisms through microglia-mediated clearance of Aβ that when lost can accelerate the onset of an AD-like phenotype in the TgCRND8 mouse.

The pro-inflammatory cytokine interleukin-1β (IL-1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1β increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1β-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv β3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1β-treated mPCNs, suggesting that established P2Y2R/αv β3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1β-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases., (© 2013 International Society for Neurochemistry.)

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

The essential requirement for copper in early development is dramatically illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by loss-of-function mutations in the gene encoding the copper transporting ATPase ATP7A. In this study, we generated mice with enterocyte-specific knockout of the murine ATP7A gene (Atp7a) to test its importance in dietary copper acquisition. Although mice lacking Atp7a protein within intestinal enterocytes appeared normal at birth, they exhibited profound growth impairment and neurological deterioration as a consequence of copper deficiency, resulting in excessive mortality prior to weaning. Copper supplementation of lactating females or parenteral copper injection of the affected offspring markedly attenuated this rapid demise. Enterocyte-specific deletion of Atp7a in rescued pregnant females did not restrict embryogenesis; however, copper accumulation in the late-term fetus was severely reduced, resulting in early postnatal mortality. Taken together, these data demonstrate unique and specific requirements for enterocyte Atp7a in neonatal and maternofetal copper acquisition that are dependent on dietary copper availability, thus providing new insights into the mechanisms of gene-nutrient interaction essential for early human development.

P2Y receptors are G protein-coupled receptors (GPCRs) that are activated by adenine and uridine nucleotides and nucleotide sugars. There are eight subtypes of P2Y receptors (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ), which activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, differentiation, phagocytosis, secretion, nociception, cell adhesion, and cell migration. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, adenylyl and guanylyl cyclases, protein kinases, and phosphodiesterases. In addition, there are numerous ion channels, cell adhesion molecules, and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response.

Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.