Stimulation of TLR3 Triggers Release of Lysosomal ATP in Astrocytes and Epithelial Cells That Requires TRPML1 Channels

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and non-targeting scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1-/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways. © 2018 The Author(s)

Stimulation of TLR3 Triggers Release of Lysosomal ATP in Astrocytes and Epithelial cells that Requires TRPML1 Channels

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and “non-targeting” scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1−/− cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways

Stimulation of TLR3 Triggers Release of Lysosomal ATP in Astrocytes and Epithelial Cells that Requires TRPML1 Channels

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and non-targeting scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1-/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways. © 2018 The Author(s)

Regulator of G Protein Signaling Protein 12 (Rgs12) Controls Mouse Osteoblast Differentiation via Calcium Channel/Oscillation and Gαi-ERK Signaling

Bone homeostasis intimately relies on the balance between osteoblasts (OBs) and osteoclasts (OCs). Our previous studies have revealed that regulator of G protein signaling protein 12 (Rgs12), the largest protein in the Rgs super family, is essential for osteoclastogenesis from hematopoietic cells and OC precursors. However, how Rgs12 regulates OB differentiation and function is still unknown. To understand that, we generated an OB-targeted Rgs12 conditional knockout (CKO) mice model by crossing Rgs12 fl/fl mice with Osterix (Osx)-Cre transgenic mice. We found that Rgs12 was highly expressed in both OB precursor cells (OPCs) and OBs of wild-type (WT) mice, and gradually increased during OB differentiation, whereas Rgs12-CKO mice (Osx Cre/+ ; Rgs12 fl/fl ) exhibited a dramatic decrease in both trabecular and cortical bone mass, with reduced numbers of OBs and increased apoptotic cell population. Loss of Rgs12 in OPCs in vitro significantly inhibited OB differentiation and the expression of OB marker genes, resulting in suppression of OB maturation and mineralization. Further mechanism study showed that deletion of Rgs12 in OPCs significantly inhibited guanosine triphosphatase (GTPase) activity and cyclic adenosine monophosphate (cAMP) level, and impaired Calcium (Ca 2+ ) oscillations via restraints of major Ca 2+ entry sources (extracellular Ca 2+ influx and intracellular Ca 2+ release from endoplasmic reticulum), partially contributed by the blockage of L-type Ca 2+ channel mediated Ca 2+ influx. Downstream mediator extracellular signal-related protein kinase (ERK) was found inactive in OBs of Osx Cre/+ ; Rgs12 fl/fl mice and in OPCs after Rgs12 deletion, whereas application of pertussis toxin (PTX) or overexpression of Rgs12 could rescue the defective OB differentiation via restoration of ERK phosphorylation. Our findings reveal that Rgs12 is an important regulator during osteogenesis and highlight Rgs12 as a potential therapeutic target for bone disorders. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Researc

Compromised Phagosome Maturation Underlies RPE Pathology in Cell Culture and Whole Animal Models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group

Endogenously expressed bestrophin-1 modulates calcium signaling in the retinal pigmented epithelium

Mutations in the BEST1 gene lead to a variety of retinal degenerations. To understand the function of the BEST1 gene product, bestrophin-1, represents the first step in the investigation of patho-mechanisms leading to BEST1-associated retinal degenerations. Bestrophin-1 is a protein expressed in the cells of the retinal pigment epithelium (RPE), a monolayer of epithelial cells in the outer retina which closely interact with the photoreceptors. Thus, it is likely that mutant bestrophin-1 changes the RPE function which in turn leads to photoreceptor degeneration. So far the properties of bestrophin-1 were identified by functional analysis of over-expressed proteins. In these studies bestrophin-1 was identified as a Ca2+-dependent Cl channel in the plasma membrane. However, this result could not explain observations made in animal models for bestrophin-1 or cellular function analysis in relation to presence of endogenously expressed bestrophin-1. The few studies on the function of endogenously expressed bestrophin-1 indicated that it may have an intracellular function in relation to Ca2+signaling. The aim of the study was to investigate the function of endogenously expressed bestrophin-1 in mouse models and in a newly developed in vitro model of porcine RPE cells. Since the retina of bestrophin-1 knock-out mice or Best+/Y227N knock-in mice showed no degeneration, the susceptibility of these retinae to light-damage at the threshold to light-damage was tested. Here, no differences between wild-type and transgenic mice were found. Also the ATP-dependent Ca2+-Signals in RPE cells from these mouse strains were not changed. In the porcine RPE cell model siRNA bestrophin-1 was investigated. Ca2+-signaling was ignited by induction of store-operated Ca2+-entry (SOCE). Pharmacological analysis and siRNA knock-down of Orai-1 expression reveled that SOCE was dependent on the activation of Orai-1 channels by stim-1 (stromal interacting molecule-1). Reduction of bestrophin-1 expression did not change the expression of stim-1 or orai-1 expression but reduced the SOCE amplitude. Measuring amplitude of Ca2+-release from intracellular Ca2+ stores in response to inhibition of sarcoplasmaic Ca2+-ATPase (SERCA) and quantification of the amount of Ca2+ releasable from ER Ca2+ stores revealed that after bestrophin-1 knock-down the amount of Ca2+ in these stores was strongly decreased. In addition to these functional data also the analysis of subcellular bestrophin-1 localization by means of immunocytochemistry or electron-microscopy revealed that bestrophin-1 is an intracellular protein which co-localizes with stim-1. However, bestrophin-1 and stim-1 did not physically interact. In summary, we found that bestrophin-1 is likely an intracellular Cl channel in the membrane of cytosolic Ca2+ stores which are localized close to the basolateral membrane. As a Cl channel bestrophin-1 can Ca2+-dependently conduct the counter-ion for Ca2+ to accumulate Ca2+ in ER stores. The reduced Ca2+ amount in Ca2+-stores decreases the activation SOCE and therefore changes intracellular Ca2+ signaling which controls RPE function. With these conclusions we can open a new route to understand the chain of events leading to retinal degeneration in BEST1-associated diseases

Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome

<p>Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.</p