Inhibition of CysLTR1 reduces the levels of aggregated proteins in retinal pigment epithelial cells.

The endosomal-lysosomal system (ELS), which carries out cellular processes such as cellular waste degradation via autophagy, is essential for cell homeostasis. ELS inefficiency leads to augmented levels of damaged organelles and intracellular deposits. Consequently, the modulation of autophagic flux has been recognized as target to remove damaging cell waste. Recently, we showed that cysteinyl leukotriene receptor 1 (CysLTR1) antagonist application increases the autophagic flux in the retinal pigment epithelial cell line ARPE-19. Consequently, we investigated the effect of CysLTR1 inhibition-driven autophagy induction on aggregated proteins in ARPE-19 cells using flow cytometry analysis. A subset of ARPE-19 cells expressed CysLTR1 on the surface (SE+); these cells showed increased levels of autophagosomes, late endosomes/lysosomes, aggregated proteins, and autophagy as well as decreased reactive oxygen species (ROS) formation. Furthermore, CysLTR1 inhibition for 24 h using the antagonist zafirlukast decreased the quantities of autophagosomes, late endosomes/lysosomes, aggregated proteins and ROS in CysLTR1 SE- and SE+ cells. We concluded that high levels of plasma membrane-localized CysLTR1 indicate an increased amount of aggregated protein, which raises the rate of autophagic flux. Furthermore, CysLTR1 antagonist application potentially mimics the physiological conditions observed in CysLTR1 SE+ cells and can be considered as strategy to dampen cellular aging.

Cysteinyl leukotriene receptor 1 is a potent regulator of the endosomal-lysosomal system in the ARPE-19 retinal pigment epithelial cell line.

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Chronobiological activity of cysteinyl leukotriene receptor 1 during basal and induced autophagy in the ARPE-19 retinal pigment epithelial cell line.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line. Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy. CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.