Abstract
The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1−/− mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.
Highlights
The thrombin protease activated receptor 1 (PAR1) is a G-protein-coupled receptor and one of four members of protease-activated receptors (PARs) [1]
To determine the distribution of PAR1 protein in mouse neuroretina, eyes were enucleated from ten C57BL/6J male mice and were paraffin embedded, and retinal sections were stained with an anti-PAR1 antibody
As previous studies suggested that PAR1 is expressed in retinal ganglion cells in vitro [17], the staining of these cells served as an internal positive control (Figure 1A,C)
Summary
The thrombin protease activated receptor 1 (PAR1) is a G-protein-coupled receptor and one of four members of protease-activated receptors (PARs) [1]. PAR1 and its main activator thrombin were found to play key roles in pathologies of the central and peripheral nervous systems, including in Parkinson’s disease, amyotrophic lateral sclerosis (ALS), glioblastoma (GBM), diabetic neuropathy (DN), and sciatic nerve injury [3,4,5,6,7]. PAR1 is expressed in the sciatic node of Ranvier, where its activation leads to conduction block [8], and its increased activation on Schwann cells following injury was suggested to limit axonal repair [9]. The PAR1/thrombin pathway has been marked as a potential target for the treatment of these diseases.
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