Targeting the Notch transcriptional activation complex with a novel PRAG1 inhibitor to attenuate reactive gliosis

Abstract

Purpose : In response to neurodegenerative diseases and injury to the retina, astrocytes and Müller glia (MG) undergo reactive gliosis. Chronic reactive gliosis exacerbates neurodegeneration by promoting inflammation and scar formation. Studies report that Notch signaling enhances gliosis and neuronal death. Current compounds that target this pathway, including non-specific inhibitors of presenilin-dependent γ-secretase, do not effectively treat central nervous system (CNS) disease and injuries. Recently, PEAK1 Related, Kinase-Activating Pseudokinase 1 (PRAG1) was identified as a novel Notch transcriptional coactivator (and given the alternate name Notch Activation Complex Kinase [NACK]). Our team identified a first-in-class PRAG1 inhibitor to target Notch1 activity directly and specifically. Given the activation of Notch1 signaling upon CNS injury and the critical role of PRAG1 in the activation of this signaling pathway, we hypothesize that directly modulating Notch1 transcriptional activity via PRAG1 inhibition will mitigate reactive gliosis and neuropathic progression.

Methods : Human and mouse MG were stimulated with Notch ligands then treated with the PRAG1 inhibitor UM74 for Western blot and real-time PCR. We also studied changes in Notch signaling and the effects of UM74 following ischemia/reperfusion (IR) injury in 2-month-old C57BL/6J mice. Retinal ischemia was induced for 1 hour by elevating intraocular pressure above systolic blood pressure with normal saline (to 120 mmHg). Intravitreal injection of UM74 was performed immediately after IR injury. Mice were sacrificed 7 days after injury and their eyes were enucleated for Western blot, real-time PCR, and immunofluorescence staining.

Results : Notch-1 signaling was upregulated in human and mouse MG after Notch ligand treatment as confirmed by an increase in activated Notch-1 and Hes1. Notch-1 signaling was also upregulated after 7 days post IR-mediated retinal gliosis. Blockade of Notch signaling with PRAG1 inhibitor UM74 attenuated astrocytic and MG hypertrophy and GFAP expression as assessed by immunofluorescence staining and Western blot, respectively. Longitudinal retinal ganglion cell functional studies are underway.

Conclusions : Notch-1 signaling is induced after IR-mediated reactive gliosis, and its direct blockage by PRAG1 inhibition may be a potential therapeutic strategy.

This is a 2021 ARVO Annual Meeting abstract.