Abstract
Purpose :
Reduced levels of neurotrophic cytokines accelerate neuronal cell death in the retina during injury, but prolonged overexpression can lead to inflammation and retinal damage. It is therefore critical to find molecular targets that regulate endogenous production of these neurotrophic factors. Müller glia (MG) upregulate Leukemia Inhibitory Factor (LIF), a predominant neurotroph, during injury. They also express Caveolin-1 (Cav1) outside of canonical caveolae which is also upregulated during retinal injury. Neuroretinal Cav1 modulates neuroprotective signaling. The objectives for this project were to determine if neuroretinal Cav1 is required for LIF production and if the sequestration of Cav1 into caveolae impacts LIF production in MG during injury.
Methods :
Neuroretinal (NR)-Cav1 knockout (KO) mice were generated by Chx10-mediated Cre recombination in Cav1 floxed mice. NR-Cav1 KO mice and wild-type (WT) littermates were challenged with NMDA (N-methyl-D-aspartate) via intravitreal injection. The uninjected contralateral eye served as a naïve control. Retinas were extracted and assessed for LIF and other neuroprotective cytokine levels at 8 hours post-injection using BioPlex suspension array. For in vitro studies, we either silenced Cav1 using Cav1-specific shRNA or sequestered Cav1 into caveolae by over-expressing Cavin1/PTRF using Adenovirus-mediated transduction in the MIO-M1 MG cell line. We subsequently used ELISA to quantitatively measure LIF secretion in Cav1-silenced or Cavin1/PTRF-transduced MIO-M1 MG with or without TNF-α or LPS stimulation.
Results :
Administration of NMDA significantly induces the expression of LIF in mouse retinas compared to naïve controls. Moreover, LIF expression was significantly reduced in NR-Cav1 KO retinas as compared to WT retinas. Similarly, both TNF-α and LPS stimulation induces the secretion of LIF from MIO-M1 MG. Both Cav1 silencing and Cavin1/PTRF overexpression significantly suppresses TNF-α and LPS- induced LIF secretion in MIO-M1 MG.
Conclusions :
Our results suggest that non-caveolar Cav1 in MG regulates the production of LIF and possibly other neuroprotective cytokines in response to neurotoxic insults and retinal degenerative conditions. Future studies will focus on the mechanistic role of Cav1 in MG response to injury and the effect of sequestration of Cav1 into caveolae by overexpressing Cavin1/PTRF in MG in situ during injury conditions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.