SUMOylation of Retinal Proteins in Aroused vs. Torpid Ground Squirrels

Abstract

Purpose: : The Small Ubiquitin-Like Modifier (SUMO) is a 100 amino acid protein that is covalently bound to target proteins by the Ubc9 enzyme. Thus, "SUMOylation" is a post-translational modification that draws upon a pre-existing pool of free SUMO. SUMOylation is hypothesized to spare protein conjugates from proteasomal degradation. SUMOylation of brain proteins is up-regulated during hibernation torpor in the 13-lined ground squirrel (GS), which naturally tolerates a repeated ischemia-reperfusion cycle over several months (Lee et al. J. Cereb. Blood Flow Metab. 2007). Protein sparing by SUMOylation might thus be a key neuroprotective strategy in the CNS. We tested the hypothesis that SUMOylation of retinal proteins would be similarly up-regulated during torpor in the cone-dominant GS retina.

Methods: : Young of the year GSs from the UW Oshkosh captive colony were used. Hibernation-naïve (aroused) GSs were sacrificed in August, October and November, whereas first-hibernation (torpid) GSs were sacrificed in October and December. Retinas were processed for in situ immunolabelling, Western blotting, and reverse transcriptase-PCR. Immunoprobes (gifts of Dr. Lee) and primers that distinguished SUMO-1 from SUMO-2/3 were used.

Results: : Messenger RNAs for SUMO-1 and SUMO-2/3 were detected in retinas from aroused and torpid GSs. Compared to blots from aroused GSs, blots from torpid GSs demonstrated down-regulated free SUMO-2/3 (M_r 16 kD) alongside up-regulated SUMO-2/3 protein conjugates (M_r 60-200 kD). A similar but less-robust response was seen on blots probed with anti-SUMO-1. Retinal SUMO conjugates were not identical to those identified in brain (Lee et al. 2007). In situ immunoreactivity (IR) for both types of SUMO in August/aroused GS retina was observed in the RPE and all retinal layers except for two regions in photoreceptors: their outer segments and ellipsoid mitochondria. SUMO-IR in November/aroused and December/torpid GS retinas was reduced, relative to that seen in August/aroused retinas, particularly sclerad of the inner plexiform layer.

Conclusions: : Our Western blot data support the hypothesis that SUMOylation of retinal proteins is upregulated during torpor when the GS brain exhibits resilience to repeated ischemia-reperfusion cycles. Reduced in situ SUMO-IR during torpor may reflect epitope masking resulting from this upregulated SUMOylation of protein targets, combined with their cross-linking by aldehyde fixation.

Keywords: neuroprotection • protein modifications-post translational • retina