Role of SERPINI1 in the retinal function and determining the effects of oxidative stress on serpin expression

Abstract

Purpose : SERPINI1 is a member of serine protease inhibitor family and is expressed in neuronal tissues such as brain and retina. SERPINI1 upregulation in retina has been shown to be neuroprotective in acute ischemic injury. This study investigated the effects of SERPINI1 neutralisation on inner retinal function in healthy animal eyes. SERPINI1 is susceptible to oxidative stress, therefore we also investigated the effects of oxidative stress on SERPINI1 expression changes in 661W photoreceptor cells and change in autophagy markers.

Methods : SERPINI1 or IgG antibody was administered intravitreally in healthy mice retina (weekly once for 8 weeks, 2µl, n=20). ERGs and positive scotopic threshold response (pSTR) was measured using Phoenix Ganzfield (-4.3log cd s/m²) after 8 weeks. The amplitude of each component was measured and analysed. 661W photoreceptor cells were cultured, incubated with hydrogen-peroxide (10µM) and harvested after 8 and 24 hours. Western blotting was evaluated SERPINI1 and expression of autophagy markers.

Results : The SERPINI1 neutralisation by intravitreal injection of anti-SERPINI1 in C57BL/6 mice retinas showed no change in ERG but significant loss of pSTR amplitude as compared to IgG injected or no-injection control counterparts: mean±SEM; control vs IgG vs anti-SERPINI; 51.2±3.42 µV vs 47.02±6.03 µV vs 30.37±4.12 µV (p<0.007). Western blot showed that SERPINI1 was expressed in 661W cells and was significantly elevated after 8 hrs (81.81±9.43 vs 227.7±0.72; p<0.0001, n=3) but its expression reduced at 24 hrs following H2O2 treatment. H2O2 also significantly enhanced Beclin1 (p<0.0008), LC3II (p<0.04), Atg3 (p<0.05), Atg7 (p<0.02) and Atg16 L1 (p<0.03) after 8 hrs and expression of some of these markers was then reduced after 24 hrs. Therefore, there was an early increase in SERPINI1 in response to oxidative stress which paralleled the change in autophagy markers.

Conclusions : Blocking SERPINI1 in healthy retinal neurons diminished inner retinal function as determined by pSTR amplitude reduction. The mechanism for this is unknown but may reflect increased excitotoxicity or synaptic dysfunction. Further studies are needed to clarify relationship between SERPINI1 and autophagy in photoreceptor cells. Long term oxidative stress may induce changes in SERPINI1 levels that could render neurons more susceptible to degenerative change.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.