Activation of Cytosolic Calpain, Not Caspase, Is Underlying Mechanism for Hypoxic RGC Damage in Human Retinal Explants

Abstract

Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often speciesspecific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. METHODS.Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. RESULTS. Activation of calpains in nerve fibersand increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945.CONCLUSIONS. An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.

Inhibition of calpain reduces cell apoptosis by suppressing mitochondrial fission in acute viral myocarditis

“…Moreover, PD150606, a nonpeptide, cell-permeable selective calpain inhibitor, inhibits calpains by binding to the calcium-binding site of the enzyme. Numerous studies have proved that calpain is a promising therapeutic target in many diseases, such as I/R injury (Zu et al 2020), cardiomyocytes hypertrophy (Li et al 2020), and renal diseases (Kobayashi-Otsugu et al 2020). Consist with other researches, our results demonstrated that the inhibition of calpain, Fig.…”

“…Moreover, PD150606, a nonpeptide, cell-permeable selective calpain inhibitor, inhibits calpains by binding to the calcium-binding site of the enzyme. Numerous studies have proved that calpain is a promising therapeutic target in many diseases, such as I/R injury (Zu et al 2020 ), cardiomyocytes hypertrophy (Li et al 2020 ), and renal diseases (Kobayashi-Otsugu et al 2020 ). Consist with other researches, our results demonstrated that the inhibition of calpain, both by calpastatin overexpression and PD150606 intervention, attenuated myocardial injury, alleviated cardiac inflammation, improved mitochondrial function, and reduced apoptosis in VMC, implying calpain activation promoted the severity of CVB3-induced myocarditis.…”

Metabolomic analysis of amino acids and organic acids in aging mouse eyes using gas chromatography–tandem mass spectrometry

“…The activity of glutamine synthase has also been reported to diminish during aging owing to oxidation (Sahakian et al, 1995; Smith et al, 1991). In this study, these findings may explain the association between physiological states and altered AA metabolism, which was similar to those in previous reports (Chua et al, 2013; Kobayashi‐Otsugu et al, 2020; Wang et al, 2018).…”

“…It has been reported organisms are more exposed to reactive oxygen species as they age and that reactive oxygen species cause the carboxylation of proteins, which reduces protein activity (Chakravarti & Chakravarti, 2007;Levine & Stadtman, 2001;Stadtman, 2006). In a previous study, the association between calpains and hypoxia-induced damage in retinal ganglion cells was reported in the human retinal explants (Kobayashi-Otsugu et al, 2020). In addition, it has been suggested that proteins can be oxidized by metal-catalyzed oxidation (Garland, 1990;Sell & Monnier, 2011;Stadtman, 2006).…”

Calpains as mechanistic drivers and therapeutic targets for ocular disease

“…One of the clinical hallmarks of AMD is drusen: protein and lipid aggregates that accumulate between the RPE and choroid. Notably, retinal explants from monkeys and humans under hypoxic stress (which models AMD in vitro) showed calpain activation due to accumulation of N-retinylidene-N-retinylethanolamine (A2E; a pyridinium bisretinoid), a principal component of drusen [120,121]. Importantly, the degenerating explants were rescued by application of a generic calpain inhibitor, SNJ-1945, suggesting that calpain activation is involved in AMD progression (Figure S5A in the supplemental information online) [122].…”