Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration

Abstract

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques.

ER Stress Response and Induction of Apoptosis in Malignant Pleural Mesothelioma: The Achilles Heel Targeted by the Anticancer Ruthenium Drug BOLD-100

“…Therefore some PLC-IP3 pathway inhibitors were utilized in Ca 2+ observations and with all these inhibitors, the [Ca 2+ ] i peak, induced by BOLD-100 treatment, undergoes an almost complete abrogation. The alteration of the redox ER environment due to the overexpression of ERO1α sensitized IP3R and consequently caused an increase of the IICR [ 27 , 28 ]. The redox-sensitive binding of ERp44, an ER chaperone belonging to the thioredoxin family, controls IP3R inhibiting the IICR and protecting cells against apoptosis.…”

“…REN cells were seeded overnight in 96-wells plate, then incubated at 37 °C for 30 min with 20 μM t-BOC (t-butoxycarbonyl)-Leu-Met-CMAC(7-amino-4-chlorometylcoumarin) fluorogenic calpain substrate. After incubation, cells were washed twice, and the fluorescence emission was monitored with a plate reader (ex: 355 nm/em: 485 nm) [ 28 ].…”