Necroptosis is an important mode of neuronal cell death and involves autophagy in a model of retinal ischemia.

TNF-alpha plays a largely deleterious role in ischemia-reperfusion injury in an in vivo model of retinal injury. Direct neutralization of this cytokine partially preserves retinal function. The diverse characteristics of TNF-alpha are attributed in part to the timing of its expression after injury. TNF-alpha receptor expression and function, along with combination treatments targeting death receptor-mediated apoptosis, should be further explored to develop neuroprotective therapeutic strategies for acute retinal ischemic disorders.

© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n . ABSTRACTAnalogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect-while TLCK had no effect-on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISPreactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase-vs FLISP-reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.

Following an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the Blood-Retinal-Barrier would provide for novel systemic intervention that protects the retina from acute injury and lost function. Here we study a novel calpain peptide inhibitor, cysteic–leucyl–argininal (CYLA), in an in vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA-prodrug was administered intraperitoneally before and/or after ischemia-reperfusion at concentrations of 20-40 mg/kg. We found that administering 20 mg/kg CYLA even only after ischemia provides significant preservation of retinal function.

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