Inhibition of CPP32-Like Proteases Rescues Axotomized Retinal Ganglion Cells from Secondary Cell DeathIn Vivo

Abstract

The majority of retinal ganglion cells (RGCs) degenerate and die after transection of the optic nerve (ON) in the adult rat. This secondary cell death can primarily be ascribed to apoptosis. Recent work strongly suggests a decisive role for a family of cysteine proteases, termed caspases, as mediators of neuronal apoptosis. In this study, we investigated whether activation of caspases contributes to delayed death of RGCs after axotomy. Intraocular application of various caspase inhibitors rescued up to 34% of RGCs that would otherwise have died 14 d after ON transection. Using a modified affinity-labeling technique, we detected a 17 kDa protease subunit upregulated after axotomy. Upregulation was prevented by caspase inhibitor treatment. The 17 kDa protein was identified as a CPP32-like protease by Western blot analysis and affinity labeling with biotinylated acetyl-Asp-Glu-Val-Asp-aldehyde, which specifically inhibits CPP32-like caspases. In vivo application of the irreversible caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone revealed CPP32-like proteases to be major mediators of caspase-induced apoptosis in axotomized RGCs, because this inhibitor showed an even higher neuroprotective potential than the irreversible wide-range inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. In summary, the data presented here provide further insight into the mechanisms of injury-induced neuronal apoptosis and could give rise to more effective therapeutic intervention strategies in CNS trauma and neurodegenerative diseases.

Reduction of Potassium Currents and Phosphatidylinositol 3-Kinase-Dependent Akt Phosphorylation by Tumor Necrosis Factor-α Rescues Axotomized Retinal Ganglion Cells from Retrograde Cell DeathIn Vivo

“…It has been shown that this retrograde cell death can be ascribed to apoptosis (Garcia-Valenzuela et al, 1994;Rabacchi et al, 1994;Isenmann et al, 1997). Caspase-3 (CPP32)-like protease has been identified as an important mediator of apoptotic RGC death in our experimental paradigm (Kermer et al, 1998(Kermer et al, , 1999b. Morphological changes common to apoptotic cells include cell shrinkage.…”

“…Surgical procedures have been described elsewhere (K löcker et al, 1997; Kermer et al, 1998). Briefly, adult female Sprague Dawley rats (200 -250 gm) purchased from Charles River Wiga (Sulzfeld, Germany) were anesthetized by intraperitoneal injection of chloral hydrate (0.42 gm / kg body weight).…”

“…Retrograde labeling of RGC s was achieved by placing a small sponge soaked in 5% FluoroGold (FG; Fluorochrome, Denver, C O) at the ocular stump of the transected ON. Fourteen days later the retinas were processed as described (Kermer et al, 1998), blinded, and counted by two independent investigators. The counting was performed under a fluorescence microscope (Axiovert 35, Z eiss, Oberkochen, Germany) using a DAPI filter (365/420 nm).…”

Lens Injury Stimulates Axon Regeneration in the Mature Rat Optic Nerve

“…cAMP, through protein kinase A activation, augments the effects of growth factors by causing their receptors to translocate to the cell surface (Meyer-Franke et al, 1998), and similar effects are seen in vivo (Shen et al, 1999). Even in the absence of polypeptide growth factors, long-term survival can be maintained in vivo by overexpressing the anti-apoptotic protein Bcl-2 (Bonfanti et al, 1996) or by limiting caspase activity (Kermer et al, 1998). Enhanced survival by itself is insufficient to assure axon regeneration, however, because Bcl-2-overexpressing mice show almost no axon growth past the site of nerve injury (Chierzi et al, 1999).…”

Transduction of axotomized retinal ganglion cells by adenoviral vector administration at the optic nerve stump: an in vivo model system for the inhibition of neuronal apoptotic cell death

“…Cutting the rat optic nerve results in apoptotic cell death of more than 80% of the retinal ganglion cells, which are the only retinal cells projecting an axon into the optic nerve, 14 days after the lesion has been set. [17][18][19] In recent studies we have successfully used the intraocular injection of recombinant BDNF peptide along with free radical scavenger 20 and inhibitory peptides for caspases 21 to prevent apoptosis of RGCs. However, due to the short half-life of the peptides repeated injections were necessary, finally resulting in overall damage of the retina.…”