Inhibition of the interleukin-1β converting enzyme family rescues neurons from apoptotic death

Abstract

The interleukin-1␤ converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH 2 F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 M) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH 2 F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH 2 F), and CPP32-like proteases (Z-DEVD-CH 2 F), failed to prevent apoptotic death. An 35 S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH 2 F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH 2 F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.

Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium

“…To investigate whether cleavage of DNA occurred in the absence of caspase-3, we performed DNA fragmentation analysis. Cleavage of DNA, which is normally observed within 8 hr of K ϩ deprivation (Yan et al, 1994;Lynch et al, 1997;D'Mello et al, 1998), is not detectable in caspase-3 -deficient neurons even at 18 hr after deprivation (Fig. 5).…”

“…In normal cerebellar granule neuron cultures from the rat, low-K ϩ -mediated death can be inhibited by boc.Asp.fmk, a pan-caspase inhibitor (Lynch et al, 1997;D'Mello et al, 1998). As is shown in Figure 6, boc.Asp.fmk also inhibits apoptosis in murine neurons, though somewhat less effectively (62% survival at 24 hr).…”

Caspase-dependent cleavage of the retinoblastoma protein is an early step in neuronal apoptosis

“…Degradation of RB is reversed by inhibitors of the DEVD-and YVAD-types (Dou and An, 1998;Janicke et al, 1996;Tan et al, 1997), suggesting that two Lynch et al, 1997;Marks et al, 1998). Cleavage of this substrate started at 3 ± 4 h of K + deprivation, with a maximum of 10-fold at 10 h. The best characterized DEVD substrate preferring caspase is CPP32, whose proteolytic cleavage by upstream caspases generates an active p20 subunit (Armstrong et al, 1997;Nicholson et al, 1995).…”

“…RB cleavage was blocked by the z-VAD.fmk inhibitor, as well as with z-DEVD.fmk (Figure 1d). The¯uorogenic substrate Ac-DEVD-amc is cleaved with a very high eciency (Figure 2, Armstrong et al, 1997;Lynch et al, 1997;Marks et al, 1998), pointing to CPP32 (caspase 3) as a good candidate for caspases involved in RB cleavage during CGN apoptosis. CPP32 has been shown to be required for developmental apoptosis in neurons, whereas it is not obligatory in some other cell types (Kuida et al, 1996).…”

“…It is documented that K + -induced apoptosis of CGN is inhibited by broad spectrum caspases inhibitors, such as Asp(D)-coupled derivatives (Dichlorobenzoyl or Fluoromethylketone) (D'Mello et al, 1998;Lynch et al, 1997;Nath et al, 1996;Tanabe et al, 1998). A panel of synthetic tetrapeptide inhibitors is now available and can be used to distinguish between activation of the dierent ICE and CED3 subfamilies of caspases (see classi®cation of caspases in Alnemri et al, 1996).…”