Activation of the CED3/ICE-Related Protease CPP32 in Cerebellar Granule Neurons Undergoing Apoptosis But Not Necrosis

Abstract

Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.

The Dietary Excitotoxins β‐N‐Methylamino‐l‐Alanine and β‐N‐Oxalylamino‐l‐Alanine Induce Necrotic‐ and Apoptotic‐Like Death of Rat Cerebellar Granule Cells

“…Transferring CGC to a low K+ -containing medium allowed the identification of a number of morphological (nuclear shrinkage and chromatin condensation) and biochemical (ohigonucleosomal DNA fragmentation, endonuclease inhibition by ATA) features of apoptosis, in agreement with previous studies (D 'Mello et al, 1993;Yan et al, 1994;Copani et al, 1995;Miller and Johnson, 1996;Armstrong et al, 1997). The morphological features of low K + -induced death were similar in many respects to those seen after exposure to BMAA or BOAA, supporting the involvement of an apoptotic-hike pathway.…”

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

“…DEVD-MCA cleavage activity was measured , as described by Armstrong et al (1997). After 12 hr in S-K5, 500,000 granule cells were washed once with PBS and lysed in 100 l of buffer A (10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM MgCl 2 , 1 mM DTT, and 1 mM PMSF, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and 1 g/ml leupeptin).…”

Role of N-terminal tau domain integrity on the survival of cerebellar granule neurons

“…As shown in Figure 6a, the caspase-3-like protease activity both in control uninfected apoptotic neurons as well as in cells overexpressing Lac-Z and undergoing apoptosis was approximately 10-fold elevated, confirming previous findings. 37 In contrast, granule neurons overexpressing tau (1-230) and tau (1-441) exhibited a progressively lower level of activation according to the MOI concentration employed. In order to determine whether tau inhibited the activity of caspase-3 protease or prevented its activation, which requires proteolytic processing of the inactive zymogens into activated p17 and p12 subunits, we examined the initial activation of the procaspase-3 in response to the apoptotic trigger.…”