Intracerebroventricular (i.c.v.) injection of gp120 in the adult rat causes microglial cell activation and enhanced expression of IL-1ß and this seems to be responsible for neuronal apoptosis in the neocortex (see Corasaniti et al., 2001). Thus, the antagonist of IL-1 receptor type I (IL-1ra) and Ac-YVAD-chloro-methylketone (Ac-YVAD-cmk), a specific inhibitor of caspase 1, afford neuroprotection (see Corasaniti et al., 2001). Here we now report that gp120 induces cyt-c translocation and this, but not elevated IL-1ß, is minimized by Ac-YVAD-dmk, a caspase-1 inhibitor (Thornberry et al., 1992).

Under chloral hydrate (400 mg/kg i.p.) anaesthesia, male Wistar rats (250-280 g) were stereotaxically implanted with a guide cannula (25 gauge) into one lateral cerebral ventricle as previously described (see Bagetta et al., 1999). The animals were allowed 4 days recovery before treatment. Then, a single dose (100 ng i.c.v.; 2 µl volume; 1µlmin^-1 rate) of gp120 was given alone or in combination with caspase 1 inhibitors to each rat with a 5 µl Hamilton syringe. Six hours after the last treatment the animals have been sacrificed and the brain cortical tissue dissected out. Immunoreactive IL-1ß was assayed in cytosolic and mitocondrial fractions from individual brain cortical tissue samples by an established, rat specific ELISA (see Corasaniti et al., 2001); cyt-c (mouse monoclonal ab Pharmingen San Diego, CA; 1:2000 dilution) was measured by western blotting (see Corasaniti et al., 2001). Cyt-c has also been studied by immunogold electron microscopy (Pharmingen San Diego, CA; 1:10 dilution). In the antagonism study, injection of gp120 was preceded (1 h beforehand) by Ac-YVAD-dmk or by Ac-YVAD-cmk. Immunoelectron microscopy (n=3) and western blot analysis (n=3) demonstrate that gp120 causes cyt-c translocation in the brain neocortex of rat. Densitometric analysis (see Corasaniti et al., 2001) of cyt-c immunoreactive band revealed that Ac-YVAD-dmk (50 pmoles i.c.v.) caused an approx. 60% reduction of cyt-c vs gp120 (103.3+8.2 vs 160.3+16.4 arbitrary units; P<0.05, ANOVA followed by Tukey-Kramer). This treatment did not affect gp120 increased mitochondrial (10.7+2.4 vs gp120 9.4+1 pmolmg^-1 protein; P>0.05) and cytosolic (12.3+4.1 vs gp120 9.1+1.2 pmolmg^-1 protein) IL-1ß. Likewise Ac-YVAD-dmk, a neuroprotective dose (100 pmoles i.c.v.) of Ac-YVAD-cmk (Bagetta et al., 1999), did not reduce enhanced IL-1ß levels by gp120 into the cytosolic (11.7+3.1 pgmg^-1 protein; n=3, P<0.05 vs control) and mitochondrial (21.3+1.7 pgmg^-1 protein; n=3, P<0.001 vs control and vs gp120 given alone) fractions. In conclusion, under the present experimental conditions, caspase 1 inhibitors seem to afford neuroprotection via inhibition of cyt-c translocation.

Bagetta G. et al. (1999) Neuroscience 89, 1051-1066.
Corasaniti M.T. et al. (2001) J. Neurochem. 79, 1-8.
Thornberry N.A. et al. (1992) Nature, 356, 768-774.

Supported by FIRB (MIUR) and II AIDS Project (ISS), Rome.