Differential regulation of Smac/DIABLO and Hsp-70 during brain maturation

Abstract

The heat shock protein (Hsp) system is a cell defense mechanism constitutively expressed at the basal state and essential for cell survival in response to damaging stimuli. Apoptosis is a physiological cell death program that preserves tissue homeostasis. We investigated the intrinsic pathway of apoptosis at various stages of brain maturation in CD-1 mice, triggered by two mitochondrial proapoptotic proteins, cytochrome c and Smac/DIABLO, and the pathway's regulation by Hsp-70. Smac/DIABLO and Hsp-70 proteins were upregulated 2-fold and 1.5-3-fold, respectively, after birth. In contrast, in the presence of cytochrome c/2'-deoxyadenosine 5'-triphosphate (dATP), caspase activity in mouse brain cell-free extracts increased 90-fold and 61-fold, at fetal and neonatal stages, whereas no activation was detected 15 days postnatally or at any subsequent times. These results indicate that the activation pattern of the intrinsic pathway of apoptosis undergoes a marked shift during postnatal maturation.

Fig. 1

Western blot analysis of Smac/DIABLO protein during brain maturation. (A) Representative Western blot of 50 µg of cell-free extracts of mouse brain tissue at various stages of maturation: fetal (14–16 days) (lane 1) and postnatal (0–1 day; lane 2), 15–30 days (lane 3), 3–4 months (lane 4) and 8–12 months (lane 5). A positive control consisted of recombinant Smac/DIABLO protein, which was observed as a single band of 25 kDa (lane c). (B) Densitometric values for relative Smac/DIABLO protein from (A) were obtained as described in Materials and Methods section. Values are mean ± SD, representative of three independent experiments. A one-way ANOVA analysis gave a P-value 0.004

Fig. 2

Activation of the intrinsic pathway of apoptosis during brain maturation. Fifty micrograms of cell-free extracts of mouse brain tissue at several stages of maturation—fetal (14–16 days) (lane 1) and postnatal (0–1 day; lane 2), 15–30 days (lane 3), 3–4 months (lane 4) and 8–12 months (lane 5)—were activated with cyt c/dATP. Control samples in the absence of cyt c/dATP were run under the same conditions. Caspase activation was measured fluorometrically as described in Materials and Methods section and expressed as rates of substrate (Ac-DEVD-AFC) hydrolysis (V). The data are presented as means ± SD, representative of three independent experiments

Fig. 3

Western blot analysis of Hsp-70 during brain maturation. (A) Representative Western blot of 50 µg of cell-free extracts of mouse brain tissue at various stages of maturation: fetal (14–16 days) (lane 1) and postnatal (0–1 day; lane 2), 15–30 days (lane 3), 3–4 months (lane 4) and 8–12 months (lane 5). A positive control consisted of 50 µg of recombinant Hsp-70 protein (lane c). (B) Densitometric values for relative Hsp-70 protein concentrations from (A) were obtained as described in Materials and Methods section. Values shown are means ± SD, and are representative of three independent experiments. A one-way ANOVA analysis gave a P-value below 0.001

Fig. 4

Model of the intrinsic pathway of apoptosis during brain maturation. In the intrinsic pathway of apoptosis, cyt c is released to the cytosol, where in the presence of dATP it assembles with Apaf-1 and procaspase-9 (pC9). Caspase 9 (C9) activates procaspase-3 (pC3) thus leading to apoptosis. Caspase-9 and -3 (C3) activities are regulated by the inhibitor of apoptotic proteins (IAPs). Smac/DIABLO counteracts IAP’s inhibitory role, thus favoring apoptosis. During brain maturation, the mechanism by which apoptosis activation by cyt c/dATP is decreased during maturity is linked to the alteration in the expression levels of Apaf-1, Hsp-70 and/or procaspase-9 and -3. Further details are provided in the main text