Regulation of Fas-Associated Death Domain (FADD) adaptor and phosphorylated FADD in brain regions of kainic acid-treated mice: Increased hippocampal p-Ser191 FADD/FADD survival ratio

B. Keller, J.A. García-Sevilla. University of the Balearic Islands, IUNICS 07122, Spain

Kainic acid (KA, a stable analog of glutamate) induces severe excitotoxicity and neurodegeneration in the mammal brain (especially in hippocampal neurons) through the activation of kainate receptors, a major subtype of ionotropic glutamate receptors. Cell death by apoptosis is mediated by the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. Apoptosis requires the formation of a death-inducing signaling complex (DISC), which includes Fas receptor, FADD adaptor, initiator caspase-8, and FLIP (a catalytically inactive caspase-8 homolog), as well as the involvement of mitochondrial factors that can dampen (Bcl-2) or amplify (Bax, cytochrome c) death receptor signals. These apoptotic pathways converge to activate executioner caspases (e.g. caspase-3) that cleave vital substrates leading to cell death. Notably, FADD is a multifunctional protein that when phosphorylated at Ser191 can also induce non-apoptotic (survival) signals. This study examined the status of canonical apoptotic pathways, the activation of pro-apoptotic c-Jun NH2-terminal protein kinase (JNK), and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) in the brain of kainic acid-treated CD1 male mice. Target proteins were quantified in the hippocampus and cerebral cortex by Western blot analysis (at least three replicates) with specific and validated antibodies. Data (mean ± SEM, n=9) were analysed by Student’s t-test. Treatment with kainic acid (45 mg/kg, i.p., n=9), compared with saline-treated mice (n=9), induced a severe behavioral syndrome with seizures (60-90 min) which subsided in a few days (24-72 h). Therefore, neurochemical assays in hippocampus and cortex were performed 90 min and 72 h after KA. In KA-treated mice (72 h; greater effects), the content of Fas aggregates was modestly reduced (–12% ± 9% to –25% ± 8%, P<0.05), that of FADD decreased (–20% ± 20% to –43% ± 15%, P<0.05), and that of p-Ser191 FADD increased (+24% ± 24% to +41% ± 17%, P<0.05). Therefore, the ratio of FADD to p-Ser191 FADD, a survival marker, was increased after KA treatment (72 h) in both brain regions (hippocampus: +5.8-fold, P<0.05; cortex: +1.6-fold, P<0.05). Other DISC components (pro-apoptotic caspase-8 and anti-apoptotic FLIP) were unaltered. In the mitochondrial pathways (KA, 72 h), anti-apoptotic Bcl-2 and pro-apoptotic Bax (including Bcl-2/Bax ratio), cytochrome c (a potent caspase-3 activator) and caspase-3/fragments were unchanged in the hippocampus and cortex. In KA-treated mice (72 h), the activation of pro-apoptotic JNK1/2 (ratio of p-Thr183/Tyr185 JNK to total JNK) was markedly increased (hippocampus: +2.3-fold, P<0.05; cortex: +3.0-fold, P<0.05). Moreover, the proteolytic cleavage of nuclear PARP-1, assessed through the quantification of 85 kDa fragment, was increased after 72 h KA (hippocampus: +114% ± 46%, P<0.05; cortex: +42% ± 21%, P<0.05). The enhanced degradation of nuclear PARP-1, a hallmark of apoptosis, clearly indicates the induction of aberrant cell death in the hippocampus and cortex of KA-treated mice. KA neurotoxicity was not primarily associated with abnormalties of key components of mitochondrial pathway. The observed downregulation of Fas–FADD receptor complex and the increased p-Ser191 FADD/FADD ratio may reflect the induction of contraregulatory (survival) adaptations of multifunctional FADD to partly counteract KA neurotoxicity.

Supported by SAF2011-29918 and RETICS RD06/0001/0003 (MINECO-FEDER, Spain).