Here we report that a novel member of the TNF-a family, TNF-related apoptosis-inducing ligand (TRAIL), contributes substantially to amyloid-induced neurotoxicity in human SH-SY5Y neuronal cell line. Involvement of TRAIL in the amyloidinduced cell death is supported by cDNA array, Northern blot, and Western blot data, demonstrating increased TRAIL expression after treatment of the cells with a neurotoxic fragment of amyloid protein (bAP). TRAIL was also found to be released in the culture media after bAP treatment with a time-course overlapping to contents of the intracellular protein. Contribution of TRAIL to bAP neurotoxicity is demonstrated by data showing that TRAIL-neutralizing monoclonal antibody protects neuronal SH-SY5Y cells from bAP neurotoxicity. Moreover, exposure of neuronal SH-SY5Y cells to TRAIL leads to cell death, indicating that this substance per se is endowed with neurotoxic properties. We also found that, similarly to bAP and TRAIL, activation of the death-domain adaptor protein FADD results in neuronal cell death. Lack of FADD function, by overexpression of its dominant negative, rescued cells from either TRAIL-or bAP-induced neurotoxicity, supporting the hypothesis that these three molecules share common intracellular pathways. Finally, we found that bAP strongly activated caspase-8, and the cell-permeable, selective caspase-8 inhibitor z-IETD-FMK prevents both bAP-and TRAIL-induced neurotoxicity. In view of TRAIL's potency in inducing neuronal death, and its role as mediator of bAP, it is plausible to hypothesize that TRAIL can be regarded as a molecule that provides substantial contribution to bAP-dependent cell death, which takes part in the progression of the neurodegenerative process and related chronic inflammatory response.

A common arginine to proline polymorphism is harboured at codon 72 of the human p53 gene. In this investigation, we found that fibroblasts and lymphocytes isolated from arginine allele homozygote centenarians and sexagenarians (Arg þ ) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro þ ). At variance, the difference in apoptosis susceptibility between Arg þ and Pro þ is not significant when cells from 30-year-old people are studied. Further, we found that Arg þ and Pro þ cells from centenarians differ in the constitutive levels of p53 protein and p53/MDM2 complex, as well as in the levels of oxidative stress-induced p53/Bcl-xL complex and mitochondria-localised p53. Consistently, all these differences are less evident in cells from 30-year-old people. Finally, we investigated the in vivo functional relevance of the p53 codon 72 genotype in a group of old patients (66-99 years of age) affected by acute myocardial ischaemia, a clinical condition in which in vivo cell death occurs. We found that Arg þ patients show increased levels of Troponin I and CK-MB, two serum markers that correlate with the extent of the ischaemic damage in comparison to Pro þ patients. In conclusion, these data suggest that p53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.

BackgroundHomeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase whose activity is fundamental in maintaining wild-type p53 function, thereby controlling the destiny of cells when exposed to DNA damaging agents. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer's Disease (AD) that led to an impaired and dysfunctional response to stressors.Methodology/Principal FindingsHere we examined the molecular mechanisms underlying the impairment of p53 activity in two cellular models, HEK-293 cells overexpressing the amyloid precursor protein and fibroblasts from AD patients, starting from recent findings showing that p53 conformation may be regulated by HIPK2. We demonstrated that beta-amyloid 1–40 induces HIPK2 degradation and alters HIPK2 binding activity to DNA, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus. Expression of HIPK2 was analysed by western blot experiments, whereas HIPK2 DNA binding was examined by chromatin immunoprecipitation analysis. In particular, we evaluated the recruitment of HIPK2 onto some target promoters, including hypoxia inducible factor-1α and metallothionein 2A.Conclusions/SignificanceThese results support the existence of a novel amyloid-based pathogenetic mechanism in AD potentially leading to the survival of injured dysfunctional cells.

The identification of biological markers of Alzheimer's disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinson's disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

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