p53 and Alzheimer’s disease: from biological marker to molecular mechanism

Alzheimer’s disease (AD) is the most common cause of dementia among the elderly population and its incidence doubles every five years between 65 and 85 years of age. The treatment of AD remains a major challenge because of an incomplete understanding of the events that lead to the selective neurodegeneration typical of Alzheimer’s brains. In view of existing and emerging therapeutic compounds, there is an increasing interest to develop tecniques allowing an accurate detection of the earliest phase of the disease. According to the current criteria, definite AD can only be diagnosed following neuropathological examination of brain samples, obtained by biopsy or autopsy. Furthermore, when evaluating the intermediate state between normal aging and established AD, commonly known as mild cognitive impairment (MCI), not all patients with MCI progress to AD and hence there is a need of a reliable prediction tool able to identify which patients with MCI will progress to AD. The current inability of clinical criteria to accurately identify this at-risk group underscores the importance of developing biomarkers able to potentially supplement the clinical approaches. Candidate biochemical markers for AD should be molecules representing some of the cerebral pathogenetic processes typical of AD or representing altered metabolic or cellular processes possibly identified either in brain or peripheral tissues from affected patients. To date, three cerebrospinal fluid (CSF) biomarkers have shown the highest diagnostic potential: total-tau, phospho-tau and Abeta 1-42. We have been studying AD biomarkers in fibroblasts since 1993. These cells can be considered a good model to study in vitro the dynamic alterations of metabolic and biochemical processes that may reflect events occurring in the AD brain. Most recently we demonstrated an abnormal response of AD fibroblasts to an acute oxidative injury. Fibroblasts from AD patients were found to be less vulnerable to the oxidative injury induced by H2O2 in comparison with fibroblasts from non-AD subjects. The protective mechanism involved an impairment of the ROS activated p53-dependent apoptosis. On the basis of immunoprecipitation studies with conformation-specific p53 antibodies, we found that in fibroblasts from AD patients a significant amount of total p53 assumes an unfolded tertiary structure; such alteration can compromise p53 response to an acute injury elicited by an excess of free radical production. Mutant p53 found in AD fibroblasts has been demonstrated to be independent from gene mutations on the basis of sequence analysis of the p53 gene, thus suggesting that one of the peripheral events associated to the disease is responsible for generating a conformationally altered p53 isoform. Consistently with this hypothesis we found that nanomolar concentrations of beta-amyloid peptide were responsible for the induction of unfolded p53 protein isoform in fibroblasts derived from non-AD subjects. Recently, we set up a rapid and easy flow-cytometric approach to investigate the expression of conformationally altered p53 in AD and non-AD subjects in peripheral blood cells and the findings were that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate Alzheimer’s cases from normal ageing, Parkinson’s disease and other dementias. In particular, the measurement of conformational mutant p53 has been demonstrated to be highly sensitive mainly in young patients with a sensitivity of 90% in subjects up to 70 years of age. The main potential advantage of our method was the development of a biological sample preparation procedure as well as of an analytical method that allows routine analysis, by starting from small blood volumes (3-4 ml) and by using a flow-cytometric method, widely diffused either in hospitals or in diagnostic laboratories. On the basis of these data, one of the aim of the current proposal is to investigate further the usefulness of this method especially for younger patients, supporting its putative application for subjects with mild cognitive impairment (MCI) and earlier in the clinical course of AD.