Daniela Uberti

Characterization of tau proteins in human neuroblastoma SH-SY5Y cell line.

Here we report three experimental paradigms in which tau proteins are differentially localized and expressed in human neuroblastoma cells SH-SY5Y. We found that in undifferentiated cells, tau proteins were predominantly localized in the nucleus. Western blot analysis of nuclear extracts revealed, among the others, a high molecular weight tau isoform and evaluation of tau mRNA levels showed a single tau isoform. After differentiation, tau immunoreactivity was detected only in cytosol and along neuritic processes. The high molecular weight tau isoform disappeared and an additional tau mRNA species was detected. Treatment of differentiated cells with doxorubicin or okadaic acid resulted in an increase of tau immunoreactivity and in a subsequent cell loss. Our results indicate that both subcellular localization and pattern of expression of tau proteins vary depending on the developmental and functional state of the cells, thus suggesting different roles in cell function.

Conformational altered p53 affects neuronal function: relevance for the response to toxic insult and growth-associated protein 43 expression

The role of p53 in neurodegenerative diseases is essentially associated with neuronal death. Recently an alternative point of view is emerging, as altered p53 conformation and impaired protein function have been found in fibroblasts and blood cells derived from Alzheimer’s disease patients. Here, using stable transfected SH-SY5Y cells overexpressing APP751wt (SY5Y-APP) we demonstrated that the expression of an unfolded p53 conformation compromised neuronal functionality. In particular, these cells showed (i) augmented expression of amyloid precursor protein (APP) and its metabolites, including the C-terminal fragments C99 and C83 and β-amyloid peptide (ii) high levels of oxidative markers, such as 4-hydroxy-2-nonenal Michael-adducts and 3-nitro-tyrosine and (iii) altered p53 conformation, mainly due to nitration of its tyrosine residues. The consequences of high-unfolded p53 expression resulted in loss of p53 pro-apoptotic activity, and reduction of growth-associated protein 43 (GAP-43) mRNA and protein levels. The role of unfolded p53 in cell death resistance and lack of GAP-43 transcription was demonstrated by ZnCl2 treatment. Zinc supplementation reverted p53 wild-type tertiary structure, increased cells sensitivity to acute cytotoxic injury and GAP-43 levels in SY5Y-APP clone.

Pharmacogenetics and Pharmagenomics, Trends in Normal and Pathological Aging Studies: Focus on p53

In spite of the fact that the aging organism is the result of complex life-long gene/environment interactions, making peculiar the susceptibility to diseases and the response to drugs, pharmacogenetics studies are largely neglected in the aged. Altered response to drugs, cardiovascular and metabolic alterations, cancer and dementia are among the age associated ailments. The latter two are the major contributors to illness burden for the aged. Aging, dementia and cancer share a critical set of altered cellular functions in the response to DNA damage, genotoxic stress, and other insults. Aging in higher animals may be influenced by the balance of cell survival versus death, a decision often governed by checkpoint proteins in dividing cells. The paper is mainly focused on one of such proteins, p53 which has been recently shown to be involved in aging and Alzheimers Disease (AD). Within this reference frame we studied p53 in aged controls and demented patients finding that with aging there is an increase of mutant like conformation state of p53 in peripheral blood cells, which is more pronounced in AD patients. As a result of such conformational change, p53 partially loses its activity and may become unable to properly activate an apoptotic program when cells are exposed to a noxious stimulus. Moreover we found that the tertiary structure of p53 and the sensitivity to p53-dependent apoptosis are affected by low concentrations of soluble beta amyloid, the peptide that accumulates in AD brain but also present in peripheral tissues. It is possible that p53 conformers may occur in the presence of misfolded molecules such as, but not limited to, beta amyloid. In particular at neuronal level the altered function of cell cycle proteins may affect synaptic plasticity rather than cell duplication.

Impaired DNA Repair Systems: Generation of Mitochondrial Dysfunction and Increased Sensitivity to Excitotoxicity

Preservation of genomic stability is an essential biological function, and cells engage efficient mechanisms to maintain DNA stability over time and prevent the generation and the persistence of impaired cells that may be detrimental to the organism. These mechanisms involve several DNA surveillance and repair proteins, such as the DNAmismatch repair system (MMR), nucleotide excision repair (NER), and base excision repair (BER) [1]. DNA damage may arise in cells as the result of endogenous and exogenous sources, such as free radical production during cellular metabolism, replication errors, ultraviolet ionizing radiation, and/or mutagenic agents. MMR has been widely investigated because mutations in MMR genes in humans are associated with hereditary nonpolyposis colorectal cancer (HNPCC) and a wide variety of sporadic tumors [2,3]. In eukaryotic cells, mismatches in DNA are recognized by the MutS homologs MSH2, MSH3, andMSH6, which initiate anMMR event. They form heterodimeric complexes, MSH2-MSH6 (MutSa) andMSH2-MSH3 (MutSb), which are required for the repair of base-base mispairs as well as single-base insertion/deletion loops (IDLs). An interaction between MutS and MutL homologs is required to activate subsequent repair events [4]. DNA repair is not the only function of theMMR system; the key proteins of MMR appear to play a dual role in (1) repairing DNA damage and (2) activating an apoptotic program in response to extensive DNA damage. MMR mutations allow improved survival of neoplastic cells, preventing the damageinduced apoptotic response [5]. Although the primary role of MMR takes place during replication, several independent observations indicate that MMR is active also in postmitotic cells, such as neurons. Little is known about the role of MMR in neurons, even if