Molecular and Translational Medicine

The neurotoxlc effect of lead has been studied measuring the function of different neurotransmitters in various rat brain areas. Dopaminergic and GABAergic systems are modified at various steps : both at presynaptlc and postsvnnDtic (receptors) levels. The changes observed, together with those re~ted with other.brain neurotransmitters, suggest the hypothesis that the behavloural and clinical changes observed in humans exposed to lead may be explained on the bases of selective actions of lead on neurotransmltters ~n discrete brain areas.

Apoptosis is now recognized as an important component in many progressive and acute neurodegenerative diseases. Extracellular signals and intracellular mechanisms triggering and regulating apoptosis in neuronal cells are still a matter of investigation. Here we review data from our and other laboratories with the aim to elucidate the nature of some proteins which are known to be involved in cell cycle regulation as well as in promoting degeneration and apoptosis of neurons. The following molecules will be taken into consideration: NF-kB, p53, p21 and MSH2. These proteins are activated by neurotoxic experimental conditions which involve the stimulation of selective receptors for the excitatory aminoacid glutamate. Thus, we hypothesize their contribution to an intracellular pathway responsible for the glutamateinduced neuronal death. Identification of such mechanisms could be relevant for understanding the apoptosis associated with various neurodegenerative diseases as well as for developing novel strategies of pharmacological intervention.

On March 19, 2008 a Symposium on Pathophysiology of Ageing and Age-Related diseases was held in Palermo, Italy. Here, the lectures of M. Racchi on History and future perspectives of Alzheimer Biomarkers and of G. Scapagnini on Cellular Stress Response and Brain Ageing are summarized. Alzheimer's disease (AD) is a heterogeneous and progressive neurodegenerative disease, which in Western society mainly accounts for clinica dementia. AD prevention is an important goal of ongoing research. Two objectives must be accomplished to make prevention feasible: i) individuals at high risk of AD need to be identified before the earliest symptoms become evident, by which time extensive neurodegeneration has already occurred and intervention to prevent the disease is likely to be less successful and ii) safe and effective interventions need to be developed that lead to a decrease in expression of this pathology. On the whole, data here reviewed strongly suggest that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate AD cases from normal ageing, Parkinson's disease and other dementias. On the other hand, available data on the involvement of curcumin in restoring cellular homeostasis and rebalancing redox equilibrium, suggest that curcumin might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation, such as AD.

Background: Homeodomain 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.

X-linked lymphoproliferative disease (XLP) is a rare and severe immune deficiency, characterized by abnormal immune responses to the Epstein-Barr virus. Recently, the gene responsible for XLP, SH2D1A, has been identified and shown to code for a small cytoplasmic protein with an SH2 domain that interacts with SLAM and 2B4, two receptorial molecules involved in signal transduction in T and NK cells, respectively. A variety of SH2D1A gene mutations have been reported thus far in XLP males. Here we describe a single-strand conformation polymorphism assay for mutation analysis in XLP. Four novel patients with SH2D1A mutations are described. These mutants, and the others previously reported in the literature, have been included in a Registry (SH2D1Abase) that is fully accessible on the World Wide Web. A three-dimensional model of the SH2 domain of the SH2D1A protein has been developed, based on homology with other SH2 domains. The structural consequences of disease-causing SH2D1A mutations are discussed.

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNPkeyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4 + T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Background: According to the current clinical criteria, definite Alzheimer's disease (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, known as mild cognitive impairment (MCI), not all MCI patients 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. Recently a role for conformationally altered p53 as a novel candidate biomarker for early onset AD has been described. The aim of our work is to investigate the usefulness of this method especially for younger patients, thus supporting its putative application for subjects with MCI and earlier in the clinical course of AD. Methods: We used a flow-cytometric approach to investigate the different expression of conformationally altered p53 among MCI, AD and non-AD subjects on peripheral blood cells. Results: We found that peripheral blood cells from MCI specifically expressed increased levels of unfolded p53 compared to age-matched controls. We found that the expression of conformationally altered p53 is age dependent. For our preliminary data analysis we have arbitrarily worked out the related cut-points by linear regression, taking as reference linear fit of controls, thus dividing the subjects in specific age interval segments. Young (< 70 years) MCI patients show levels of conformationally altered p53 comparable to those measured in AD patients, but significantly different from subjects of control group. Conclusions: The highest levels of conformationally altered p53 have been observed in amnestic MCI patients, which represent an early stage of AD, thus supporting the promise of this protein measurement to accurately identify this at-risk group.

This study points out different behaviour between HEK cells overexpressing wild-type or mutant APP when exposed to oxidative insult. Although apparently both APPwt and APPmut overexpression conferred resistance to oxidative insult, some differences in terms of degree of protection was observed in the two clones. We found that the two clones differed, especially, in terms of redox profile. HEK-APPmut cells were characterized by higher levels of oxidative markers in comparison with HEK-APPwt. In addition, SOD activity appeared more efficient in HEK-APPwt than in HEK-APPmut, thus justifying the differences in terms of cell survival in the two clones. We suggest that, according to ''hormesis theory'', in HEK-APPwt cells low amount of oxidative stress can exert a beneficial effect that at a higher intensity results harmful. In contrast, HEK-APPmut cells lost this stress resistance probably because the degree of oxidative stress is too high and the antioxidant enzymes are themselves compromised.

We previously demonstrated that the dopaminergic agonist pergolide, independently from its DA agonist activity, can exert neuroprotective effects against cell death induced in SH-SY5Y neural cells by H 2 O 2 treatment. Since oxidative stress in SH-SY5Y neural cells is known to activate the NF-kB pathway we tested the hypothesis that pergolide may interfere with NF-kB activity. Based on Western blot analysis and immunocytochemistry, pergolide was found to prevent H 2 O 2 -induced apoptosis by inhibiting NF-kB nuclear translocation and activation of p53 signalling pathway. Similarly, the cell-permeable SN50 peptide, which is known to block NF-kB nuclear translocation, prevented both H 2 O 2 -induced p53 expression and apoptosis. The mechanism of action of pergolide responsible for neuroprotection differed from that of antioxidants. In fact, Vitamin E, contrary to pergolide and SN50, rescued neuronal cells from H 2 O 2 -induced apoptosis acting upstream NF-kB activation, as demonstrated by the prevention of H 2 O 2 -induced IkB degradation. These data suggest a novel site of action of pergolide that may account for additional pharmacological properties of this drug. # 2004 Elsevier Inc. All rights reserved.

Background: Pramipexole exists as two isomers. The S(-) enantiomer is a potent D 3 /D 2 receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. Very limited studies are available to characterize the pharmacological spectrum of the R(+) enantiomer of pramipexole. Results: Using differentiated SH-SY5Y neuroblastoma cells as an experimental model, here we show that S(-) and R (+) pramipexole are endowed with equipotent efficacy in preventing cell death induced by H 2 O 2 and inhibiting mitochondrial reactive oxygen species generation. Both pramipexole enantiomers prevented mitochondrial ROS generation with a potency about ten times higher then that elicited for neuroprotection. Conclusions: These results support the concept of both S(-) and R(+) pramipexole enantiomers as mitochondriatargeted antioxidants and suggest that the antioxidant, neuroprotective activity of these drugs is independent of both the chiral 6-propylamino group in the pramipexole molecule and the DA receptor stimulation.

Early onset generalized dystonia is a severe form of primary dystonia linked to a mutation of the DYT1(TOR1A) gene on chromosome 9q34. DYT1 gene codifies for human torsinA, an AAA+ ATPase associated with the membranes of endoplasmic reticulum (ER) and the synaptic vesicles and proposed to be involved in trafficking of tubular -vesicular membrane through neuronal processes. In this study, the presence and the intracellular distribution of torsinA protein in SH-SY5Y neuroblastoma cells were evaluated by immunofluorescence and Western blot analysis following differentiation with retinoic acid and BDNF. Protein expression was then inhibited by transient antisense transfection and the possible effect on neurite outgrowth was observed. In SH-SY5Y cells torsinA, with an apparent MW of 38 kDa, is endogenously present and distributed, with a punctate pattern, in the cytosol and along the neurites. The protein showed high intensity of immunoreactivity in the neurite varicosities and was partially co-localized with vesicles markers. Terminally differentiated cells showed an increase of protein expression. Oligonucleotide antisense treatment induced a significant response to differentiating stimuli, lead to sprouting of longer neurites and increase in growth cone areas. A relationship between torsinA and tau protein, which is involved in axon elongation and establishment of neuronal polarity, was demonstrated by co-immunoprecipitation experiments. These findings suggest that torsinA, throughout the interaction with microtubule associated proteins, may contribute to control neurite outgrowth and could be involved in maintaining cell polarity. D

The molecular mechanism(s) responsible for the differential expression of various tau protein isoforms as well as their functional role in morphogenesis, neurofibrillary tangle formation and neurodegeneration have not been completely clarified.