Cristina Lanni

Depression and antidepressants: molecular and cellular aspects

Clinical depression is viewed as a physical and psychic disease process having a neuropathological basis, although a clear understanding of its ethiopathology is still missing. The observation that depressive symptoms are influenced by pharmacological manipulation of monoamines led to the hypothesis that depression results from reduced availability or functional deficiency of monoaminergic transmitters in some cerebral regions. However, there are limitations to current monoamine theories related to mood disorders. Recently, a growing body of experimental data has showed that other classes of endogenous compounds, such as neuropeptides and amino acids, may play a significant role in the pathophysiology of affective disorders. With the development of neuroscience, neuronal networks and intracellular pathways have been identified and characterized, describing the existence of the interaction between monoamines and receptors in turn able to modulate the expression of intracellular proteins and neurotrophic factors, suggesting that depression/antidepressants may be intermingled with neurogenesis/neurodegenerative processes.

p53 at the crossroads between cancer and neurodegeneration.

Aging, dementia, and cancer share a critical set of altered cellular functions in response to DNA damage, genotoxic stress, and other insults. Recent data suggest that the molecular machinery involved in maintaining neural function in neurodegenerative disease may be shared with oncogenic pathways. Cancer and neurodegenerative diseases may be influenced by common signaling pathways regulating the balance of cell survival versus death, a decision often governed by checkpoint proteins. This paper focuses on one such protein, p53, which represents one of the most extensively studied proteins because of its role in cancer prevention and which, furthermore, has been recently shown to be involved in aging and Alzheimer disease (AD). The contribution of a conformational change in p53 to aging and neurodegenerative processes has yet to be elucidated. In this review we discuss the multiple functions of p53 and how these correlate between cancer and neurodegeneration, focusing on various factors that may have a role in regulating p53 activity. The observation that aging and AD interfere with proteins controlling duplication and cell cycle may lead to the speculation that, in senescent neurons, aberrations in proteins generally dealing with cell cycle control and apoptosis could affect neuronal plasticity and functioning rather than cell duplication.

White matter lesions in the elderly: Pathophysiological hypothesis on the effect on brain plasticity and reserve

The effect of white matter lesions (WMLs) on the brain of elderly individuals is unclear. Most debate has focused on the clinical effect of WMLs on cognitive impairment. Large cross-sectional and longitudinal clinic- and population-based studies suggest that the effect of WMLs on global cognitive performance is relatively small, only individuals with the most severe degrees of WMLs having clinically relevant effects. Here, we review recent data suggesting that WMLs might affect brain function through impairment of brain plasticity and reserve. The clinical effect consists in inability of the brain to respond to interventions such as psychotropic drug medications or rehabilitative interventions.

Conformationally altered p53 : a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimers 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 Parkinsons 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.

β-Amyloid precursor protein metabolism: focus on the functions and degradation of its intracellular domain

Alzheimers disease (AD) is a neurodegenerative disorder that represents the most common type of dementia in the elderly. One of the hallmarks of this disease is a progressive accumulation of amyloid fibrils in senile plaques (SPs), which are composed principally of amyloid-beta peptides (Abeta). The 4-kDa beta-amyloid peptides are produced from the beta-amyloid precursor protein (APP) through sequential processing by beta- and gamma-secretase enzymes in the amyloidogenic pathway. By an alternative non-amyloidogenic pathway, mediated by alpha- and gamma-secretases enzymes, APP is processed within the Abeta domain. Both processing pathways may result in the generation of a fragment called APP intracellular C-terminal domain (AICD) which is hypothesized to contribute to the pathophysiology of AD. Experimental evidence highlights that biological functions of AICD are mediated by interactions between its YENPTY motif and specific binding factors. We critically reviewed literature concerning physiological function of this proteolitic fragment, mainly focusing on their degradation by the two best characterized systems, proteasome and IDE (insulin degrading enzyme). Our work is aimed to analyse the functional role of AICD, integrating also the AICD degradation processes, to better define a potential role of AICD in signal transduction.

Unfolded p53: a potential biomarker for Alzheimer's disease.

The identification of biological markers of AD can improve diagnostic accuracy and therapy follow-up as well as provide information on the pathogenesis of the disease. We recently found that fibroblasts derived from AD patients expressed an altered conformational status of p53 and were less sensitive to p53-dependent apoptosis compared to fibroblasts from non-AD subjects. When investigating the mechanism of such alteration, we found that the exposure to nanomolar concentrations of amyloid-beta (Abeta) 1-40 peptide induced the expression of an unfolded p53 protein isoform in fibroblasts derived from non-AD subjects. These data suggest that the tertiary structure of p53 and the sensitivity to p53-dependent apoptosis is influenced by low concentrations of soluble Abeta. On this basis, we hypothesized that low amounts of soluble Abeta induce early pathological changes at cellular level that may precede the amyloidogenic cascade. One of these changes is the induction of a novel conformational state of p53. If low amounts of Abeta peptide, not resulting in cytotoxic effects, are responsible for p53 structure changes, it could be possible to consider the unfolded p53 both as an agent participating to the early pathogenesis and as a specific marker of the early stage of AD.

Identification of a mutant-like conformation of p53 in fibroblasts from sporadic Alzheimer's disease patients.

Here we show that fibroblasts from sporadic Alzheimers disease (AD) patients specifically express an anomalous and detectable conformational state of p53 that makes these cells distinct from fibroblasts of age-matched non-AD subjects. In particular, we found that, in contrast to non-AD fibroblasts, p53 in AD fibroblasts is expressed at higher levels in resting condition, and presents a significant impairment of its DNA binding and transcriptional activity. All together, these findings figured out the presence of a mutant-like p53 phenotype. However, gene sequencing of the entire p53 gene from either AD or non-AD did not unravel point mutations. Based on immunoprecipitation studies with conformation-specific p53 antibodies (PAb1620 and PAb240), which discriminated folded versus unfolded p53 tertiary structure, we found that a significant amount of p53 assumed an unfolded tertiary structure in fibroblasts from AD patients. This conformational mutant-like p53 form was virtually undetectable in fibroblasts from non-AD patients. These data, independently from their relevance in understanding the etiopathogenesis of AD, might be useful for supporting AD diagnosis.

Conformational altered p53 as an early marker of oxidative stress in Alzheimer's disease.

In order to study oxidative stress in peripheral cells of Alzheimers disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named “unfolded p53”, was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients.

Nuclear Factor κB-Dependent Neurite Remodeling Is Mediated by Notch Pathway

In this study, we evaluated whether a cross talk between nuclear factor κB (NF-κB) and Notch may take place and contribute to regulate cell morphology and/or neuronal network in primary cortical neurons. We found that lack of p50, either induced acutely by inhibiting p50 nuclear translocation or genetically in p50−/− mice, results in cortical neurons characterized by reduced neurite branching, loss of varicosities, and Notch1 signaling hyperactivation. The neuronal morphological effects found in p50−/− cortical cells were reversed after treatment with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl 1]-S-phenylglycine t-butyl ester) or Notch RNA interference. Together, these data suggested that morphological abnormalities in p50−/− cortical neurons were dependent on Notch pathway hyperactivation, with Notch ligand Jagged1 being a major player in mediating such effect. In this line, we demonstrated that the p50 subunit acts as transcriptional repressor of Jagged1. We also found altered distribution of Notch1 and Jagged1 immunoreactivity in the cortex of p50−/− mice compared with wild-type littermates at postnatal day 1. These data suggest the relevance of future studies on the role of Notch/NF-κB cross talk in regulating cortex structural plasticity in physiological and pathological conditions.

CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity

Soluble and toxic oligomers of amyloid β (Aβ) protein have been identified as the true neurotoxic species involved in Alzheimers disease and considering them as targets to inhibit Aβ aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of Aβ protein‐containing 42 amino acids (Aβ1–42) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on Aβ oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well‐known anticancer drugs, as well as suramin and a suramin‐like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block Aβ1–42 oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces Aβ1–42 neurotoxicity.