A Conformational Variant of p53 (U-p53AZ) as Blood-Based Biomarker for the Prediction of the Onset of Symptomatic Alzheimer's Disease.

BACKGROUND: Ongoing research seeks to identify blood-based biomarkers able to predict onset and progression of Alzheimer's disease (AD). OBJECTIVE: The unfolded conformational variant of p53 (U-p53AZ), previously observed in AD individuals, was evaluated in plasma samples from individuals participating in the Australian Imaging, Biomarkers and Lifestyle (AIBL) cohort for diagnostic and prognostic assessment, validated on a neuropsychological-based diagnosis, over the course of six years. DESIGN: Retrospective Longitudinal Prognostic biomarker study. SETTING: Single-center study based on the AIBL cohort. PARTICIPANTS: 482 participants of the AIBL cohort, aged 60-85 years, without uncontrolled diabetes, vascular disease, severe depression or psychiatric illnesses. MEASUREMENTS: The AlzoSure® Predict test, consisting of immunoprecipitation (IP) followed by liquid chromatography (LC) tandem mass spectrometry (MS/MS), was performed to quantify the AZ 284® peptide as readout of U-p53AZ and compared with an independent neuropsychological diagnosis. The amyloid load via amyloid ß-positron emission tomography (Aß-PET) and supporting clinical information were included where possible. RESULTS: U-p53AZ diagnostic and prognostic performance was assessed in both time-independent and time-dependent (36, 72 and 90 months following initial sampling) analyses. Prognostic performance of Aß-PET and survival analyses with different risk factors (gender, Aß-PET and APOE ε4 allele status) were also performed. U-p53AZ differentiated neuropsychologically graded AD from non-AD samples, and its detection at intermediate/high levels precisely identified present and future symptomatic AD. In both time-independent and time-dependent prognostic analyses U-p53AZ achieved area under the curve (AUC) >98%, significantly higher than Aß-PET AUCs (between 84% and 93%, P respectively <0.0001 and <0.001). As single factor, U-p53AZ could clearly determine the risk of AD neuropsychological diagnosis over time (low versus intermediate/high U-p53AZ hazard ratio=2.99). Proportional hazards regression analysis identified U-p53AZ levels as a major independent predictor of AD onset. CONCLUSIONS: These findings support use of U-p53AZ as blood-based biomarker predicting whether individuals would reach neuropsychologically-defined AD within six years prior to AD diagnosis. Integration of U-p53AZ in screening processes could support refined participant stratification for interventional studies.

Methylglyoxal affects cognitive behaviour and modulates RAGE and Presenilin-1 expression in hippocampus of aged mice.

Methylglyoxal (MG), a potent glycotoxin that can be found in the diet, is one of the main precursors of Advanced glycation end products (AGEs). It is well known that modifications in lifestyle such as nutritional interventions can be of great value for preventing brain deterioration. This study aimed to evaluate in vivo how an oral MG treatment, that mimics a high MG dietary intake, could affect brain health. From our results, we demonstrated that MG administration affected working memory, and induced neuroinflammation and oxidative stress by modulating the Receptor for Advanced glycation end products (RAGE). The gene and protein expressions of RAGE were increased in the hippocampus of MG mice, an area where the activity of glyoxalase 1, one of the main enzymes involved in MG detoxification, was found reduced. Furthermore, at hippocampus level, MG mice showed increased expression of proinflammatory cytokines and increased activities of NADPH oxidase and catalase. MG administration also increased the gene and protein expressions of Presenilin-1, a subunit of the gamma-secretase protein complex linked to Alzheimer's disease. These findings suggest that high MG oral intake induces alteration directly in the brain and might establish an environment predisposing to AD-like pathological conditions.

Mitochondria and cellular redox state on the route from ageing to Alzheimer's disease.

Several theories have been postulated, trying to explain why and how living organisms age. Despite some controversies and still huge open questions, a growing body of evidence suggest alterations of mitochondrial functionality and redox-homeostasis occur during the ageing process. Oxidative damage and mitochondrial dysfunction do not represent the cause of ageing per se but they have to be analyzed within the complexity of those series of processes occurring during lifespan. The establishment of a crosstalk among them is a shared common feature of many chronic age-related diseases, including neurodegenerative disorders, for which ageing is a major risk factor. The challenge is to understand when and how the interplay between these two systems move towards from normal ageing process to a pathological phenotype. Here in this review, we discuss the crosstalk between mitochondria and cytosolic-ROS. Furthermore, through a visual data mining approach, we attempt to describe the dynamic interplay between mitochondria and cellular redox state on the route from ageing to an AD phenotype.

Monitoring Caco-2 to enterocyte-like cells differentiation by means of electric impedance analysis on printed sensors.

BACKGROUND: Colorectal adenocarcinoma cells (Caco-2) are a widely used model of intestinal barrier to study cancer development, toxicological assessments, absorption and metabolism in food science or drug discovery. Caco-2 spontaneously differentiate into a monolayer expressing several specific characteristics, typically showed by mature enterocytes. For in vitro experiments, it is crucial to identify non-invasive and non-destructive techniques able to evaluate the integrity and differentiation of the cells monolayer. Thus, we aimed to assess these properties by analyzing electrical impedance measurements. METHODS: Caco-2 cells were differentiated for 21 days. The monolayer integrity and differentiation were primarily evaluated by means of morphological, biochemical and molecular data. Impedance measurements in a range of frequencies from 400 Hz to 50 kHz were performed using a dedicated set up, including customized Aerosol Jet Printed carbon-based sensors. RESULTS: The trends of RI observed at three different frequencies were able to describe cell growth and differentiation. In order to evaluate which frequencies better correlate with cell differentiation, Principal Component Analysis have been employed and the concordance analysis between RI magnitude and morphological, biochemical and molecular data, highlighted 40 kHz as the optimal frequency to assess Caco-2 cells differentiation process. CONCLUSION: We demonstrated the feasibility and reliability of applying impedance-based measurements not only to provide information about the monolayer status, but also for cell differentiation monitoring. GENERAL SIGNIFICANCE: This study underlined the possibility to use a dedicated sensor to assess the integrity and differentiation of Caco-2 monolayer, as a reliable non-destructive alternative to conventional approaches.

Characterization of the Antioxidant Effects of γ-Oryzanol: Involvement of the Nrf2 Pathway.

γ-Oryzanol (ORY) is well known for its antioxidant potential. However, the mechanism by which ORY exerts its antioxidant effect is still unclear. In this paper, the antioxidant properties of ORY were investigated for its potential effects as a reactive oxygen and nitrogen species (ROS/RNS) scavenger and in activating antioxidant-promoting intracellular pathways utilizing the human embryonic kidney cells (HEK-293). The 24 h ORY exposure significantly prevented hydrogen peroxide- (H2O2-) induced ROS/RNS production at 3 h, and this effect was sustained for at least 24 h. ORY pretreatment also enhanced the activity of antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidase (GPX). Interestingly, ORY induced the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation and upregulation of Nrf2-dependent defensive genes such as NAD(P)H quinone reductase (NQO1), heme oxygenase-1 (HO-1), and glutathione synthetase (GSS) at mRNA and protein levels in both basal condition and after H2O2 insult. Thus, this study suggested an intriguing effect of ORY in modulating the Nrf2 pathway, which is also involved in regulating longevity as well as age-related diseases.

Mitochondrial Alterations in Peripheral Mononuclear Blood Cells from Alzheimer's Disease and Mild Cognitive Impairment Patients.

It is well recognized that mitochondrial dysfunction contributes to neurodegeneration occurring in Alzheimer's disease (AD). However, evidences of mitochondrial defects in AD peripheral cells are still inconclusive. Here, some mitochondrial-encoded and nuclear-encoded proteins, involved in maintaining the correct mitochondria machine, were investigated in terms of protein expression and enzymatic activity in peripheral blood mononuclear cells (PBMCs) isolated from AD and Mild Cognitive Impairment (MCI) patients and healthy subjects. In addition mitochondrial DNA copy number was measured by real time PCR. We found some differences and some similarities between AD and MCI patients when compared with healthy subjects. For example, cytochrome C and cytochrome B were decreased in AD, while MCI showed only a statistical reduction of cytochrome C. On the other hand, both AD and MCI blood cells exhibited highly nitrated MnSOD, index of a prooxidant environment inside the mitochondria. TFAM, a regulator of mitochondrial genome replication and transcription, was decreased in both AD and MCI patients' blood cells. Moreover also the mitochondrial DNA amount was reduced in PBMCs from both patient groups. In conclusion these data confirmed peripheral mitochondria impairment in AD and demonstrated that TFAM and mtDNA amount reduction could be two features of early events occurring in AD pathogenesis.

Sulphurous thermal water increases the release of the anti-inflammatory cytokine IL-10 and modulates antioxidant enzyme activity.

The beneficial effects of hot springs have been known for centuries and treatments with sulphurous thermal waters are recommended in a number of chronic pathologies as well as acute recurrent infections. However, the positive effects of the therapy are often evaluated in terms of subjective sense of wellbeing and symptomatic clinical improvements. Here, the effects of an S-based compound (NaSH) and of a specific sulphurous thermal water characterized by additional ions such as sodium chloride, bromine and iodine (STW) were investigated in terms of cytokine release and anti-oxidant enzyme activity in primary human monocytes and in saliva from 50 airway disease patients subjected to thermal treatments. In vitro, NaSH efficiently blocked the induction of pro-inflammatory cytokines and counterbalanced the formation of ROS. Despite STW not recapitulating these results, possibly due to the low concentration of S-based compounds reached at the minimum non-toxic dilution, we found that it enhanced the release of IL-10, a potent anti-inflammatory cytokine. Notably, higher levels of IL-10 were also observed in patients' saliva following STW treatment and this increase correlated positively with salivary catalase activity (r2 = 0.19, *p less than 0.01). To our knowledge, these results represent the first evidence suggesting that S-based compounds and STW may prove useful in facing chronic inflammatory and age-related illness due to combined anti-inflammatory and anti-oxidant properties.

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 ß-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 ZnCl(2) treatment. Zinc supplementation reverted p53 wild-type tertiary structure, increased cells sensitivity to acute cytotoxic injury and GAP-43 levels in SY5Y-APP clone.

Increased CD44 gene expression in lymphocytes derived from Alzheimer disease patients.

In this study, we demonstrated for the first time an increased CD44 gene expression in lymphocytes derived from Alzheimer's disease (AD) patients in comparison with healthy subjects. CD44 is a surface antigen expressed by cells of the immune and central nervous system as well as in a variety of other tissues. Functioning as adhesion molecule, CD44 is furthermore involved in driving immune response into infected tissues, including the CNS. We also found that lymphocytes of the same patients expressed significant levels of unfolded p53 isoform, confirming what we already demonstrated in fibroblasts and lymphocytes derived from other cohorts of AD patients. A correlation between p53 and CD44 expression has been well demonstrated in cancer cells, suggesting that CD44 could be a target gene of mutant p53, or either mutant p53 could lack its ability to negatively regulate CD44 expression. The contemporaneous increased expression of unfolded p53 and CD44 in AD lymphocytes may suggest that these two molecules cross-talk together participating in peripheral immune response during the development of the disease.

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 Alzheimer's 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.

Microtubule stabilizing effect of notch activation in primary cortical neurons.

The appropriate level of microtubule stability is fundamental in neurons to assure correct polarity, migration, vesicles transport and to prevent axonal degeneration. In the present study, we have identified Notch pathway as an endogenous microtubule stabilizer. Stimulation of Notch receptors by exposure of mouse cortical neurons to the Notch ligand Jagged1 resulted in increased microtubule stability, as measured by using antibodies against post-translationally modified alpha tubulin, and changes in axonal morphology and branching, with varicosity loss, thicker neurites and enlarged growth cones. Similar effects were found after exposure of the cells to different doses of Taxol. However, contrary to Taxol, Jagged1 induced downregulation of the microtubule severing protein Spastin. We suggest that a fine-tuned manipulation of Notch signaling may represent a novel approach to modulate neuronal cytoskeleton plasticity.

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

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.

The different apoptotic potential of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death.

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+) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro+). At variance, the difference in apoptosis susceptibility between Arg+ and Pro+ is not significant when cells from 30-year-old people are studied. Further, we found that Arg+ and Pro+ 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+ 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+ 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.

A novel mechanism for pergolide-induced neuroprotection: inhibition of NF-kappaB nuclear translocation.

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-kappaB pathway we tested the hypothesis that pergolide may interfere with NF-kappaB activity. Based on Western blot analysis and immunocytochemistry, pergolide was found to prevent H(2)O(2)-induced apoptosis by inhibiting NF-kappaB nuclear translocation and activation of p53 signalling pathway. Similarly, the cell-permeable SN50 peptide, which is known to block NF-kappaB 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-kappaB activation, as demonstrated by the prevention of H(2)O(2)-induced IkappaB degradation. These data suggest a novel site of action of pergolide that may account for additional pharmacological properties of this drug.

Neutralization of TRAIL death pathway protects human neuronal cell line from beta-amyloid toxicity.

Here we report that a novel member of the TNF-alpha 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 amyloid-induced 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 (betaAP). TRAIL was also found to be released in the culture media after betaAP treatment with a time-course overlapping to contents of the intracellular protein. Contribution of TRAIL to betaAP neurotoxicity is demonstrated by data showing that TRAIL-neutralizing monoclonal antibody protects neuronal SH-SY5Y cells from betaAP 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 betaAP 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 betaAP-induced neurotoxicity, supporting the hypothesis that these three molecules share common intracellular pathways. Finally, we found that betaAP strongly activated caspase-8, and the cell-permeable, selective caspase-8 inhibitor z-IETD-FMK prevents both betaAP- and TRAIL-induced neurotoxicity. In view of TRAIL's potency in inducing neuronal death, and its role as mediator of betaAP, it is plausible to hypothesize that TRAIL can be regarded as a molecule that provides substantial contribution to betaAP-dependent cell death, which takes part in the progression of the neurodegenerative process and related chronic inflammatory response.

Expression of cell-cycle-related proteins and excitoxicity.

Previous work from our laboratory has suggested the functional contribution of p53 to the cascade of events triggered by excitatory amino acids and leading to cell death in primary neurons. Here we show that this paradigm can be extended to cortical neurons treated with NMDA. We found that exposure of the cells to either 300 microM or 2 mM NMDA induced an enhancement of p53 protein levels which was already significant at 60 min after the lesion, while very low staining of the protein was observed in untreated cells. The effect was time- and concentration-dependent, reaching the maximal induction at 3 h. NMDA treatment also resulted in an increase of gadd45 protein levels which was evident in both treatment at 3 h, the time when p53 was maximally induced. Our data give further evidence suggesting that a repertoire of events typical of proliferating cells is activated in degenerating neurons.

p53 is dispensable for apoptosis but controls neurogenesis of mouse dentate gyrus cells following gamma-irradiation.

Mammalian cells respond to DNA insults by activating cell-cycle checkpoints. This may result in a temporary cell growth arrest which allows DNA repair before proliferation or induces apoptosis. p53 is one of the main contributors in regulating these activities. To get a better insight on the molecular mechanism underlying these activities we studied the role of p53 in apoptosis and neurogenesis of brain cells from adult p53(+/+) or p53(-/-) mice exposed to gamma-irradiation. Apoptosis and neurogenesis were assessed up to 14 days following the injury. Five-ten hours following gamma-irradiation, cells with TUNEL positive nuclei were identified within the subgranular zone of dentate gyrus (DG) of both p53(+/+) and p53(-/-) mice. At the same time-points, pyknotic and shrinking nuclei were visualized by Hoechst 33258 staining. Furthermore, gamma-irradiation increased the number of proliferating cell nuclear antigen (PCNA) positive cells with a peak at 5-10 h in both animal groups. PCNA immunoreactivity was detected in cells exhibiting condensed nuclei as visualized by Hoechst 33258 staining. Neurogenesis, assessed by mitotic marker p34(cdc2) immunoreactivity, showed a biphasic response to gamma-irradiation both in p53(+/+) and p53(-/-) mice which was characterized by an early inhibition and a delayed stimulation. In p53(-/-) mice, the time required by DG granule cells to recover from the lesion and to stimulate proliferation was significantly shortened in comparison with wild-type mice thus resulting in an accelerated neurogenesis. Our data indicate that following gamma-radiation p53 plays a role in regulating cell-cycle progression rate but it is dispensable for promoting apoptosis of DG granule cells.

Estrogen derivative relaxes rabbit aorta via the endothelial receptor system.

BACKGROUND: It is well known that sexual hormones, in particular estrogens, may influence the cardiovascular system. Experimental and clinical studies have shown that estrogen directly or indirectly modulates the reactivity of vascular smooth muscle but at present the mechanism of action of this hormone has yet to be clarified. The aim of this study was to evaluate the vascular effects of a synthetic non-steroid estrogen, diethylstilbestrol, and the possible involvement of endothelial function. METHODS: We investigated, on aortic strips of a female rabbit, the inhibitory effects of diethylstilbestrol on the contractions induced by different spasmogenic agents, noradrenaline (10(-6) M), angiotensin II (10(-6) M), serotonin (10(-6) M), and KCl (10(-1) M). Some experiments were performed in high K+, Ca++-free solution. In some experiments endothelial function was abolished by mechanical ablation. Another series of experiments was incubated (30 min) with N(G)-monomethyl-L-arginine, which inhibits nitric oxide synthase or with tamoxifen, a specific antagonist of estrogen receptors. RESULTS: At doses from 10(-6) M to 10(-4) M, diethylstilbestrol showed an evident spasmolytic action on contractions induced by noradrenaline, angiotensin II and serotonin but no significant effect was observed on KCl spasm. The inhibitory response of diethylstilbestrol to increased vascular tone induced by noradrenaline disappeared when the endothelial function, validated by the acetylcholine test, was abolished by mechanical ablation. When tested in high K+, Ca++-free solution, diethylstilbestrol did not significantly shift the cumulative dose-response curve of calcium. In the experiments performed with N(G)-monomethyl-L-arginine, diethylstilbestrol failed to induce vasodilation suggesting that its action may be related to synthesis of nitric oxide. Moreover, in the presence of tamoxifen, diethylstilbestrol was unable to induce vasodilation. CONCLUSIONS: The early occurrence of vasodilation is in favor of a direct effect and seems to exclude a regulation of gene expression. These results suggest that estrogens may directly regulate vascular tone interacting with its specific endothelial cell receptors through the release of nitric oxide.

Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path?

Cell-cycle-related proteins, such as cyclins or cyclin-dependent kinases, are re-expressed in neurons committed to death in response to a variety of insults, including excitotoxins, hypoxia and ischemia, loss of trophic support, or beta-amyloid peptide. In some of these conditions events that are typical of the mid-G1 phase, such as cyclin-dependent kinase 4/6 activation, are required for the induction of neuronal death. In other cases, the cycle must proceed further and recruit steps that are typical of the G1/S transition for death to occur. Finally, there are conditions in which cell-cycle proteins might be re-expressed, but do not contribute to neuronal death. We hypothesize that cell-cycle signaling becomes a mandatory component of neuronal demise when other mechanisms are not enough for neurons to reach the threshold for death. Under this scheme, the death threshold is set by the extent of DNA damage. Whenever the extent of DNA damage is below this threshold, a cell-cycle signaling becomes crucial for the induction of neuronal death through p53-dependent or -independent pathways.

Presenilin-1 regulates the neuronal threshold to excitotoxicity both physiologically and pathologically.

A direct pathophysiological role of Familial Alzheimer's Disease (FAD)-associated Presenilin 1 (PS1) mutations in neuronal vulnerability remains a controversial matter. We evaluated the relationship between PS1 and excitotoxicity in four different experimental models of neurotoxicity by using primary neurons from (i) transgenic (tg) mice overexpressing a human FAD-linked PS1 variant (L286V mutation), (ii) tg mice overexpressing human wild-type (wt) PS1, (iii) PS1 knockout mice, and (iv) wt mice in which PS1 gene expression was knocked down by antisense treatment. We found that primary neurons overexpressing mutated PS1 showed an increased vulnerability to both excitotoxic and hypoxic-hypoglycemic damage when compared with neurons obtained from either mice overexpressing human wt PS1 or in wt mice. In addition, reduced excitotoxic damage was obtained in neurons in which PS1 expression was absent or diminished. Data obtained in in vivo experimental models of excitotoxicity partially supported the in vitro observations. Accelerated neuronal death was demonstrated in the hippocampus of mice overexpressing mutated PS1 after peripheral administration of kainic acid in comparison with wt animals. However, measurement of the infarct volume after middle cerebral artery occlusion did not show significant difference between the two animal groups. The results altogether suggest that expression of FAD-linked PS1 variants increases the vulnerability of neurons to a specific type of damage in which excitotoxicity plays a relevant role. In addition, they support the view that reduction of endogenous PS1 expression results in neuroprotection.