Antonella Vitali

Oxidative stress increases expression and activity of BACE in NT2 neurons.

Recently an aspartyl protease with beta-secretase activity called BACE was identified. In the present paper we showed that BACE is modulated by the oxidative stress product 4-hydroxynonenal (HNE). Exposure of NT(2) neurons to the two classical pro-oxidant stimuli ascorbate/FeSO(4) and H(2)O(2)/FeSO(4) resulted in a significant generation of HNE, which is temporally followed by an increased production of BACE protein levels. HNE mediated BACE induction is accompanied by a proportional elevation of carboxy-terminal fragments of amyloid precursor protein. Moreover, the direct relationship between BACE induction and lipid peroxidation products was strongly confirmed by the protection exerted by a short pretreatment with alpha-tocopherol, the most important antioxidant known to prevent the formation of aldehydic end-products of lipid peroxidation, including HNE. Our results support the hypothesis that oxidative stress and A beta production are strictly interrelated events and suggest that inhibition of BACE may have a therapeutic effect synergic with antioxidant compounds.

JNK and ERK1/2 pathways have a dual opposite effect on the expression of BACE1

The activity of beta-secretase (BACE1), the endo-protease essential for the production of amyloid beta (Abeta) peptides, is increased in brain of late-onset sporadic Alzheimers disease (AD), and oxidative stress is the potential cause of this event. Oxidative stress up-regulates the expression and the activity of BACE1 in cellular and animal models, through a mechanism that involves the increase of gamma-secretase cleavage on APP and the activation of c-jun N-terminal kinase/activator protein 1 (JNK/AP1) pathway. We further characterized the cellular pathways that control BACE1 expression under oxidative stress. We investigated the involvement of extracellular signal regulated MAP kinase (ERK1/2) pathway in the regulation of BACE1 expression, since it has been recently shown that ERK1/2 is an endogenous regulator of the gamma-secretase activity. We found that ERK1/2 pathway negatively modulates BACE1 expression and activity. Moreover, we observed that conditions that abrogate the gamma-secretase activity favor the activation of signalling pathways that promote cell survival, such as ERK1/2 and the serine/threonine kinase Akt/protein kinase B (Akt). These data suggest that the positive or negative cellular responses to oxidative stress parallel the activities of the beta- and the gamma-secretase. ERK1/2 and JNK pathways are involved in this bipartite response, which can lead to neurodegeneration or neuroprotection depending on the cellular and environmental conditions or cooperation with other signalling pathways such as Akt cascade.

Glutathione depletion induces apoptosis of rat hepatocytes through activation of protein kinase C novel isoforms and dependent increase in AP-1 nuclear binding

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.

Amyloid-β42 plasma levels are elevated in amnestic mild cognitive impairment

Amnestic mild cognitive impairment (aMCI) is considered a prodromal stage of Alzheimers disease (AD). We measured plasma levels of amyloid-beta40 (Abeta40) and Abeta42 in 191 subjects with aMCI. Seventy-nine of them were clinically followed for two years. In the total cohort of aMCI cases, the average level of Abeta42, as well as the Abeta42/Abeta40 ratio, was significantly higher than those of the 102 cognitively normal age-matched subjects. The aMCI cases that converted to probable AD within 2 years had higher levels of Abeta42 and, to a lesser extent, Abeta40 than the stable cases. However the large variability of measured values indicates that plasma Abeta is not a suitable marker of incipient AD.

Mechanisms of inactivation of hepatocyte protein kinase C isoforms following acute ethanol treatment

Acute ethanol exposure of rat isolated hepatocytes leads to a significant decrease (-30%) in cytosolic enzymatic activity of classic protein kinase C (PKC) isoforms, while immunoreactive protein level measured by Western Blot remains unaffected. The inactivation of classic cytosolic isoforms appears dependent on the modification of the enzyme function, probably due to ethanol metabolism. In fact, pretreatment with 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, fully prevented such damage. After ethanol treatment, a decrease of about 40% in both enzymatic activity and immunoreactive protein level of novel PKC isoforms was evident both in the soluble and particulate fractions. Even if 4MP cell pre-treatment afforded protection in this case too, the inhibitory action of ethanol on novel PKC hepatocyte isoforms involves a proteolytic mechanism as shown by Western Blot analysis. The reproduction of PKC inactivation by ethanol in hepatocyte lysate excluded a role of peroxisomal hydrogen peroxide in the pathogenesis of the damage investigated. This damage was not reduced by addition of catalase to the lysate model system.

Fibroblasts from FAD-linked presenilin 1 mutations display a normal unfolded protein response but overproduce Aβ42 in response to tunicamycin

Many patients affected by early onset familial Alzheimers disease (FAD), carry mutations in the presenilin 1 (PS1) gene. Since it has been suggested that FAD-linked PS1 mutations impair the unfolded protein response (UPR) due to endoplasmic reticulum (ER) stress, we analyzed the UPR and amyloid beta-protein processing in fibroblasts bearing various PS1 mutations. Neither in normal conditions nor after induction of ER stress with DTT or tunicamycin were the mRNA levels of UPR-responsive genes (BiP and PDI) significantly different in control and FAD fibroblasts. DTT, which blocked APP transport to the Golgi, caused a 30% decrease of secreted Abeta42 in wild type and PS1 mutant fibroblasts. In contrast, tunicamycin, which allowed exit of APP from the ER, increased secreted Abeta42 only in PS1 mutant fibroblasts. Our findings suggest that, although the UPR is active in fibroblasts from FAD patients, mutant PS1 may selectively increase Abeta42 secretion when N-glycosylation is impaired.

Soluble amyloid β-protein is increased in frontotemporal dementia with tau gene mutations

The relationship between senile plaques and neurofibrillary tangles, the main pathologic lesions of Alzheimers disease, is not completely understood. We addressed this issue examining the type and amount of amyloid beta-protein (Abeta) associated with the soluble and insoluble tissue fractions in the frontal cortex of 8 cases with frontotemporal dementia with parkinsonism caused by mutations of the Tau gene (FTDP-17), in which the intracellular accumulation of polymerised tau is definitely the primary cause of neurodegeneration. As control, we examined 7 cases with frontotemporal dementia lacking distinctive histopathology (DLDH) as well as 8 pathologically normal subjects. In all cases the presence of Abeta deposits was ruled out using immunocytochemistry on sections adjacent to those used for biochemical analysis. ELISA analysis showed a 2.7 and 2.1 fold (p < 0.01) increase of soluble Abeta42 and Abeta40 in FTDP-17, compared to normal and DLDH brains, both of which had comparable levels of Abeta species. Furthermore, the immunoreactivity of the intracellular Abeta42 was significantly increased in cortical neurons of subjects affected with FTDP-17. The results demonstrate that the aggregation of tau protein produces an accumulation of Abeta, which, however, does not reach the critical concentration needed for Abetaplaques formation.

Ethanol-induced effects on expression level, activity, and distribution of protein kinase C isoforms in rat liver Golgi apparatus

Acute ethanol administration induces significant modifications both in secretive and formative membranes of rat liver Golgi apparatus. The decrease in glycolipoprotein secretion and their retention into the hepatocyte contribute to the pathogenesis of alcohol-induced fatty liver. Molecular and cellular mechanisms behind the ethanol-induced injury of the liver secretory pathway are not yet completely defined. In this study on intact livers from ethanol-treated rats, the involvement of the Golgi compartment in the impairment of hepatic glycolipoprotein secretion has been correlated with changes in the expression level, subcellular distribution and enzymatic activity of protein kinase C (PKC) isoforms. Acute ethanol exposure determined a translocation of classic PKCs and delta isoform from the cytosol to cis and trans Golgi membranes, the site of glycolipoprotein retention in the hepatic cell. A marked stimulation of cytosolic epsilon PKC activity was observed throughout the period of treatment. The presence of activated PKC isozymes at the Golgi compartment of alcohol-treated rat livers may play a role in hepatic secretion and protein accumulation. Direct and indirect effects of ethanol consumption on PKC isozymes and Golgi function are discussed.

A novel presenilin 1 L166H mutation in a pseudo-sporadic case of early-onset Alzheimer's disease.

We report a 44-year-old woman presenting at 33 years with memory loss, followed by progressive dementia. Her family history was negative for dominant genetic disorders at high penetrance. Analysis of presenilin-1 gene revealed a missense mutation at codon 166, leading to the substitution from leucine to histidine. The mutation occurs in the third transmembrane domain of presenilin-1, at the position of two different mutations previously described, associated with an atypical phenotype. The present case has two implications: (1) mutations of presenilin-1 have to be searched also in apparently sporadic cases of dementia beginning in the third decade of life; (2) as yet unidentified factors, besides the γ-secretase complex, influence the phenotype of presenilin-1 mutations.

Neuronal apoptosis is accompanied by amyloid β-protein accumulation in the endoplasmic reticulum

A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimers disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.