Alessandra Piccini

β‐Site APP cleaving enzyme up‐regulation induced by 4‐hydroxynonenal is mediated by stress‐activated protein kinases pathways

4‐Hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, up‐regulates expression of the β‐site APP cleaving enzyme (BACE‐1), an aspartyl protease responsible for the β‐secretase cleavage of amyloid precursor protein (AβPP), and results in increased levels of amyloid β (Aβ) peptide. The mechanisms underlying this remain unclear but are of fundamental importance because prevention of BACE‐1 up‐regulation is viewed as an important therapeutic strategy. In this study, we exposed NT2 neurons to a range of HNE concentrations (0.5–5 µm) that elicited an up‐regulation of BACE‐1 expression, a significant increase in intracellular and secreted levels of Aβ peptides as well as apoptosis involving poly‐ADP ribose polymerase cleavage and activation of caspase 3. To delineate the molecular events involved in HNE‐mediated BACE‐1 activation, we investigated the involvement of stress‐activated protein kinases (SAPK), signal transducers and activators of transcription (STAT) and serine–threonine kinase B/phosphatidylinositol phosphate 3 kinase (Akt/PtdIns3K). Using specific pharmacological inhibitors, our results show that activation of c‐Jun N‐terminal kinases and p38MAPK., but not STAT or Akt/PtdIns3K, pathways mediate the HNE‐dependent up‐regulation of BACE‐1 expression. Therefore, HNE, an oxidative stress mediator detected in vivo in the brains of Alzheimers disease patients, may play a pathogenetic role in Alzheimers disease by selectively activating SAPK pathways and BACE‐1 that regulate the proteolytic processing of AβPP.

Neprylisin decreases uniformly in Alzheimer's disease and in normal aging.

The proteolysis of β‐amyloid (Aβ) requires neprylisin, an enzyme that has been shown as reduced in Alzheimers disease (AD). We investigated whether a decrease in neprilysin levels contributes to the accumulation of amyloid deposits not only in AD but also in the normal aging. We analyzed neprilysin mRNA and protein levels in cerebral cortex from 10 cognitively normal elderly subjects with amyloid plaques (NA), 10 cases of AD, and 10 control cases free of amyloid plaques. We found a significant decrease in neprilysin mRNA levels in both AD and NA compared to control cases. Thereby, the defect of neprilysin appears to correlate with Aβ deposition but not with degeneration and dementia.

The increased activity of BACE1 correlates with oxidative stress in Alzheimer's disease.

We evaluated expression, protein levels and activity of the Beta-site cleaving enzyme (BACE1) as well as the amount of products of lipid peroxidation in frontal cortex of three groups of cases: sporadic Alzheimers disease (AD); control subjects (CTR); cognitively normal subjects with abundant amyloid plaques (NA). We found a significant increase of BACE1 activity and products of lipid peroxidation in brain tissue of AD cases, with normal gene expression, and non-significant elevation of protein levels. CTR and NA samples showed similar levels of BACE1 activity and oxidative products. BACE1 activity and the amount of oxidative products were significantly correlated in all cases.Moreover, both BACE1 activity and the level of 4-hydroxynonenal were correlated with the amount of Beta-amyloid pyroglutamated 3-42, the more toxic Beta-amyloid peptide that is characteristic of AD. These findings suggest that BACE1 activity reflects the type of ABeta species, rather than the Beta-amyloid plaques load. Hence, the increase of BACE1 activity occurring in sporadic AD is likely the effect, rather the cause, of ABeta accumulation and oxidative stress.

Mutant presenilin 1 increases the expression and activity of BACE1

Mutations of the presenilin 1 (PS1) gene are the most common cause of early onset familial Alzheimer disease (FAD). PS1 mutations alter the activity of the γ-secretase on the β-amyloid precursor protein (APP), leading to selective overproduction of β-amyloid (Aβ) 42 peptides, the species that forms oligomers that may exert toxic effects on neurons. Here we show that PS1 mutations, expressed both transiently and stably, in non-neuronal and neuronal cell lines increase the expression and the activity of the β-secretase (BACE1), the rate-limiting step of Aβ production. Also, BACE1 expression and activity are elevated in brains of PS1 mutant knock-in mice compared with wild type littermates as well as in cerebral cortex of FAD cases bearing various PS1 mutations compared with in sporadic AD cases and controls. The up-regulation of BACE1 by PS1 mutations requires the γ-secretase cleavage of APP and is proportional to the amount of secreted Aβ42. Aβ42, and not AICD (APP intracellular domain), is indeed the APP derivative that mediates the overexpression of BACE1. The effect of PS1 mutations on BACE1 may contribute to determine the wide clinical and pathological phenotype of early onset FAD.

Role of water‐soluble amyloid‐β in the pathogenesis of Alzheimer's disease

Water‐soluble amyloid‐β (wsAβ) is present in cerebral cortex of subjects at risk of Alzheimers disease (AD) as well as in normal elderly subjects as a mixture of three major amyloid‐β (Aβ) species: 1–42, py3–42 and py11–42. The three wsAβ species are nondetectable in brains of young people, free of immunohistochemically detectable amyloid plaques. In the brains of Downs syndrome and APP‐mutant transgenic mice, wsAβ appears long time before amyloid deposition, indicating that it represent the first form of Aβ aggregation and accumulation. In normal brain, wsAβ is bound to apolipoprotein E that favours its degradation by proteases. The composition of wsAβ, in terms of the ratio between the full‐length 1–42 and the py3–42 peptides, correlates with the severity of clinical and pathological phenotype in familial early onset AD. Water‐soluble Aβ is the native counterpart of the Aβ small aggregates (soluble oligomers) that show in vitro an early and high neuronal toxicity.

Elevation of β-Amyloid 1-42 Autoantibodies in the Blood of Amnestic Patients With Mild Cognitive Impairment

OBJECTIVE To develop a blood-based test for screening populations at risk for Alzheimer disease. DESIGN Case-control study. Subjects A total of 180 patients with mild cognitive impairment (MCI) and 105 age-matched, cognitively normal controls. INTERVENTIONS The titer of beta-amyloid 1-42 autoantibodies in the plasma was obtained at the time of diagnosis and evaluated by enzyme-linked immunosorbent assay before and after dissociation of the antigen-antibody complexes. A total of 107 patients with MCI were followed up for 36 months; 70 of the 107 cases progressed to Alzheimer disease. RESULTS The average level of beta-amyloid 1-42 plasma autoantibodies in patients with MCI that progressed to Alzheimer disease, but not that of the stable cases, was significantly higher than in cognitively normal controls (P < .001). CONCLUSIONS The results suggest that the plasma beta-amyloid 1-42 autoantibodies parallel beta-amyloid 42 deposition in the brain, which is known to precede by several years the clinical onset of Alzheimer disease. The evaluation of beta-amyloid 1-42 autoantibodies after dissociation of the complexes is a simple and inexpensive method that can be used to predict the occurrence of Alzheimer disease.

Endogenous APP derivatives oppositely modulate apoptosis through an autocrine loop

&NA; We have recently shown that, in rat cerebellar granule cells, apoptosis triggered by KCl deprivation is associated with an amyloidogenic shift in the processing of amyloid precursor protein (APP) resulting in an increase of amyloid β‐protein (Aβ) secretion. To further investigate this issue we studied the relationship between secretion of APP metabolites (Aβ, APPs) and neuronal degeneration. We postulated that the endogenous products of the APP metabolism may modulate neuronal survival by an autocrine loop. Treatment of cerebellar granule cells with various antibodies raised against different epitopes of APPs and Aβ oppositely modulates low potassium apoptotic cell death. Antibodies specific for the N‐terminal of Aβ (4G8, 6E10, R3659) increased neuronal survival by 30% over controls. On the contrary, treatment of cultures undergoing apoptosis with the monoclonal antibody 22C11 directed against the APP N‐terminus reduced neuronal survival by 53%, suggesting that endogenous α‐APPs contribute to neuronal survival. Moreover low KCl culture medium, conditioned by cerebellar granule cells, attenuated the apoptotic process. This anti‐apoptotic effect was abolished by removal of APPs from the conditioned medium. Western blotting of APPs removed from the conditioned medium confirmed the presence of α‐APPs. These data indicate that APP cleavage products oppositely modulate neuronal survival through an autocrine loop and further strengthen an Alzheimers disease pathogenetic scheme based on altered metabolism of APP.

β-Amyloid 1-42 induces physiological transcriptional regulation of BACE1

J. Neurochem. (2012) 122, 1023–1031.

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.

Plasma levels of insulin and amyloid β42 are correlated in patients with amnestic Mild Cognitive Impairment

Epidemiological and experimental data suggest that type 2 diabetes (DM2) and sporadic late-onset Alzheimers disease (AD) share a common mechanism, that is able to produce accumulation of insulin and amyloid beta 42 (Abeta42), the major pathogenic events respectively of the two conditions. In 71 non diabetic patients with amnestic mild cognitive impairment we found a significant linear correlation between fasting plasma levels of insulin and Abeta42 (R = +0.25, P < 0.05). The levels of both peptides were elevated in comparison to 48 age-matched cognitively normal controls. The correlation of insulin and Abeta42 plasma levels suggests a pathogenic link between DM2 and sporadic AD.