Martina Zimmermann

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimers Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well‐known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha‐secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH‐SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH‐SY5Y cells released significantly more sAPPα into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPα levels. Pre‐incubation with atropine decreased release of sAPPα significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.

Platelet APP, ADAM 10 and BACE alterations in the early stages of Alzheimer disease

Amyloid precursor protein (APP), ADAM 10, and β-site-APP cleaving enzyme (BACE) alterations were evaluated in platelets of 31 patients with Alzheimer disease (AD) and 15 age-matched controls. A significant modification of these proteins and enzymes involved in the amyloid cascade was detected from the earliest clinically detectable disease stage. This observation suggests that AD is associated with an early metabolic derangement toward amyloidogenic pathways and supports the potential value of APP and secretase measurements for early diagnosis of AD.

Amyloid precursor protein metabolism is regulated toward alpha-secretase pathway by Ginkgo biloba extracts

Clinical trials report that Ginkgo biloba extracts (e.g., EGb761) reduce cognitive symptoms in age-associated memory impairment and dementia, including Alzheimer disease (AD). However, the mechanisms behind their neuroprotective ability remain to be fully established. In this study, the effect of EGb761 on the amyloid precursor protein (APP) metabolism has been investigated by both in vitro and in vivo models. To this aim, alpha-secretase, the enzyme regulating the non-amyloidogenic processing of APP and the release of alphaAPPs, the alpha-secretase metabolite, were studied in superfusates of hippocampal slices after EGb761 incubation, and in hippocampi and cortices of EGb761-treated rats. PKC translocation state was evaluated as well. EGb761 increases alphaAPPs release through a PKC-independent manner. This effect is not accompanied by a modification of either APP forms or alpha-secretase expression. Moreover, EGb761 influence on alphaAPPs release was strictly dependent on treatment dosage. Our findings suggest that the benefit of EGb761 reported by previous clinical studies is underscored by a specific biological mechanism of this compound on APP metabolism, directly affecting the release of the non-amyloidogenic metabolite. Additional research will be needed to clearly define the effective clinical relevance, thus considering EGb761 as a possible supplementary treatment in dementing diseases.

Cholinesterase inhibitors influence APP metabolism in Alzheimer disease patients

Platelets mirror pathogenic alterations in the central nervous system of Alzheimer disease (AD) patients: an alteration of the Amyloid Precursor Protein (APP) forms pattern and decreased alpha-secretase activity--the non-amyloidogenic APP processing enzyme--were demonstrated. Platelets were analysed at baseline and after 30 days of cholinesterase inhibitor (ChEI) treatment (T30). ADAM10 levels, alpha- and beta-secretase activity were assessed measuring ADAM10 immunoreactivity, sAPPalpha release and the membrane-attached C-terminal fragments produced by beta- and alpha-secretase cleavage, that is, CTF99 and CTF83, respectively. ChEIs treatment rescues impaired APP metabolism increasing significantly ADAM10 levels (T30 vs. T0, P < 0.05), alpha-secretase activity (T30 vs. T0, P < 0.05) and reducing beta-secretase cleavage (T30 vs. T0, P < 0.05). Restoration of the balance between the mutually exclusive alpha- and beta-secretase pathway in APP processing caused by short-term ChEIs treatment potentially represents a key event in AD therapy linking in vivo cholinergic effect to APP metabolism. The use of platelets may represent a useful tool to follow molecular aspects of pharmacological response in AD patients.

Molecular Rationale for the Pharmacological Treatment of Alzheimer’s Disease

Cerebral deposition of amyloid plaques containing amyloid β-peptide (Aβ) has traditionally been considered the central feature of Alzheimer’s disease (AD). Aβ is derived from amyloid precursor protein (APP), which is cleaved by several different proteases: α-, β- and γ-secretase. In the past decade, however, the molecular pathogenesis of AD has been shown to involve alterations in several neurotransmitter, inflammatory, oxidative, and hormonal pathways that represent potential targets for AD prevention and treatment.Much research has shown a direct link between cholinergic impairment and altered APP processing as a major pathogenetic event in AD. Three highly probable mechanisms of APP regulation through inhibition of acetylcholinesterase are thus current topics of investigation. Indeed, acetylcholinesterase inhibitors appear to cause selective muscarinic activation of α-secretase and to induce the translation of APP mRNA; they may also restrict amyloid fibre assembly.Activation of N-methyl-d-aspartate receptors is considered a probable cause of chronic neurodegeneration in AD, and memantine has been widely used in some countries in AD patients to block cerebral N-methyl-d-aspartate receptors that normally respond to glutamate.Further studies are needed to determine whether antioxidants such as vitamins C and E are effective, through various mechanisms, in patients with mild-to-moderate AD. Additional data are also required for non-steroidal anti-inflammatory drugs, some of which appear to possess experimental effects that may ultimately prove favourable in AD patients. Statins also warrant further investigation, since they have activated α-secretase and they reduced Aβ generation and amyloid accumulation in a transgenic mouse model.β-Secretase would seem to be an ideal target for anti-amyloid therapy in AD, but potential clinical and pharmacological issues, such as ensuring selectivity of inhibition, stability, and ease of blood-brain barrier penetration and cellular uptake, remain to be addressed for β-secretase inhibitors. γ-Secretase is not an easy candidate for pharmacological manipulation. Immunotherapeutic strategies have targeted Aβ directly; however, intensive investigation of indirect approaches to the management of AD with immunotherapy is now underway.

Dramatic increase in readthrough acetylcholinesterase in a cellular model of oxidative stress

J. Neurochem. (2011) 116, 1088–1096.

Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68.

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.

Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

Background The alpha-7 nicotinic acetylcholine receptor (α7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimers disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the α7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the α7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of α7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance The results reported here demonstrate a hitherto unknown relationship between the α7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

Artificial neural networks allow the use of simultaneous measurements of Alzheimer Disease markers for early detection of the disease

BackgroundPrevious studies have shown that in platelets of mild Alzheimer Disease (AD) patients there are alterations of specific APP forms, paralleled by alteration in expression level of both ADAM 10 and BACE when compared to control subjects. Due to the poor linear relation among each key-element of beta-amyloid cascade and the target diagnosis, the use of systems able to afford non linear tasks, like artificial neural networks (ANNs), should allow a better discriminating capacity in comparison with classical statistics.ObjectiveTo evaluate the accuracy of ANNs in AD diagnosis.Methods37 mild-AD patients and 25 control subjects were enrolled, and APP, ADM10 and BACE measures were performed. Fifteen different models of feed-forward and complex-recurrent ANNs (provided by Semeion Research Centre), based on different learning laws (back propagation, sine-net, bi-modal) were compared with the linear discriminant analysis (LDA).ResultsThe best ANN model correctly identified mild AD patients in the 94% of cases and the control subjects in the 92%. The corresponding diagnostic performance obtained with LDA was 90% and 73%.ConclusionThis preliminary study suggests that the processing of biochemical tests related to beta-amyloid cascade with ANNs allows a very good discrimination of AD in early stages, higher than that obtainable with classical statistics methods.

Neuronal AChE splice variants and their non‐hydrolytic functions: redefining a target of AChE inhibitors?

AChE enzymatic inhibition is a core focus of pharmacological intervention in Alzheimers disease (AD). Yet, AChE has also been ascribed non‐hydrolytic functions, which seem related to its appearance in various isoforms. Neuronal AChE presents as a tailed form (AChE‐T) predominantly found on the neuronal synapse, and a facultatively expressed readthough form (AChE‐R), which exerts short to medium‐term protective effects. Notably, this latter form is also found in the periphery. While these non‐hydrolytic functions of AChE are most controversially discussed, there is evidence for them being additional targets of AChE inhibitors. This review aims to provide clarification as to the role of these AChE splice variants and their interplay with other cholinergic parameters and their being targets of AChE inhibition: AChE‐R is particularly involved in the mediation of (anti‐)apoptotic events in cholinergic cells, involving adaptation of various cholinergic parameters and a time‐dependent link to the expression of neuroprotective factors. The AChE‐T C‐terminus is central to AChE activity regulation, while isolated AChE‐T C‐terminal fragments mediate toxic effects via the α7 nicotinic acetylcholine receptor. There is direct evidence for roles of AChE‐T and AChE‐R in neurodegeneration and neuroprotection, with these roles involving AChE as a key modulator of the cholinergic system: in vivo data further encourages the use of AChE inhibitors in the treatment of neurodegenerative conditions such as AD since effects on both enzymatic activity and the enzymes non‐hydrolytic functions can be postulated. It also suggests that novel AChE inhibitors should enhance protective AChE‐R, while avoiding the concomitant up‐regulation of AChE‐T.