Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.     

ApoE genotype influences the biological effect of donepezil on APP metabolism in Alzheimer disease: evidence from a peripheral model.

Three major amyloid precursor protein (APP) forms with apparent molecular weight ranging from 106 to 130 kDa are present in human platelets. Alzheimer disease (AD) is associated with a decreased APP forms ratio (APPr) between the three major forms. A total of 25 mild to moderate AD patients were investigated. Platelet APPr was studied before and after 30 days of acetylcholinesterase-inhibitor treatment (donepezil, 5 mg daily). Patients were grouped into non-epsilon4 carriers and epsilon4 carriers according to apolipoprotein E (ApoE) genotype. At baseline, all patients showed low APPr levels and no significant difference was found between the two ApoE subgroups. After treatment, although a marked improvement in APPr was observed in most patients, non-epsilon4 carriers displayed a higher increase compared to epsilon4 carriers (P<0.0001). The present study provides evidence that donepezil influences APP metabolism in platelets, and suggests that ApoE genotype might be an important modulating factor for drug responsiveness in AD.     

β-Amyloid peptide as a target for treatment of Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative disorder characterised by a series of biochemical and histological changes although the net of relations and its initial cause is far from being fully understood. The amyloid hypothesis points out the pathological processing of a physiologically normal protein, the amyloid precursor protein, to neurotoxic forms of amyloid β-peptide as the origin of the cascade of biochemical changes that lead to Alzheimer’s disease. Normal APP processing involves three proteases, α-, β- and γ-secretase, to yield physiological amyloid fragments. Familial Alzheimer’s disease patients exhibit an increased activity of β- and γ-secretases, resulting in higher than average levels of small amyloid fragments, of 40 or 42 amino acids (Aβ₄₀ and Aβ₄₂, respectively). These newly formed Aβ₄₀ and Aβ₄₂ may suffer a conformational change followed by aggregation into fibrils and finally deposition as senile plaques in a complex process named fibrillogenesis, which is associated with neurotoxicity. Modulation of this multistep process is a reasonably hopeful approach for the treatment of Alzheimer’s disease. In a general sense, this approach can be divided in three groups: first, modulating the production of Aβ promoting the non-amyloidogenic route; second, inhibiting fibrillogenesis and third, by immunisation techniques, enhancing the formation of anti-Aβ antibodies in order to mark fibrils and plaques as targets for microglial cells.     

Statine-derived tetrapeptide inhibitors of β-secretase

Alzheimer’s disease (AD) is characterised by the accumulation of amyloid β-peptide (Aβ) within senile plaques in the brain. β-secretase is one of the enzymes necessary for the production of amyloid β-peptide from the amyloid precursor protein (APP), the other being γ-secretase. β-Secretase was recently characterised as a novel aspartyl protease. Statine-derived tetrapeptide inhibitors of this enzyme, described in this patent, may have therapeutic applications in AD.     

Processing of amyloid precursor protein as a biochemical link between atherosclerosis and Alzheimer's disease

Macrophage activation in atherosclerotic plaques plays a role in plaque destabilization, rupture and subsequent atherothrombosis. Platelet phagocytosis that occurs within human atherosclerotic plaques can activate macrophages and it has been suggested that the platelet constituent amyloid precursor protein (APP) is involved. Recent studies show that amyloid beta (Abeta), a peptide extensively studied in Alzheimer's disease and that is cleaved from APP by beta- and gamma-secretase, and/or Abeta-like peptides are also present in human atherosclerotic plaques, in particular in activated, inducible nitric oxide synthase (iNOS) expressing perivascular macrophages that had phagocytized platelets. In vitro studies confirm that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Abeta-like peptides, resulting in iNOS induction. In addition, non-steroidal anti-inflammatory drugs (NSAIDs) and HMG-CoA reductase inhibitors (statins), two classes of drugs reported to affect APP processing and Abeta formation in Alzheimer's disease, have been evaluated for their capacity to inhibit macrophage activation evoked by platelet phagocytosis. Remarkably, the same NSAIDs reported to alter gamma-secretase activity in Alzheimer's disease also reduce macrophage activation after platelet phagocytosis and inhibit formation of Abeta-containing peptides. From the statins investigated (fluvastatin, atorvastatin, simvastatin, pravastatin, lovastatin and rosuvastatin) only fluvastatin and atorvastatin selectively inhibit macrophage activation after platelet phagocytosis, possibly through inhibition of Rho activity. Taken together, these new findings point to the involvement of platelet-derived APP in macrophage activation in atherosclerosis and suggest a biochemical link between atherosclerosis and Alzheimer's disease. Accordingly, drugs interfering with APP processing might have an impact on both diseases.     

神经传递与β-淀粉样蛋白前体的加工调控

淀粉样蛋白沉积是阿尔采末病（Ａｌｚｈｅｉｍｅｒｓｄｉｓｅａｓｅ,ＡＤ）中重要的组织病理学特征,其前体ＡＰＰ在加工代谢过程中生成两类作用相反的产物。有些神经递质受体参与调控这一加工过程,提示神经传递活动在ＡＤ发生发展机制中起了作用。     

Gamma-secretase as a pharmacological target in Alzheimer disease research: when, why and how?

Alzheimer disease (AD) is characterized by excessive deposition of amyloid beta-peptides (Abeta peptides) in the form of senile plaques as well as neurofibrillary tangles (NFTs) in the brain. In the amyloidogenic pathway, the amyloid-beta precursor protein (APP) is cleaved by beta-secretase first, followed by gamma-secretase cleavage producing therefore Abeta. This review summarizes the recent findings in the AD field and focuses on the different gamma-secretase inhibitors that have been developed as a therapeutic approach toward AD.     

Inhibition of BACE, a promising approach to Alzheimer's disease therapy

The first proteolytic step in the processing of amyloid precursor protein (APP) to amyloid-beta (Abeta) in the brain is performed by beta-site APP cleaving enzyme (BACE1). This enzyme is a membrane bound aspartic protease with high homology of the catalytic domain to renin and pepsin and of yet unknown physiologic function. It is a primary drug discovery target for Alzheimer s disease therapy. The first potent inhibitors are based on the sequence of APP around the beta-secretase cleavage site EVNL/DAEF, with the scissile Leu-Asp amide bond being replaced by a hydroxyethylene transition state analogue isostere. In addition, lipophilic sidechains have been incorporated and a crystal structure of such an octapeptidic inhibitor bound in the active site is already available. Recent progress in the field of BACE inhibition is reviewed.     

Glucose metabolism and insulin receptor signal transduction in Alzheimer disease

Nosologically, Alzheimer disease is not a single disorder in spite of a common clinical phenotype. Etiologically, two different types or even more exist. (1) In a minority of about 5% or less of all cases, Alzheimer disease is due to mutations of three genes, resulting in the permanent generation of βA4. (2) The great majority (95% or more) of cases of Alzheimer disease are sporadic in origin, with old age as main risk factor, supporting the view that susceptibility genes and aging contribute to age-related sporadic Alzheimer disease. However, disturbances in the neuronal insulin signal transduction pathway may be of central pathophysiological significance. In early-onset familial Alzheimer disease, the inhibition of neuronal insulin receptor function may be due to competitive binding of amyloid beta (Aβ) to the insulin receptor. In late-onset sporadic Alzheimer disease, the neuronal insulin receptor may be desensitized by inhibition of receptor function at different sites by noradrenaline and/or cortisol, the levels of which both increase with increasing age. The consequences of the inhibition of neuronal insulin signal transduction may be largely identical to those of disturbances of oxidative energy metabolism and related metabolism, and of hyperphosphorylation of tau-protein. As far as the metabolism of amyloid precursor protein (APP) in late-onset sporadic Alzheimer disease is concerned, neuronal insulin receptor dysfunction may result in the intracellular accumulation of Aβ and in subsequent cellular damage. In this context, the desensitization of the neuronal insulin receptor in late-onset sporadic Alzheimer disease is different from that occurring in normal aging and early-onset familial Alzheimer disease. In late-onset sporadic Alzheimer disease changes in the brain are similar to those caused by non-insulin-dependent diabetes mellitus.     

β-Amyloid therapies in Alzheimer’s disease

Neurones in the brain produce β-amyloid (Aβ) fragments from a larger precursor molecule termed the amyloid precursor protein (APP). When released from the cell, these protein fragments may accumulate in extracellular amyloid plaques and consequently hasten the onset and progression of Alzheimer’s disease (AD). β-Amyloid fragments are generated through the action of specific proteases within the cell. Two of these enzymes, β- and γ-secretase, are particularly important in the formation of Aβ as they cleave within the APP protein to give rise to the N-terminal and C-terminal ends of the Aβ fragment, respectively. Consequently, many researchers are investigating therapeutic approaches that inhibit either β- or γ-secretase activity, with the ultimate goal of limiting Aβ production. An alternative AD therapeutic approach that is being investigated is to employ anti-Aβ antibodies to dissolve plaques that have already formed. Both of these approaches focus on the possibility that accrual of Aβ leads to neuronal degeneration and cognitive impairment characterised by AD and test the hypothesis that limiting Aβ deposition in neuritic plaques may be an effective treatment for AD.     

Effect of non-steroidal anti-inflammatory drugs on amyloid-beta formation and macrophage activation after platelet phagocytosis

Recently, we showed that platelet phagocytosis occurs in human atherosclerotic plaques and leads to foam cell formation. Platelet phagocytosis, resulting in macrophage activation and iNOS induction, was associated with the formation of amyloid-beta peptide (Abeta) via proteolytic cleavage of platelet-derived amyloid precursor protein (APP), possibly by secretases. To test the involvement of gamma-secretase in this process, we used indomethacin, ibuprofen, and sulindac sulfide, non-steroidal anti-inflammatory drugs (NSAIDs) known to alter the gamma-secretase cleaving site of APP, on their ability to inhibit macrophage activation evoked by platelet phagocytosis. J774 macrophages were incubated with human platelets or lipopolysaccharide (LPS) with or without NSAIDs. Nitrite was quantified as a measure for inducible nitric oxide synthase (iNOS) activity. Indomethacin, ibuprofen, sulindac sulfide, and meloxicam concentration-dependently reduced nitrite production by macrophages incubated with platelets, but did not alter LPS-induced iNOS activity or platelet uptake. However, acetylsalicylic acid and naproxen, two NSAIDs without effect on the gamma-secretase cleaving site of APP, did not affect nitrite production in either platelet- or LPS-stimulated macrophages. Surface-enhanced laser desorption/ionization time-of-flight mass-spectrometry demonstrated time-dependent formation of Abeta-containing peptides after platelet phagocytosis, which could be inhibited by indomethacin. In conclusion, these results point to the involvement of gamma-secretase in macrophage activation following platelet phagocytosis.     

Oestrogen and nerve growth factor – neuroprotection and repair in Alzheimer’s disease

The neurogenetics and neuropathology of Alzheimer’s disease (AD) are still largely unknown, even though recent work has clarified some genetic components in this common and devastating neurodegenerative disease. Most of the genetic mutations have been shown to be, at least in the early onset type of AD, related to the function of a large transmembrane protein, amyloid precursor protein (APP). This protein is cleaved into various smaller fragments that are either soluble or aggregating. It is thought that this processing of APP is inherently important for the initiation and progression of AD. Recent animal models have suggested that it is not the formation of β-amyloid plaques per se, but the altered processing of APP and the subsequent loss of soluble APP, that sets the stage for the massive neuronal cell loss which occurs in AD. We would like to propose a three-way relationship between oestrogen, APP and nerve growth factor (NGF) in the neural pathways of the brain which are involved in learning and memory – the limbic system. The degeneration of the cholinergic innervation from the basal forebrain to the hippocampal formation in the temporal lobe is thought to be one of the factors determining the progression of memory decay, both during normal ageing and AD. Oestrogen and NGF are among the neuroprotective agents that have shown some potential for the treatment of AD. Previous results of treatment with these two agents and their relationship to the amyloid proteins, will be discussed in this review.     

The search for alpha-secretase and its potential as a therapeutic approach to Alzheimer s disease

In the nonamyloidogenic processing pathway the Alzheimer s amyloid precursor protein (APP) is proteolytically cleaved by alpha-secretase. As this cleavage occurs at the Lys16-Leu17 bond within the amyloid beta domain, it prevents deposition of intact amyloidogenic peptide. In addition, the large ectodomain (sAPP(alpha)) released by the action of alpha-secretase has several neuroprotective properties. Studies with a range of hydroxamic acid-based compounds, such as batimastat, indicate that alpha-secretase is a zinc metalloproteinase, and members of the adamalysin family of proteins, TACE, ADAM10 and ADAM9, all fulfil some of the criteria required of alpha-secretase. APP is constitutively cleaved by alpha-secretase in most cell lines. However, on stimulation with muscarinic agonists or activators of protein kinase C, such as phorbol esters, the alpha-secretase cleavage of APP is up-regulated. The constitutive alpha-secretase activity is primarily at the cell surface, while the regulated activity is predominantly located within the Golgi. The beneficial action of cholinesterase inhibitors may in part be due to activation of muscarinic receptors, resulting in an up-regulation of alpha-secretase. Other agents can also increase the nonamyloidogenic cleavage of APP including estrogen, testosterone, various neurotransmitters and growth factors. As the alpha-secretase cleavage of APP both precludes the deposition of the amyloid beta peptide and releases the neuroprotective sAPP(alpha), pharmacological up-regulation of alpha-secretase may provide alternative therapeutic approaches for Alzheimer s disease.     

Small interfering RNA delivery to the neurons near the amyloid plaques for improved treatment of Alzheimer׳s disease

Gene therapy represents a promising treatment for the Alzheimer׳s disease (AD). However, gene delivery specific to brain lesions through systemic administration remains big challenge. In our previous work, we have developed an siRNA nanocomplex able to be specifically delivered to the amyloid plaques through surface modification with both CGN peptide for the blood–brain barrier (BBB) penetration and QSH peptide for β-amyloid binding. But, whether the as-designed nanocomplex could indeed improve the gene accumulation in the impaired neuron cells and ameliorate AD-associated symptoms remains further study. Herein, we prepared the nanocomplexes with an siRNA against β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), the rate-limiting enzyme of Aβ production, as the therapeutic siRNA of AD. The nanocomplexes exhibited high distribution in the Aβ deposits-enriched hippocampus, especially in the neurons near the amyloid plaques after intravenous administration. In APP/PS1 transgenic mice, the nanocomplexes down-regulated BACE1 in both mRNA and protein levels, as well as Aβ and amyloid plaques to the level of wild-type mice. Moreover, the nanocomplexes significantly increased the level of synaptophysin and rescued memory loss of the AD transgenic mice without hematological or histological toxicity. Taken together, this work presented direct evidences that the design of precise gene delivery to the AD lesions markedly improves the therapeutic outcome.     

Can statins put the brakes on Alzheimer’s disease?

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which initiates the syntheses of cholesterol and isoprenoid lipids that are needed to provide amyloid peptides for the amyloid cascade. This cascade is believed to induce sporadic or late-onset Alzheimer’s disease, which accounts for 90 – 95% of Alzheimer’s disease sufferers. Cholesterol is also the prime driver of cerebrovascular disease that (along with amyloid peptides) increasingly appears to be linked to the cognitive deterioration of Alzheimer’s disease. Cholesterol is needed to make the lipid rafts that are the platforms for isoprenoid-dependent assembly and activation of raftophilic β- and γ-secretases that work in tandem to excise dangerous 40 and 42 amino acid amyloid-β (Aβ) fragments from amyloid precursor protein, the transmembrane amyloid precursor glycoprotein. When they are excessively produced and can no longer be effectively destroyed or otherwise cleared from the hypoperfused ageing brain, the Aβ42 fragments released from the active synaptic terminals of normally busy neurons (and from stressed neurons unsuccessfully trying to proliferate and producing disruptive tangles of hyperphosphorylated τ-proteins) aggregate into neuritic plaques, which activate glial cells. The pro-inflammatory cytokines and growth factors from the glial cells further damage and kill neurons. As statins strike at several parts of the Alzheimer’s disease mechanism (such as the infliction of cholesterol-dependent cerebrovascular damage) by inhibiting HMG-CoA reductase, their long-term use (starting as early as possible during Alzheimer’s disease development) should slow or even prevent the progression of Alzheimer’s disease. Indeed, there is some evidence of a significantly reduced incidence of Alzheimer’s disease among people who have been using statins to reduce hypercholesterolaemia and its cardiovascular effects. To be certain of this, there must be more multi-year trials to specifically assess the effects of statins on sporadic Alzheimer’s disease.     

Rodent models for Alzheimer’s disease drug discovery

Introduction: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and personality changes, leading to dementia. Histopathological hallmarks are represented by aggregates of beta-amyloid peptide (Aβ) in senile plaques and deposition of hyperphosphorylated tau protein in neurofibrillary tangles in the brain. Rare forms of early onset familial Alzheimer’s disease are due to gene mutations. This has prompted researchers to develop genetically modified animals that could recapitulate the main features of the disease. The use of these models is complemented by non-genetically modified animals.

Areas covered: This review summarizes the characteristics of the most used transgenic (Tg) and non-Tg models of AD. The authors have focused on models mainly used in their laboratories including amyloid precursor protein (APP) Tg2576, APP/presenilin 1, 3xAD, single h-Tau, non-Tg mice treated with acute injections of Aβ or tau, and models of physiological aging.

Expert opinion: Animal models of disease might be very useful for studying the pathophysiology of the disease and for testing new therapeutics in preclinical studies but they do not reproduce the entire clinical features of human AD. When selecting a model, researchers should consider the various factors that might influence the phenotype. They should also consider the timing of testing/treating animals since the age at which each model develops certain aspects of the AD pathology varies.     

The role of amyloid-beta in the regulation of memory

In this review there is evidence that amyloid-beta peptide is a memory enhancer at physiological (picomolar) concentrations. Pathological overproduction of amyloid-beta leads to impaired memory, oxidative damage, damage to the blood brain barrier, neurofibrillary tangles and amyloid plaque formation. Antisenses to amyloid precursor protein (APP) can reverse these effects in mice when they lower amyloid-beta protein to physiological levels. Data suggests that overproduction of APP leads to oxidative stress producing a vicious cycle of neuronal damage. For these reasons we have revised the “amyloid cascade hypothesis” removing emphasis from the plaque to amyloid-beta overproduction and suggest that an “amyloid-beta mitochondrial vicious cycle” hypothesis may be a better pathophysiological model for understanding Alzheimer's disease.     

Genetic basis of neurodegeneration in familial Alzheimer's disease

Alzheimer's disease (AD), the most common form of dementia, is characterized by two types of brain lesions, referred to as senile plaques and neurofibrillary tangles. Moreover, neuronal cell loss and synaptic degeneration appear in affected regions of the brain. A series of endoproteolytic cleavages of the amyloid precursor protein (APP) controlled by alpha, beta, and gamma-secretases leads to a formation of non-amyloidogenic (the alpha-secretase pathway) and amyloidogenic (the beta-secretase pathway) products which are essential for neurodegeneration. According to the "amyloid cascade hypothesis", the accumulation of amyloid beta (Abeta) peptides in the brain is a primary event in the pathogenesis of AD. One of the strong pieces of evidence supporting this hypothesis was the identification of pathogenic mutations within APP, presenilin 1 and presenilin 2 genes responsible for familial autosomal dominant AD. These mutations affect APP processing causing overproduction of Abeta42. Finding specific inhibitors of the Abeta42 generation is a major goal of AD drug development programs now and the key challenge for the treatment of the most devasting disease of human brain.     

Neuroprotective profiles of anti-aging gene Klotho in Alzheimer disease mouse model

OBJECTIVE Alzheimer disease(AD) is the most common type of senile dementia. The anti-aging gene Klotho is reported to decline in the brain of patients and animals with AD. However, the role of Klotho in the progression of AD remains elusive. The present study explored the effects and underlying mechanism of Klotho in amyloid precursor protein/presenilin 1(APP/PS1) transgenic mice. METHODS The upregulation of cerebral Klotho expression was mediated by intracerebroventricular administration of a lentiviral vector that encoded Klotho(LV-KL) in APP/PS1 transgenicmice.RESULTS LV-KL significantly increased Klotho overexpression and effectively ameliorated cognitive deficits and AD-like pathology in aged AD mice. LV-KL might induce autophagy activation and protein kinase B/mammalian target of rapamycin inhibition in both AD mice and cultured BV2 murine microglia. Meanwhile, LV-KL altered the expression of low density lipoprotein receptor-related protein 1(LRP-1), receptor for advanced glycation end products, P-glycoprotein and ABCA1 both at the brain microvascular and choroid plexus as well as the contents of plasma s LRP-1 in aged AD mice.CONCLUSION The current results indicate that Klotho plays a crucial role in the clearance of cerebral amyloid β protein and the progression of AD in mice. These findings highlight the preventive and therapeutic potential of Klotho for the treatment of AD.