The mechanism of A beta production and its inhibition as a therapeutic strategy for the prevention of Alzheimer's disease

Deposition of amyloid beta peptide (A beta) as senile plaques or cerebrovascular amyloid characterizes the brains of patients with Alzheimer's disease (AD). A beta are composed of 40-42 amino acids that are proteolytically produced from its precursor beta APP. We have shown that the deposition of A beta ending at the 42nd residue (A beta 42) is one of the earliest pathological changes in AD brains. Genetic and cell biological evidence strongly suggest that mutations in beta APP or presenilin (PS) 1 and 2 genes cause AD through increase in production of A beta 42. Recently, PS1 and PS2 are shown to be the catalytic subunits of gamma-secretase that cleaves the intramembrane segments of beta APP and Notch. beta-amyloid hypothesis that emphasizes the primacy of A beta in the pathogenesis of AD is currently being verified by the new experimental therapeutic approaches, e.g., A beta vaccine therapy or administration of inhibitors of beta- or gamma-secretases. The mechanistic roles of newly identified co-factor proteins of PS (i.e., APH-1 and PEN-2) in gamma-secretase function, as well as recent advances in the development of gamma- or beta-secretase inhibitors will be discussed.     

Long-term deprivation of oestrogens by ovariectomy potentiates beta-amyloid-induced working memory deficits in rats.

1 In the present study, we examined whether deprivation of oestrogens by ovariectomy could modify learning and memory deficits caused by a continuous intracerebroventricular (i.c.v.) infusion of amyloid beta-peptide (Abeta), the major constituent of senile plaques in AD. 2 Neither long-term (3 months) nor short-term (1 month), deprivation of oestrogens by ovariectomy caused a significant impairment in spatial learning and memory in a water maze and spontaneous alternation behaviour in a Y-maze. 3 A continuous i.c.v. infusion of Abeta-(1-42) caused spatial learning and memory deficits in both ovariectomized and sham-operated rats. 4 The Abeta-induced working memory deficits were significantly potentiated in ovariectomized rats compared with sham-operated rats when mnemonic ability was examined 3 months after ovariectomy. 5 These results suggest that long-term deprivation of oestrogens induced by ovariectomy increases susceptibility to memory deficits produced by Abeta-(1-42) in rats.     

Cholesterol and Alzheimer's disease: clinical and experimental models suggest interactions of different genetic, dietary and environmental risk factors

Alzheimer's disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39-42 residues known as amyloid beta-peptide (Abeta) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Abeta precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1/2) and apolipoprotein E (APOE) also play a critical role in increased Abeta deposition. Several biochemical and molecular studies using transfected cultured cells and transgenic animals point to mechanisms by which Abeta is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as 'secretases' participate in APP processing leading to the generation of either Abeta or non-amyloid proteins. However, the mechanisms of neurotoxicity of Abeta and the role of APP function in AD remain important unanswered questions. Although early studies recognized the loss of cholesterol and other lipids in the brain, these findings have been poorly connected with AD pathogenesis, despite the identification of the epsilon4 allele of APOE as a major risk factor in AD. The recent finding that cholesterol can modulate the yield of potentially toxic Abeta has boosted research on its role in AD. Consequently, several cholesterol-reducing drugs are currently being evaluated for the treatment of AD. The present review summarizes our current understanding of the relationship of AD pathogenesis with cholesterol, lipids and other genetic and environmental risk factors.     

Targeting the alpha 7 nicotinic acetylcholine receptor to reduce amyloid accumulation in Alzheimer's disease pyramidal neurons

Although there is still no known effective preventative treatment or cure for Alzheimer's disease (AD), the development of new drugs that target pathological features that appear early in the course of this disease and alleviate some of the early cognitive and memory symptoms is a laudable goal that may be one step closer. To date, the acetylcholinesterase inhibitors have been the most widely used AD drugs and have been somewhat successful in slowing loss of cognition. In the last few years, a number of studies have demonstrated that amyloid beta (1-42) (Abeta42), the predominant Abeta peptide species in amyloid plaques, first accumulates in vulnerable neurons prior to plaque formation. Recently, we have shown that many (if not most) amyloid plaques in the entorhinal cortex of AD brains are actually the lysis remnants of degenerated, Abeta42-overburdened neurons. Furthermore, the most vulnerable neurons appear to be those that abundantly express the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), and internalization of Abeta42 appears to be facilitated by the high-affinity binding of Abeta42 to the alpha7nAChR on neuronal cell surfaces, followed by endocytosis of the resulting complex and its accumulation within the lysosomal compartment. This mechanism provides a reasonable explanation for the selective vulnerability of cholinergic and cholinoceptive neurons in AD brains and for the fact that Abeta42 is the dominant Abeta peptide species in both intraneuronal accumulations and amyloid plaques. In view of the pathophysiological consequences of Abeta42 binding to alpha7nAChR on neuronal surfaces that stem from excessive intraneuronal Abeta42 accumulation, the alpha7nAChR could be an important therapeutic target for treatment of AD. In addition, it further emphasizes the potential merits of new and effective therapeutic strategies pointed towards the goal of lowering of Abeta42 levels in the blood and cerebrospinal fluid as well as blocking Abeta42 in the blood from penetrating the blood-brain barrier and entering into the brain parenchyma.     

Beta-secretase inhibition for the treatment of Alzheimer's disease--promise and challenge

As the number of cases of Alzheimer's disease (AD) rises in all developed countries, the unmet medical need for disease-modifying pharmacotherapy continues to grow. Much of AD research has been focused on the amyloid cascade hypothesis, which states that amyloid-beta-42 (A beta 42), a proteolytic derivative of the large transmembrane protein amyloid precursor protein (APP), plays an early and crucial role in all cases of AD. Consequently, blocking the production of A beta 42 by specific inhibition of the key proteases required for A beta 42 generation is a major focus of research into AD therapy. The identification of beta-secretase, the aspartic protease that generates the N-terminus of A beta 42, has triggered a race to develop drug-like inhibitors of this enzyme, which has become one of the major AD targets. Although the biology of beta-secretase holds great promise, it will be challenging to generate drug-like inhibitors of this unusual enzyme.     

Improvement by nefiracetam of beta-amyloid-(1-42)-induced learning and memory impairments in rats

1. We have previously demonstrated that continuous i.c.v. infusion of amyloid beta-peptide (A beta), the major constituent of senile plaques in the brains of patients with Alzheimer's disease, results in learning and memory deficits in rats. 2. In the present study, we investigated the effects of nefiracetam [N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, DM-9384] on A beta-(1-42)-induced learning and memory deficits in rats. 3. In the A beta-(1-42)-infused rats, spontaneous alternation behaviour in a Y-maze task, spatial reference and working memory in a water maze task, and retention of passive avoidance learning were significantly impaired as compared with A beta-(40-1)-infused control rats. 4. Nefiracetam, at a dose range of 1-10 mg kg(-1), improved learning and memory deficits in the A beta-(1-42)-infused rats when it was administered p.o. 1 h before the behavioural tests. 5. Nefiracetam at a dose of 3 mg kg(-1) p.o. increased the activity of choline acetyltransferase in the hippocampus of A beta-(1-42)-infused rats. 6. Nefiracetam increased dopamine turnover in the cerebral cortex and striatum of A beta-(1-42)-infused rats, but failed to affect the noradrenaline, serotonin and 5-hydroxyindoleacetic acid content. 7. These results suggest that nefiracetam may be useful for the treatment of patients with Alzheimer's disease.     

Determination of guinea-pig cortical gamma-secretase activity ex vivo following the systemic administration of a gamma-secretase inhibitor

(2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide (compound E) is a gamma-secretase inhibitor capable of reducing amyloid beta-peptide (1-40) and amyloid beta-peptide (1-42) levels. In this study we investigated the effect of in vivo administration of compound E on guinea-pig plasma, CSF and cortical amyloid beta-peptide (1-40) concentration. Using repeated sampling of CSF, compound E (30 mg/kg p.o.) was shown to cause a time-dependent decrease in CSF amyloid beta-peptide (1-40) levels, which was maximal at 3 h (70% inhibition), compared to baseline controls. After 3 h administration, compound E (3, 10 and 30 mg/kg p.o.), reduced plasma, CSF and DEA-extracted cortical amyloid beta-peptide (1-40) levels by 95, 97 and 99%; 26, 48 and 78%; 32, 33, and 47%, respectively, compared to vehicle control values. In the same animals, compound E (3, 10 and 30 mg/kg p.o.) inhibited cortical gamma-secretase activity, determined ex vivo using the recombinant substrate C100Flag, by 40, 71 and 79% of controls, respectively. These data demonstrate the value of determining not only the extent by which systemic administration of a gamma-secretase inhibitor reduces amyloid beta-peptide, but also the inhibition of brain gamma-secretase activity, as a more direct estimate of enzyme occupancy.     

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.     

Amyloid precursor protein,presenilins, and alpha-synuclein: molecular pathogenesis and pharmacological applications in Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia that arises on a neuropathological background of amyloid plaques containing beta-amyloid (A beta) derived from amyloid precursor protein (APP) and tau-rich neurofibrillary tangles. To date, the cause and progression of both familial and sporadic AD have not been fully elucidated. The autosomal-dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding APP, presenilin-1 (chromosome 14), and presenilin-2 (chromosome 1). APP is processed by several different proteases such as secretases and/or caspases to yield A beta and carboxyl-terminal fragments, which have been implicated in the pathogenesis of Alzheimer's disease. Alzheimer's disease and Parkinson's disease are associated with the cerebral accumulation of A beta and alpha-synuclein, respectively. Some patients have clinical and pathological features of both diseases, raising the possibility of overlapping pathogenic pathways. Recent studies have strongly suggested the possible pathogenic interactions between A beta, presenilins, and/or alpha-synuclein. Therefore, treatments that block the accumulation of A beta and alpha-synuclein might benefit a broad spectrum of neurodegenerative disorders. This review covers the trafficking and processing of APP, amyloid cascade hypothesis in AD pathogenesis, physiological and pathological roles of presenilins, molecular characteristics of alpha-synuclein, their interactions, and therapeutic strategies for AD.     

Selective Contrast Enhancement of Individual Alzheimer’s Disease Amyloid Plaques Using a Polyamine and Gd-DOTA Conjugated Antibody Fragment Against Fibrillar Aβ42 for Magnetic Resonance Molecular Imaging

PURPOSE: The lack of an in vivo diagnostic test for AD has prompted the targeting of amyloid plaques with diagnostic imaging probes. We describe the development of a contrast agent (CA) for magnetic resonance microimaging that utilizes the F(ab')2 fragment of a monoclonal antibody raised against fibrillar human Abeta42 METHODS: This fragment is polyamine modified to enhance its BBB permeability and its ability to bind to amyloid plaques. It is also conjugated with a chelator and gadolinium for subsequent imaging of individual amyloid plaques RESULTS: Pharmacokinetic studies demonstrated this 125I-CA has higher BBB permeability and lower accumulation in the liver and kidney than F(ab')2 in WT mice. The CA retains its ability to bind Abeta40/42 monomers/fibrils and also binds to amyloid plaques in sections of AD mouse brain. Intravenous injection of 125I-CA into the AD mouse demonstrates targeting of amyloid plaques throughout the cortex/hippocampus as detected by emulsion autoradiography. Incubation of AD mouse brain slices in vitro with this CA resulted in selective enhancement on T1-weighted spin-echo images, which co-register with individual plaques observed on spatially matched T2-weighted spin-echo image CONCLUSIONS: Development of such a molecular probe is expected to open new avenues for the diagnosis of AD.     

Mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in Alzheimer's disease

Alzheimer's disease (AD) is the most common neurodegenerative disorder of late life characterized by insidious, chronic, and progressive memory impairment in association with the accumulation of senile plaques, neurofibrillary tangles, and massive loss of neurons. Apoptosis is believed to be an important contributor to progression and pathology of neurodegeneration in AD. There is considerable evidence that amyloid beta-peptide, a major component of senile plaques, has the capacity to activate intracellular apoptosis pathways leading to neuronal cell death. AD-related mutations in genes coding presenilins are also shown to cause neuronal apoptosis, by directly and indirectly regulating apoptotic signaling cascades. Recent evidence suggests that two intrinsic pathways, mitochondrial dysfunction and endoplasmic reticulum stress, are central in the execution of apoptosis in AD. This review summarizes recent progress of research in this field focused on the molecular mechanisms involved in neuronal apoptosis mediated by organelle dysfunction.     

An overview of the current and novel drugs for Alzheimer's disease with particular reference to anti-cholinesterase compounds

Several cellular processes could be targeted if the complex nature of Alzheimer's disease (AD) was already understood. Most of AD treatments have been focused on the inhibition of acetylcholinesterase (AChE) in order to raise the levels of its substrate, i.e. the neurotransmitter acetylcholine (ACh), to augment cognitive functions of affected patients. Effectiveness in AChE inhibition and side-effect issues of clinical (tacrine, donepezil, galanthamine and rivastigmine) as well as of novel inhibitors is reviewed here. Novel design methods for the inhibition of AChE include the use of in silico tools to predict the interactions between AChE and the desired compound, both at the active site of the enzyme, responsible of hydrolysing ACh and with the peripheral anionic site (PAS), which has been described as a promoting agent of the amyloid beta-peptide (A beta) aggregation present in the senile plaques of the brain of AD individuals.     

Human beta-secretase (BACE) and BACE inhibitors: progress report

A key step in the processing of the integral membrane protein APP, or Amyloid Precursor Protein is through the proteolytic cleavage by the enzyme beta-Secretase (BACE). The proteolysis of APP by BACE, followed by subsequent C-terminal cleavage(s) by gamma-secretase, results in the formation of the amyloid beta (Abeta) peptide. The principal component of the neuritic plaque found in the brains of Alzheimer's Disease (AD) patients is Abeta which is a neurotoxic and highly aggregatory peptide segment of APP. The amyloid hypothesis holds that the neuronal dysfunction and clinical manifestation of AD is a consequence of the long term deposition and accumulation of 40-42 amino-acid long Abeta peptides, and that this process leads to the onset and progression of AD. Due to the apparent causal relationship between Abeta and AD, the so-called "secretases" that produce Abeta have been targeted for development of inhibitors that might serve as therapeutic agents for treatment of this dreaded, and ever more prevalent disease. Herein will be discussed our current understanding of BACE, its role in the formation of neuritic plaques and the known inhibitors of the enzyme.     

Molecular rationale for the pharmacological treatment of Alzheimer's disease

Cerebral deposition of amyloid plaques containing amyloid beta-peptide (Abeta) has traditionally been considered the central feature of Alzheimer's disease (AD). Abeta is derived from amyloid precursor protein (APP), which is cleaved by several different proteases: alpha-, beta- and gamma-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 alpha-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 alpha-secretase and they reduced Abeta generation and amyloid accumulation in a transgenic mouse model. beta-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 beta-secretase inhibitors. gamma-Secretase is not an easy candidate for pharmacological manipulation. Immunotherapeutic strategies have targeted Abeta directly; however, intensive investigation of indirect approaches to the management of AD with immunotherapy is now underway.     

Ginkgo biloba extract and its flavonol and terpenelactone fractions do not affect beta-secretase mRNA and enzyme activity levels in cultured neurons and in mice

Numerous clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer's disease (AD). Although neuroprotective properties of EGb761 have been consistently reported, the molecular mechanisms of EGb761 and the specific role of its major constituents, the flavonols and terpenlactones, are largely unknown. One major hallmark of AD is the deposition of amyloid-beta (A beta) as amyloid plaques in the brain. A beta is a cleavage product of amyloid precursor protein (APP). Certain proteases, called beta-secretases (BACE), are crucial in the formation of A beta. The purpose of the present study was to investigate the efficacy of EGb761 and its flavonol and terpenelactone fraction to modulate BACE-1 enzyme activity and mRNA levels in vitro and in vivo. Neither EGb761 nor its fractions affected BACE-1 activity in vitro. Furthermore, also in Neuro-2a cells and wild-type as well as transgenic (Tg2576) laboratory mice, no significant effect of EGb761 on BACE-1 enzyme activity and mRNA levels were observed. Current findings suggest that BACE-1 may not be a major molecular target of EGb761 and its flavonol and terpenelactone fraction.     

Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.     

Muscarinic agonists as preventative therapy for Alzheimer's disease

Overproduction of the peptide amyloid beta (Abeta) is a critical pathogenic event in Alzheimer's disease (AD), leading to the formation of amyloid plaques, neurofibrillary tangles, synaptic loss and dementia. Decreasing Abeta production may therefore slow or halt the progression of AD. Recent animal experiments suggest that Abeta overproduction in aging and sporadic AD may be due to age-related loss of cortical cholinergic innervation. Muscarinic agonists, particularly M1-selective agents, have been shown to decrease the production of Abeta in vitro and in vivo; these compounds may be uniquely suited to a preventative role in AD therapy.     

Radioiodinated flavones for in vivo imaging of beta-amyloid plaques in the brain

In vivo imaging of beta-amyloid (A beta) peptide aggregates in the brain may lead to early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of AD treatment. The purpose of this study was to develop novel amyloid imaging agents based on flavone as a core structure. Radioiodinated flavone derivatives were designed and synthesized. The binding affinities of flavone derivatives for A beta aggregates varied from 13 to 77 nM. When in vitro plaque labeling was carried out using post-mortem AD brain sections, all flavones intensely stained not only amyloid plaques but also cerebrovascular amyloids. In biodistribution studies using normal mice, they displayed high brain uptakes ranging from 3.2 to 4.1% ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (0.5-1.9% ID/g at 30 min), which is highly desirable for amyloid imaging agents. The results in the study suggest that these classes of radioiodinated flavones may be useful candidates as potential imaging agents for amyloid plaques.     

Amyloid beta-peptide [1-42]-associated free radical-induced oxidative stress and neurodegeneration in Alzheimer's disease brain: mechanisms and consequences

In addition to synapse loss, neurofibrillary tangles, and neurodegeneration, oxidative stress and amyloid beta-peptide [Abeta] deposition are hallmarks of Alzheimer's disease [AD] brain. Our laboratory coupled these two characteristics of AD into a comprehensive model to account for the synapse loss and neurodegeneration in AD brain. This model combines much of the extant studies on AD and is based on oxidative stress associated with amyloid beta-peptide. This review presents evidence in support of this model and provides insight into the molecular basis of this devastating dementing disorder.     

Beta-amyloid therapies in Alzheimer's disease

Neurones in the brain produce beta-amyloid 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). A beta fragments are generated through the action of specific proteases within the cell. Two of these enzymes, beta- and gamma-secretase, are particularly important in the formation of A beta as they cleave within the APP protein to give rise to the N-terminal and C-terminal ends of the A beta fragment, respectively. Consequently, many researchers are investigating therapeutic approaches that inhibit either beta- or gamma-secretase activity, with the ultimate goal of limiting A beta; production. An alternative AD therapeutic approach that is being investigated is to employ anti-A beta antibodies to dissolve plaques that have already formed. Both of these approaches focus on the possibility that accrual of A beta leads to neuronal degeneration and cognitive impairment characterised by AD and test the hypothesis that limiting A beta deposition in neuritic plaques may be an effective treatment for AD.