Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.     

Mechanisms of A beta mediated neurodegeneration in Alzheimer's disease

Development of a comprehensive therapeutic treatment for the neurodegenerative Alzheimer's disease (AD) is limited by our understanding of the underlying biochemical mechanisms that drive neuronal failure. Numerous dysfunctional mechanisms have been described in AD, ranging from protein aggregation and oxidative stress to biometal dyshomeostasis and mitochondrial failure. In this review we discuss the critical role of amyloid-beta (A beta) in some of these potential mechanisms of neurodegeneration. The 39-43 amino acid A beta peptide has attracted intense research focus since it was identified as a major constituent of the amyloid deposits that characterise the AD brain, and it is now widely recognised as central to the development of AD. Familial forms of AD involve mutations that lead directly to altered A beta production from the amyloid-beta A4 precursor protein, and the degree of AD severity correlates with specific pools of A beta within the brain. A beta contributes directly to oxidative stress, mitochondrial dysfunction, impaired synaptic transmission, the disruption of membrane integrity, and impaired axonal transport. Further study of the mechanisms of A beta mediated neurodegeneration will considerably improve our understanding of AD, and may provide fundamental insights needed for the development of more effective therapeutic strategies.     

雌激素对淀粉样β蛋白代谢的调节和毒性缓解

以淀粉样β蛋白为主的老年斑胞外沉积和神经元内神经原纤维缠结,是阿尔采末病(AD)特征性的病理学改变。近来,人们逐渐认可淀粉样蛋白假说,即认为淀粉样蛋白沉积是AD最初起因。研究人员正在寻找针对淀粉样β蛋白沉积的药物,雌激素是其中之一。初步的工作证明,雌激素能够调节淀粉样β蛋白前体代谢,减少淀粉样β蛋白生成,也能够减轻淀粉样B蛋白引起的免疫炎症反应、氧应激对细胞造成的损伤,和对抗细胞凋亡。     

Oligomers on the brain: the emerging role of soluble protein aggregates in neurodegeneration

Extracellular fibrous amyloid deposits or intracellular inclusion bodies containing abnormal protein fibrils characterize many different neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, Huntington's disease, and the transmissible 'prion' dementias. There is strong evidence from genetic, transgenic mouse and biochemical studies to support the idea that the accumulation of protein aggregates in the brain plays a seminal role in the pathogenesis of these diseases. How monomeric proteins ultimately convert to highly polymeric deposits is unknown. However, studies employing, synthetic, cell-derived and purified recombinant proteins suggest that amyloid proteins first come together to form soluble low n-oligomers. Further association of these oligomers results in higher molecular weight assemblies including so-called 'protofibrils' and 'ADDLs' and these eventually exceed solubility limits until, finally, they are deposited as amyloid fibrils. With particular reference to AD and PD, we review recent evidence that soluble oligomers are the principal pathogenic species that drive neuronal dysfunction.     

Human amyloid-beta causes changes in the levels of endothelial protein kinase C and its alpha isoform in vitro

Amyloid-beta (A(beta)) deposits and neurofibrillary pathology are characteristic features of Alzheimer's disease (AD). The association of A(beta) with cerebral vessels is an intriguing feature of AD. While there is considerable evidence of altered activities of the major isoforms of protein kinase C (PKC) in the vasculature and neurons of AD brains, little is known about the relationship between the Abeta toxicity and the altered PKC levels in cerebral endothelial cells.In this study, cultured brain endothelial cells exposed to A(beta)1-40 revealed a translocation of PKC from the membrane fraction to the cytosol. The content of the isoform PKC(alpha), involved in the regulation of amyloid precursor protein (APP) secretion, was decreased in the membrane-bound fraction of rat endothelial cells and increased in the cytosol after A(beta)1-40 treatment. These data suggest that the accumulation of A(beta) peptide in the cerebral vasculature may play a significant role in the down-regulation of PKC seen in the AD cerebral vasculature.     

Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease

The formation of extracellular or intracellular deposits of amyloid-like protein fibrils is a prominent pathological feature of many different neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). In AD, the beta-amyloid peptide (A(beta)) accumulates mainly extracellularly at the center of senile plaques, whereas, in PD, the alpha-synuclein protein accumulates within neurons inside the Lewy bodies and Lewy neurites. We have shown recently that solutions of A(beta) 1-40, A(beta) 1-42, A(beta) 25-35, alpha-synuclein and non-A(beta) component (NAC; residues 61-95 of alpha-synuclein) all liberate hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). These hydroxyl radicals were readily detected by means of electron spin resonance spectroscopy, employing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. Hydroxyl radical formation was inhibited by the inclusion of catalase or metal-chelators during A(beta) or alpha-synuclein incubation. Our results suggest that hydrogen peroxide accumulates during the incubation of A(beta) or alpha-synuclein, by a metal-dependent mechanism, and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD and PD, and possibly other neurodegenerative or amyloid diseases, could be the direct production of hydrogen peroxide during formation of the abnormal protein aggregates.     

Alzheimer beta-amyloid peptides: normal and abnormal localization

Alzheimer's disease (AD) neuropathology is characterized by accumulation of "senile" plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are principally composed of aggregates of up to 42/43 amino acid beta-amyloid (A beta) peptides. The discovery of familial AD (FAD) mutations in the genes for the amyloid precursor protein (APP) and presenilins (PSs), all of which increase A beta42 production, support the view that A beta is centrally involved in the pathogenesis of AD. A beta42 aggregates readily, and is thought to seed the formation of fibrils, which then act as templates for plaque formation. A beta is generated by the sequential intracellular cleavage of APP by beta-secretase to generate the N-terminal end of A beta, and intramembranous cleavage by gamma-secretase to generate the C-terminal end. Cell biological studies have demonstrated that A beta is generated in the ER, Golgi, and endosomal/lysosomal system. A central question involving the role of A beta in AD concerns how A beta causes disease and whether it is extracellular A beta deposition and/or intracellular A beta accumulation that initiates the disease process. The most prevalent view is that SPs are composed of extracellular deposits of secreted A beta and that A beta causes toxicity to surrounding neurons as extracellular SP. The recent emphasis on the intracellular biology of APP and A beta has led some investigators to consider the possibility that intraneuronal A beta may directly cause toxicity. In this review we will outline current knowledge of the localization of both intracellular and extracellular A beta.     

The multiple mechanisms of amyloid deposition: The role of parkin

Amyloid deposition is one of the central neuropathological abnormalities in Alzheimer’s disease (AD) but it also takes places in many neurodegenerative diseases such as prionic disorders, Huntington’s disease (HD) and others. Up to very recently amyloid formation was considered a very slow process of deposition of an abnormal protein due to genetic abnormalities or post-translational modification of the deposited protein. Recent data suggest that the process of amyloidogenesis may be much more rapid in many cases and due to multiple mechanisms.

We have found a mouse model of progressive neurodegeneration that resemble motor, behavioral and pathological hallmarks of parkinsonism and tauopathies, but surprisingly, also present amyloid deposits in brain and peripheral organs. Here we review some of these recent works which may provide new insight into the process of formation of amyloid and, perhaps, new ideas for its treatment.     

Amyloid-beta-peptide reduces copper(II) to copper(I) independent of its aggregation state

Alzheimer's disease (AD) is characterized by the deposition of amyloid beta-peptide (A beta) and neuronal degeneration in brain regions involved in learning and memory. One of the leading etiologic hypotheses regarding AD is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Recent evidence suggests that metals concentrated in amyloid deposits may contribute to the oxidative insults observed in AD-affected brains. We hypothesized that A beta peptide in the presence of copper enhances its neurotoxicity generating free radicals via copper reduction. In the present study, we have examined the effect of the aggregation state of amyloid-beta-peptide on copper reduction. In independent experiments we measured the copper-reducing ability of soluble and fibrillar A beta(1-40) forms by bathocuproine assays. As it was previously observed for the amyloid precursor protein (APP), the A beta peptide showed copper-reducing ability. The capacity of A beta to reduce copper was independent of the aggregation state. Finally, the A beta peptide derived from the human sequence has a greater effect than the A beta peptide derived from the rat sequence, suggesting that histidine 13 may play a role in copper reduction. In agreement with this possibility, the A beta peptide reduces less copper in the presence of exogenous histidine.     

Links Between the Pathology of Alzheimer's Disease and Vascular Dementia

The major neuropathological lesions defining Alzheimer's disease (AD) include neurofibrillary tangles and amyloid plaques, which are mainly composed of abnormally phosphorylated tau and amyloid-beta (A beta), respectively. Numerous neuropathological and neuroimaging studies indicate that at least one-third of AD cases are complicated by some degree of vascular pathology, whereas in a similar proportion of patients clinically diagnosed with vascular dementia, AD pathology is also present. Many classical vascular risk factors such as hypertension, diabetes mellitus, and hypercholesterolemia have recently been shown also to increase the risk of AD. Growing evidence suggests that vascular pathology lowers the threshold for the clinical presentation of dementia at a given level of AD-related pathology and potentially directly promotes AD lesions such as A beta plaques. Cerebral ischemia, chronically up-regulates expression of the amyloid precursor protein (APP), which is the precursor to the amyloid beta peptide and damages the blood-brain barrier (BBB), affecting A beta peptide clearance from the brain. Recognition of the importance of these vascular risk factors for AD-related dementia and their treatment will be beneficial not only for preventing cardiac, cerebral, and peripheral complications of vascular disease, but also will likely have a direct impact on the occurrence of sporadic AD in older subjects. In this paper, we review some of the links between vascular risk factors and AD pathology and present data on the direct effect of ischemia on cognitive function and A beta deposition in a mouse model of AD.     

Alzheimer''s disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695-amino acid-long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.     

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.     

The evolution of A beta peptide burden in the APP23 transgenic mice: implications for A beta deposition in Alzheimer disease.

BACKGROUND: High levels of A beta in the cerebral cortex distinguish demented Alzheimer's disease (AD) from nondemented elderly individuals, suggesting that decreased amyloid-beta (A beta) peptide clearance from the brain is a key precipitating factor in AD. MATERIALS AND METHODS: The levels of A beta in brain and plasma as well as apolipoprotein E (ApoE) in brain were investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting at various times during the life span of the APP23 transgenic (Tg) and control mice. Histochemistry and immunocytochemistry were used to assess the morphologic characteristics of the brain parenchymal and cerebrovascular amyloid deposits and the intracellular amyloid precursor protein (APP) deposits in the APP23 Tg mice. RESULTS: No significant differences were found in the plasma levels of A beta between the APP23 Tg and control mice from 2-20 months of age. In contrast, soluble A beta levels in the brain were continually elevated, increasing 4-fold at 2 months and 33-fold in the APP23 Tg mice at 20 months of age when compared to the control mice. Soluble A beta42 was about 60% higher than A beta40. In the APP23 Tg mice, insoluble A beta40 remained at basal levels in the brain until 9 months and then rose to 680 microg/g cortex by 20 months. Insoluble A beta40 was negligible in non-Tg mice at all ages. Insoluble A beta42 in APP23 Tg mice rose to 60 microg/g cortex at 20 months, representing 24 times the control A beta42 levels. Elevated levels of ApoE in the brain were observed in the APP23 Tg mice at 2 months of age, becoming substantially higher by 20 months. ApoE colocalized with A beta in the plaques. Beta-amyloid precursor protein (betaAPP) deposits were detected within the neuronal cytoplasm from 4 months of age onward. Amyloid angiopathy in the APP23 Tg mice increased markedly with age, being by far more severe than in the Tg2576 mice. CONCLUSIONS: We suggest that the APP23 Tg mouse may develop an earlier blockage in A beta clearance than the Tg2576 mice, resulting in a more severe accumulation of A beta in the perivascular drainage pathways and in the brain. Both Tg mice reflect decreased A beta elimination and as models for the amyloid cascade they are useful to study AD pathophysiology and therapy.     

P2Y receptors in Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta‐amyloid peptides (Aβ) and neuronal death in brain regions involved in learning and memory. Although Aβ is neurotoxic, the primary mechanisms by which Aβ affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aβ have revealed that Aβ has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aβ and contributes to AD pathology. In recent years, Aβ has been shown to affect ATP release from brain and blood cells and alter the expression of G protein‐coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aβ production and elimination, neuroinflammation, neuronal function and cerebral blood flow.     

Stereochemical specificity of Alzheimer's disease beta-peptide assembly

The formation and growth of insoluble amyloid deposits composed primarily of the human beta-amyloid peptide (A beta) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of A beta in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble A beta peptide onto preexisting A beta aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-A beta aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-A beta aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-A beta isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble A beta onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.     

The multiple mechanisms of amyloid deposition: The role of parkin

Amyloid deposition is one of the central neuropathological abnormalities in Alzheimer disease (AD) but it also takes places in many neurodegenerative diseases such as prionic disorders, Huntington''s disease (HD) and others. Up to very recently amyloid formation was considered a very slow process of deposition of an abnormal protein due to genetic abnormalities or post-translational modification of the deposited protein. Recent data suggest that the process of amyloidogenesis may be much more rapid in many cases and due to multiple mechanisms.We have found a mouse model of progressive neurodegeneration that resemble motor, behavioral and pathological hallmarks of parkinsonism and tauopathies, but surprisingly, also present amyloid deposits in brain and peripheral organs. Here we review some of these recent works which may provide new insight into the process of formation of amyloid and, perhaps, new ideas for its treatment.Key words: □-amyloid, Alzheimer disease, chaperones, fronto-temporal dementia, parkin, Parkinson disease, PSP, proteosome, tau protein, tauopathies, autophagy, transgenic mice     

Role of the cell membrane interface in modulating production and uptake of Alzheimer's beta amyloid protein

The beta amyloid protein (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis and its interaction with cell membranes in known to promote mutually disruptive structural perturbations that contribute to amyloid deposition and neurodegeneration in the brain. In addition to protein aggregation at the membrane interface and disruption of membrane integrity, growing reports demonstrate an important role for the membrane in modulating Aβ production and uptake into cells. The aim of this review is to highlight and summarize recent literature that have contributed insight into the implications of altered membrane composition on amyloid precursor protein (APP) proteolysis, production of Aβ, its internalization in to cells via permeabilization and receptor mediated uptake. Here, we also review the various membrane model systems and experimental tools used for probing Aβ-membrane interactions to investigate the key mechanistic aspects underlying the accumulation and toxicity of Aβ in AD.     

Amyloid beta.

Amyloid beta (A beta) is a 39-43 residue amyloidogenic peptide that is deposited into the extracellular amyloid plaques which characterize an Alzheimer's disease (AD) brain. A beta is derived from the amyloid precursor protein (APP) and undergoes a toxic conformational change (gain of toxic function). The length of the A beta peptide dramatically influences its properties with the longer 42 and 43 residue species being more amyloidogenic. The genetics of familial AD (FAD) supports a central role for A beta in AD since mutations in the FAD causing genes APP and the presenilins (PS1 and PS2) increase the formation of A beta 42,43. Considerable activity is directed towards A beta as a therapeutic target. These strategies aim to inhibit A beta synthesis, A beta fibril formation, its toxic actions on cells or promote its clearance from the brain.     

Brains of rhesus monkeys display Aβ deposits and glial pathology while lacking Aβ dimers and other Alzheimer's pathologies

Cerebral amyloid beta (Aβ) deposits are the main early pathology of Alzheimer's disease (AD). However, abundant Aβ deposits also occur spontaneously in the brains of many healthy people who are free of AD with advancing aging. A crucial unanswered question in AD prevention is why AD does not develop in some elderly people, despite the presence of Aβ deposits. The answer may lie in the composition of Aβ oligomer isoforms in the Aβ deposits of healthy brains, which are different from AD brains. However, which Aβ oligomer triggers the transformation from aging to AD pathogenesis is still under debate. Some researchers insist that the Aβ 12‐mer causes AD pathology, while others suggest that the Aβ dimer is the crucial molecule in AD pathology. Aged rhesus monkeys spontaneously develop Aβ deposits in the brain with striking similarities to those of aged humans. Thus, rhesus monkeys are an ideal natural model to study the composition of Aβ oligomer isoforms and their downstream effects on AD pathology. In this study, we found that Aβ deposits in aged monkey brains included 3‐mer, 5‐mer, 9‐mer, 10‐mer, and 12‐mer oligomers, but not 2‐mer oligomers. The Aβ deposits, which were devoid of Aβ dimers, induced glial pathology (microgliosis, abnormal microglia morphology, and astrocytosis), but not the subsequent downstream pathologies of AD, including Tau pathology, neurodegeneration, and synapse loss. Our results indicate that the Aβ dimer plays an important role in AD pathogenesis. Thus, targeting the Aβ dimer is a promising strategy for preventing AD.     

Visualizing interaction of proteins relevant to Alzheimer's disease in intact cells

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer's disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.