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Defined Neurofilament, , and Amyloid Precursor Protein Epitopes Distinguish Alzheimer From Non-Alzheimer Senile Plaques
Abstract
Eight antisera and one monoclonal antibody to synthetic peptides that corresponded to domains extending over the entire length of the beta-amyloid precursor protein (beta-APP), and an antiserum to the full-length 695-amino acid form of the beta-APP, were raised to probe the composition of the core and corona of senile plaques (SPs). We localized distinct beta-APP domains, including the beta-amyloid protein or A4 region, within the SPs of 13 end-stage Alzheimer disease (AD) and 13 age-matched control samples of hippocampus and entorhinal cortex. The composition of SPs also was probed with antibodies to defined epitopes in tau (tau) as well as the large and mid-size neurofilament (NF) proteins. The most important observations were that beta-APP domains outside the A4 region were largely restricted to SP coronas in the AD samples, together with tau and NF determinants, whereas the same epitopes were absent from A4-positive blood vessels and exceptionally rare in non-AD SPs. Indeed, samples from a subset of the non-AD cases contained a considerable number of A4-positive SPs totally devoid of any of the other beta-APP, tau, and NF epitopes. These observations suggest that the deposition of the A4 protein in AD SPs results from the local processing of beta-APPs in association with tau and NF protein fragments. It is unclear whether this association is fortuitous or linked by common mechanisms. However, differences between the complement of beta-APP, tau, and NF protein epitopes in AD versus non-AD brains implicate a defect involving one or more steps in the posttranslational modification, degradation, or elimination of these proteins in AD brains, and this may account for the massive numbers of SPs that characterize AD.
... To develop model systems for studying interactions between different plaque and tangle components, we showed that injections of purified PHFT into the rodent brain induced long-lived co-deposits of A@, ubiquitin, and ACT at the injection site (Shin et al., 1993). Thus, the convergence of PHFT and AP in authentic cortical SPs and NFTs (Hyman et al., 1989;Shin et al., 1989;Arai et al., 1990;Spillantini et al., 1990;Perry et al., 1992) may not reflect the stochastic outcome of separate pathological processes. Instead, it is possible that interactions of PHFT with other tangle components, including AP and other A@ precursor protein domains (Arai et al., 1990;Gollin et al., 1992;Perry et al., 1993), induce the aggregation ofPHFs into perikaryal NFTs. ...
... Thus, the convergence of PHFT and AP in authentic cortical SPs and NFTs (Hyman et al., 1989;Shin et al., 1989;Arai et al., 1990;Spillantini et al., 1990;Perry et al., 1992) may not reflect the stochastic outcome of separate pathological processes. Instead, it is possible that interactions of PHFT with other tangle components, including AP and other A@ precursor protein domains (Arai et al., 1990;Gollin et al., 1992;Perry et al., 1993), induce the aggregation ofPHFs into perikaryal NFTs. Further, the release of PHF7 from degenerating neurons in the AD brain may contribute to the formation of SPs by interacting with A@ secreted by neural cells (Shin et al., 1993). ...
... Normal adult, human CNS T and fetal 7 from late gestational age normal fetuses (Tohyama et al., 199 1;Lee et al., 1993;Yachnis et al., 1993) were prepared as described (Bramblett et al., 1992. The normal adult and fetal brains as well as the AD brains were evaluated histopathologically and assigned to the control or AD group using consensus criteria for the diagnosis of AD (Khachaturian, 1985) as previously described (Kosik et al., 1988;Arai et al., 1990;Schmidt et al., 1990Schmidt et al., , 1991Tohyama et al., 1991;Bramblett et al., 1992;Yachnis et al., 1993). Human high-molecular-weight neurofilament proteins (NF-H) were purified as described (Balin et al.,199 l), and recombinant human ACT was a generous gift of H. Rubin and B. Cooperman, while purified HSPGs and human ApoE (representing a mixture of ApoE2, ApoE3, and ApoE4) were obtained from Becton Dickinson and Chemicon, respectively. ...
Hyperphosphorylated adult human CNS tau (PHF tau or A68) forms paired helical filaments (PHFs) in neurofibrillary tangles (NFTs), neuropil threads, and dystrophic neurites associated with senile plaques (SPs) during the progression of Alzheimer's disease (AD). While amyloid fibrils in SPs are composed of beta-amyloid (A beta), NFTs and SPs contain similar associated components such as ubiquitin, alpha 1- antichymotrypsin (ACT), apolipoprotein E (ApoE), heparan sulfate proteoglycans (HSPGs), and aluminum salts. Thus, SPs and NFTs may result from specific interactions between PHF tau, A beta, and these other components. In fact, intracerebral injections of PHF tau induce co-deposits of A beta, ACT, and ubiquitin (Shin et al., 1993). To examine this issue further, we probed interactions between PHF tau, aluminum salts, and other plaque and tangle components. We investigated in vivo interactions of PHF tau and aluminum chloride (AlCl3) with other plaque and tangle components by injecting PHF tau with and without AlCl3 into the rodent brain. PHF tau co-injected with AlCl3 formed aggregates that persisted much longer in the rat brain, and induced longer-lived co-deposits of A beta, ubiquitin, ACT, and ApoE than PHF tau alone. Injections of PHF tau with AlCl3 also induced neurons near the injection site to acquire PHF tau-like properties as monitored with antibodies (AT8, T3P, PHF1) that recognize defined PHF tau epitopes containing phosphoserine residues (Ser202, Ser396, Ser404). Injections of AlCl3 alone as well as injections of normal adult and fetal CNS tau, several different synthetic peptides, neurofilament proteins, ACT, HSPGs, or ApoE with and without AlCl3 failed to induce co-deposits of A beta or alter the immunoreactivity of tau in rodent neurons. To determine if aluminum salts interact directly and specifically with PHF tau in situ, we pretreated sections of AD hippocampus with 10 mM AlCl3 and then probed these sections by immunohistochemistry with antibodies to PHF tau as well as to a number of other plaque and tangle components. Preincubation of these sections with AlCl3 diminished PHF tau immunoreactivity in NFTs and SPs using the PHF tau-specific antibodies AT8, T3P, and PHF1, while the immunoreactivity of other plaque and tangle proteins (A beta, ubiquitin, ACT, HSPGs, ApoE) was not abolished. We also examined the effects of AlCl3 on PHF tau and normal adult human CNS tau in vitro. AlCl3 had no effect on normal adult human CNS tau, while increasing concentrations of AlCl3 (from 0.1 to 1.0 mM) induced PHF tau to aggregate at the top of the stacking gel, and at high concentrations (0.3 and 1.0 mM) of AlCl3, PHF tau completely failed to enter the gel. These studies suggest that aluminum binds to PHF tau, induces these proteins to aggregate, and retards their proteolysis. Further, since intracerebral injections of PHF tau with and without AlCl3 in rats appear uniquely capable of inducing co-deposits of a number of proteins found in authentic AD SPs and NFTs (including A beta, ubiquitin, ACT, and ApoE), we speculate that the contributions of PHF tau to plaque and tangle formation in AD may be modulated by aluminum.
... The presence of amyloid in the brain is central to defining whether individuals are on the AD continuum [41][42][43][44][45][46][47][48][49][50]. While EVs have been reported to carry Alzheimer's Aβ peptides [10,11,14,15,17,18,21,23,33,[51][52][53][54][55] little is known about the specificity of this loading and thus whether it is of physiological consequence. ...
Alzheimer’s disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aβ42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aβ42 is detectable in the plasma, a phenomenon thought to result from Aβ becoming more aggregated in the brain and less Aβ42 and Aβ40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse “cargos” of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aβ42 and Aβ40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aβ and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aβ42 and Aβ40 bind to EVs in manner that is sequence specific, saturable, and endothermic. In addition, Aβ incubation with EVs overnight yielded larger amounts of bound Aβ peptide that was fibrillar in structure. These findings point to a specific amyloid–EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.
... In the first immunohistochemical staining with antibodies for both A and amyloid- protein precursor (APP) in the late 1980s, a subset of neurons displayed elevated staining for both precursor and product [2][3][4][5][6]. More recent studies have shown that neuritic plaques arise from neurons that have accumulated intraneuronal (iA), nuclear, and perinuclear A in aggregated and fibrillar forms, based on its reaction with aggregation-specific monoclonal antibodies like mOC78 and electron microscopy [7,8]. ...
Background:
Many identified mechanisms could be upstream of the prominent amyloid-β (Aβ) plaques in Alzheimer's disease (AD).
Objective:
To profile the progression of pathology in AD.
Methods:
We monitored metabolic signaling, redox stress, intraneuronal amyloid-β (iAβ) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan.
Results:
Intracellular accumulation of aggregated Aβ in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAβ. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAβ and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAβ accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine.
Conclusion:
The order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by an pAkt/tAkt metabolic shift in CA1, iAβ accumulation in CA1, and extracellular Aβ deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes.
... The presence of neuritic accumulations of APP in AD brains was reported in early studies. 36 Here, we aimed at investigating APP-related proteinopathy in human brain samples with regard to presynaptic proteins, APP proteases, and histopathological features of the AD brain. ...
In Alzheimer's disease (AD), the distribution of the amyloid precursor protein (APP) and its fragments other than amyloid beta, has not been fully characterized. Here, we investigate the distribution of APP and its fragments in human AD brain samples and in mouse models of AD in reference to its proteases, synaptic proteins, and histopathological features characteristic of the AD brain, by combining an extensive set of histological and analytical tools. We report that the prominent somatic distribution of APP observed in control patients remarkably vanishes in human AD patients to the benefit of dense accumulations of extra-somatic APP, which surround dense-core amyloid plaques enriched in APP-Nter. These features are accentuated in patients with familial forms of the disease. Importantly, APP accumulations are enriched in phosphorylated tau and presynaptic proteins whereas they are depleted of post-synaptic proteins suggesting that the extra-somatic accumulations of APP are of presynaptic origin. Ultrastructural analyses unveil that APP concentrates in autophagosomes and in multivesicular bodies together with presynaptic vesicle proteins. Altogether, alteration of APP distribution and its accumulation together with presynaptic proteins around dense-core amyloid plaques is a key histopathological feature in AD, lending support to the notion that presynaptic failure is a strong physiopathological component of AD.
... Interestingly, neurites expressing phosphorylated-Tau were arranged sometimes in islands, for example in the IML (Fig. 5D) likely corresponding to dystrophic neurites around senile plaques. 60 The area covered by neurites expressing phosphorylated-Tau was not larger in OML compared to other molecular layers of the hippocampus (Fig. 5E). Moreover, in OML, the presynaptic alterations were not proportional to the burden in phosphorylated-Tau (Fig. 5F). ...
Synaptic degeneration has been reported as one of the best pathological correlates of cognitive deficits in Alzheimer’s disease. However, the location of these synaptic alterations within hippocampal sub-regions, the vulnerability of the presynaptic versus postsynaptic compartments, and the biological mechanisms for these impairments remain unknown. Here, we performed immunofluorescence labelling of different synaptic proteins in fixed and paraffin-embedded human hippocampal sections and report reduced levels of several presynaptic proteins of the neurotransmitter release machinery (complexin-1, syntaxin-1A, synaptotagmin-1 and synaptogyrin-1) in Alzheimer’s disease cases. The deficit was restricted to the outer molecular layer of the dentate gyrus, whereas other hippocampal sub-fields were preserved. Interestingly, standard markers of postsynaptic densities (SH3 and multiple ankyrin repeat domains protein 2) and dendrites (microtubule-associated protein 2) were unaltered, as well as the relative number of granule cells in the dentate gyrus, indicating that the deficit is preferentially presynaptic. Notably, staining for the axonal components, myelin basic protein, SMI-312 and Tau, was unaffected, suggesting that the local presynaptic impairment does not result from axonal loss or alterations of structural proteins of axons. There was no correlation between the reduction in presynaptic proteins in the outer molecular layer and the extent of the amyloid load or of the dystrophic neurites expressing phosphorylated forms of Tau. Altogether, this study highlights the distinctive vulnerability of the outer molecular layer of the dentate gyrus and supports the notion of presynaptic failure in Alzheimer’s disease.
... It is characterized by two classic neuropathological hallmarks: senile plaques consisting of a progressive accumulation of amyloid-β peptide (Aβ) and neurofibrillary tangles (NFTs). NFTs are formed by the aggregation of paired helical filaments (PHFs) composed of truncated and hyperphosphorylated microtubule-associated protein tau (Arai et al., 1990;Braak and Braak, 1991;Citron, 2010). The latter type of lesion characterizes a variety of human neurodegenerative diseases collectively termed "tauopathies" (Murray et al., 2014;Kovacs, 2015), including AD, progressive supranuclear palsy, corticobasal degeneration, and Pick's disease (Spillantini and Goedert, 2013;Wang and Mandelkow, 2016). ...
Alzheimer’s disease (AD) is a destructive and burdensome neurodegenerative disease, one of the most common characteristics of which are neurofibrillary tangles (NFTs) that are composed of abnormal tau protein. Animal studies have suggested that dl-3-n-butylphthalide (dl-NBP) alleviates cognitive impairment in mouse models of APP/PS1 and SAMP8. However, the underlying mechanisms related to this remain unclear. In this study, we examined the effects of dl-NBP on learning and memory in P301S transgenic mice, which carry the human tau gene with the P301S mutation. We found that dl-NBP supplementation effectively improved behavioral deficits and rescued synaptic loss in P301S tau transgenic mice, compared with vehicle-treated P301S mice. Furthermore, we also found that it markedly inhibited the hyperphosphorylated tau at the Ser262 site and decreased the activity of MARK4, which was associated with tau at the Ser262 site. Finally, dl-NBP treatment exerted anti-inflammatory effects and reduced inflammatory responses in P301S mice. In conclusion, our results provide evidence that dl-NBP has a promising potential for the therapy of tauopathies, including AD.
... This central role attributed to a by-product of APP degradation, Apeptides, also contrasts with the mechanisms involved in the other NDs in which full-length proteins are misfolded. Yet, the presence of APP around plaques was reported soon after the cloning of APP [1,10,11,71], but the relevance of this localization to AD pathology has been mainly overlooked. Notably, these early reports were mostly descriptive and their interpretation was limited by the absence of immunological controls and by the lack of quantitative analysis. ...
In Alzheimer's disease (AD), a central role is given to the extracellular deposition of Aβ peptides, remotely produced by the proteolysis of the amyloid precursor protein (APP). This contrasts with other neurodegenerative diseases which are characterized by the intraneuronal aggregation of full-length proteins such as huntingtin, α-synuclein or TDP-43. Importantly, the distribution of APP around amyloid plaques is poorly characterized. Here, we combined an extensive set of methodological and analytical tools to investigate neuropathological features of APP in the human AD hippocampus and in two mouse models of AD. We report that APP remarkably accumulates in the surrounding of dense-core amyloid plaques together with the secretases necessary to produce Aβ peptides. In addition, the Nter domain, but not the Cter domain of APP is enriched in the core of amyloid plaques uncovering a potential pathological role of the secreted APP-Nter in dense-core plaques. To investigate the subcellular compartment in which APP accumulates, we labelled neuritic and synaptic markers and report an enrichment in presynaptic proteins (Syt1, VAMP2) and phosphorylated-Tau. Ultrastructural analysis of APP accumulations reveals abundant multivesicular bodies containing presynaptic vesicles proteins and autophagosomal built-up of APP. Altogether, our data supports a role of presynaptic APP in AD pathology and highlights APP accumulations as a potential source of Aβ and Nter peptides to fuel amyloid plaques.
... Intracellular accumulation of tau forming neurofibrillary tangles is one of the two hallmarks in Alzheimer's disease (AD), the most common neurodegenerative disorder in the elderly [1,2]. Abnormal tau accumulation is positively correlated with neurodegeneration and memory deterioration [3,4], and the total tau level in cerebrospinal fluids has an inverse correlation with memory score in AD patients [5,6]. ...
Intracellular tau accumulation forming neurofibrillary tangles is hallmark pathology of Alzheimer's disease (AD), but how tau accumulation induces synapse impairment is elusive. By overexpressing human full-length wild-type tau (termed hTau) to mimic tau abnormality as seen in the brain of sporadic AD patients, we find that hTau accumulation activates JAK2 to phosphorylate STAT1 (signal transducer and activator of transcription 1) at Tyr701 leading to STAT1 dimerization, nuclear translocation, and its activation. STAT1 activation suppresses expression of N-methyl-D-aspartate receptors (NMDARs) through direct binding to the specific GAS element of GluN1, GluN2A, and GluN2B promoters, while knockdown of STAT1 by AAV-Cre in STAT1 flox/flox mice or expressing dominant negative Y701F-STAT1 efficiently rescues hTau-induced suppression of NMDAR expression with amelioration of synaptic functions and memory performance. These findings indicate that hTau accumulation impairs synaptic plasticity through JAK2/STAT1-induced suppression of NMDAR expression, revealing a novel mechanism for hTau-associated synapse and memory deficits.
... In many cases, mutations in genes encoding these proteins have been linked to neurodegenerative diseases. The client proteins tested include Tau, a microtubule binding protein implicated in Alzheimer's disease 17 , polyglutamine-containing ataxin3 known to be involved in spinocerebellar ataxia 3 (SCA3) 18 , amyotrophic lateral sclerosis-associated SOD1 19 and TDP43 20,21 . To facilitate the detection of these proteins, they were expressed transiently as tagged proteins in HEK293T cells in the absence or presence of Flag-USP19. ...
Cell-to-cell transmission of misfolded proteins propagates proteotoxic stress in multicellular organisms when transmitted polypeptides serve as a seeding template to cause protein misfolding in recipient cells, but how misfolded proteins are released from cells to initiate this process is unclear. Misfolding-associated protein secretion (MAPS) is an unconventional protein-disposing mechanism that specifically exports misfolded cytosolic proteins including various neurodegenerative disease-causing proteins. Here we establish the HSC70 co-chaperone DNAJC5 as an essential mediator of MAPS. USP19, a previously uncovered MAPS regulator binds HSC70 and acts upstream of HSC70 and DNAJC5. We further show that as a membrane-associated protein localized preferentially to late endosomes and lysosomes, DNAJC5 can chaperone MAPS client proteins to the cell exterior. Intriguingly, upon secretion, misfolded proteins can be taken up through endocytosis and eventually degraded in the lysosome. Collectively, these findings suggest a transcellular protein quality control regulatory pathway in which a deubiquitinase-chaperone axis forms a “triaging hub”, transferring aberrant polypeptides from stressed cells to healthy ones for disposal.
... Clinico-pathological correlative studies in dementia research have provided significant contributions into understanding how pathological protein aggregations in the brain correspond to the clinical manifestation of dementia. Seminal studies have demonstrated features of senile plaque accumulation and neurofibrillary tangle formation are essential to the neuropathological diagnosis of Alzheimer's disease (AD) (Tomlinson et al. 1970) and more specifically emphasizing the importance of dystrophic neurites positive for hyperphosphorylated tau in neuritic plaques in AD cases compared to controls (Dickson et al. 1988;Probst et al. 1989;Arai et al. 1990). However, as patients were historically dichotomized as AD or normally aged controls, these studies lacked detail required to track disease progression. ...
A tissue microarray (TMA) has previously been developed for use in assessment of neurodegenerative diseases. We investigated the variation of pathology loads in semi-quantitative score categories and how pathology load related to disease progression. Post-mortem tissue from 146 cases were used; Alzheimer’s disease (AD) (n = 36), Lewy body disease (LBD) (n = 56), mixed AD/dementia with Lewy bodies (n = 14) and controls (n = 40). TMA blocks (one per case) were constructed using tissue cores from 15 brain regions including cortical and subcortical regions. TMA tissue sections were stained for hyperphosphorylated tau (HP-T), β amyloid and α-synuclein (αsyn), and quantified using an automated image analysis system. Cases classified as Braak stage VI displayed a wide variation in HP-T pathology in the entorhinal cortex (interquartile range 4.13–44.03%). The interquartile range for β amyloid in frontal cortex in cases classified as Thal phase 5 was 6.75–17.03% and for αsyn in the cingulate in cases classified as McKeith neocortical LBD was 0.04–0.58%. In AD and control cases, HP-T load predicted the Braak stage (p < 0.001), β amyloid load predicted Thal phase (p < 0.001) and αsyn load in LBD cases predicted McKeith type of LBD (p < 0.001). Quantitative data from TMA assessment highlight the range in pathological load across cases classified with ‘severe’ pathology and is beneficial to further elucidate the heterogeneity of neurodegenerative diseases. Quantifying pathology in multiple brain regions may allow identification of novel clinico-pathological phenotypes for the improvement of intra vitam stratification of clinical cohorts according to underlying pathologies.
... Alzheimer's disease (AD) was the most common age-related progressive neurodegenerative disorder in the aging population. The neuropathological hallmarks of AD was characterized by abundant deposits of beta-amyloid (Aβ) in senile plaques (SP), massive accumulation of abnormal phosphorylated intracellular tau filaments in neurofibrillary tangles (NFTs), and neuronal dysfunction/death and synapse loss in the cortex and hippocampus [1][2][3][4]. The main clinical manifestations of AD were progressive deterioration of selective memory, cognitive dysfunction, language disorder, personality change, and other mental symptoms. ...
Fluoxetine, a selective serotonin reuptake inhibitor, is neuroprotective; therefore, it has been applied to treat some neurodegenerative disorders. For instance, chronic fluoxetine exposure has short-term effects on Alzheimer's disease (AD). However, the long-term ameliorative effects of fluoxetine exposure on AD have not been reported. In the present study, 6-month-old 3 × TgAD mice were treated with fluoxetine for 15 days, and then the influence of fluoxetine was detected at 20 days after the drug withdrawal. We found that chronic fluoxetine treatment ameliorated cognitive deficits of 3 × TgAD mice and increased the volume of the hippocampal CA1 and dentate gyrus (DG) with increased neuron number and dendritic spine density. Meanwhile, fluoxetine exposure also stimulated the long-term potentiation (LTP) in hippocampal DG. The synaptic-related protein expression increased via activation of the cyclic AMP response element binding (CREB) protein/brain-derived neurotrophic factor (BDNF) signaling pathway induced by fluoxetine exposure. Lastly, we found that fluoxetine treatment decreased beta-amyloid (Aβ) levels. These results further certified that fluoxetine may be a potent effective drug for AD.
... Alzheimer's disease (AD) is a neurodegenerative condition with progressive decline in cognition, due to the formation of extracellular senile plaques and intracellular neurofibrillary tangles in brain, particularly the hippocampus (Arai et al. 1990;Braak and Braak 1991). Senile plaques are the deposits of Aβ (Glenner 1988) formed from Aβ precursor protein (APP) by the β and γ secretase (Yankner 1996). ...
Autophagy, the process of self-degradation of cellular components, has an important role in neurodegenerative diseases, such as Alzheimer's disease. In this study, we investigated the effects of SP600125 as c-Jun N-terminal kinase (JNK) inhibitor and bucladesine as a cyclic adenosine 3',5'-monophosphate (cAMP) analog on spatial memory and expression of autophagic factors in Aβ-injected rats. Male Wistar rats were used. Rats were randomly allocated into five groups as following: amyloid beta (Aβ)-only group, Aβ + SP600125 (30 μg/1 μ/side, n = 7) and/or bucladesine (100 μM/1 μl/side, n = 7), and the normal control (vehicle only) group. The treatments were administered bilaterally to the CA1 sub-region of the hippocampus stereotaxically. Spatial reference memory was performed using Morris Water Maze 21 days later. The expression of authophagy markers (beclin1, Atg7, Atg12, and LC3 II/LC3 I) in the hippocampus was evaluated using western blotting. Compared to the vehicle group, Aβ administration reduced spatial reference learning (P < 0.001) and memory (P < 0.01) and upregulated the expression of beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001). Compare to Aβ-only group, the administration of SP600125 and/or bucladesine improved spatial reference learning (P < 0.001) and memory (P < 0.01). Compared to the Aβ-only group, the treatment with SP600125 and/or bucladesine also reduced beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001) which was similar to amount of normal rats. In summary, it seems that the improvement of spatial memory by SP600125 and/or bucladesine in Aβ-injected rats is in relation with normalizing of autophagy to the physiologic level, possibly through neuroprotection and/or neuroplasticity.
... This suggests that some form of unseen Aß which may accumulate prior to extracellular plaque deposition is more closely associated with pathogenesis. Soon after the discovery of Aß, it was observed that APP and Aß immunoreactivity accumulates in the perinuclear area of a subset of neurons and in the corona of dystrophic neurites surrounding neuritic plaques and it was proposed that this APP represents the penultimate source of Aß accumulating in the core of the neuritic plaques (Selkoe, Podlisny et al. 1988;Grundke-Iqbal, Iqbal et al. 1989;Ikeda, Allsop et al. 1989;Perry, Siedlak et al. 1989;Stern, Otvos L et al. 1989;Arai, Lee et al. 1990;Cummings, Su et al. 1992). However, with the discovery of soluble secreted Aß, this interpretation fell out of favor (Joachim, Games et al. 1991). ...
... Aβ is a byproduct of Aβ precursor protein (APP) metabo lism that is generated by nearly all cells, and amyloid plaques are the result of the deposition of mainly Aβ 1-40 and Aβ 1-42 in the brain, although other species of Aβ are also present [6]. Th e mechanism leading to Aβ deposition diff ers in subjects for whom this occurs on a genetic basis, leading to familial AD (FAD), versus those who develop sporadic AD. ...
Cerebrospinal fluid and positron emission tomography biomarkers accurately predict an underlying Alzheimer's disease (AD) pathology; however, they represent either invasive or expensive diagnostic tools. Therefore, a blood-based biomarker like plasma amyloid beta (Aβ) that could correlate with the underlying AD pathology and serve as a prognostic biomarker or an AD screening strategy is urgently needed as a cost-effective and non-invasive diagnostic tool. In this paper we review the demographic, biologic, genetic and technical aspects that affect plasma Aβ levels. Findings of cross-sectional and longitudinal studies of plasma Aβ, including autosomal dominant AD cases, sporadic AD cases, Down syndrome cases and population studies, are also discussed. Finally, we review the association between cerebrovascular disease and Aβ plasma levels and the responses observed in clinical trials. Based on our review of the current literature on plasma Aβ, we conclude that further clinical research and assay development are needed before measures of plasma Aβ can be interpreted so they can be applied as trait, risk or state biomarkers for AD.
Senile plaques have been studied in postmortem brains for more than 120 years and the resultant knowledge has not only helped us understand the etiology and pathogenesis of Alzheimer disease (AD), but has also pointed to possible modes of prevention and treatment. Within the last 15 years, it has become possible to image plaques in living subjects. This is arguably the single greatest advance in AD research since the identification of the Aβ peptide as the major plaque constituent. The limitations and potentialities of amyloid imaging are still not completely clear but are perhaps best glimpsed through the perspective gained from the accumulated postmortem histological studies. The basic morphological classification of plaques into neuritic, cored and diffuse has been supplemented by sophisticated immunohistochemical and biochemical analyses and increasingly detailed mapping of plaque brain distribution. Changes in plaque classification and staging have in turn contributed to changes in the definition and diagnostic criteria for AD. All of this information continues to be tested by clinicopathological correlations and it is through the insights thereby gained that we will best be able to employ the powerful tool of amyloid imaging.
High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the “Kunitz” protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.
Huntington disease is an inherited neurodegeneration, for which the associated mutation was isolated in 1993. The mutation is an expansion of a CAG trinucleotide repeat, which translates to give a polyglutamine tract at the N‐terminus of a large protein, huntingtin. Neither the normal nor the pathogenic functions of this protein have been identified, but it is clear that pathogenesis is mediated through the expanded polyglutamine tract within the protein, and that polyglutamine is toxic to cells. A number of proteins which interact with the N‐terminal region of huntingtin have been isolated, but this has not, so far, yielded a rationale for pathogenesis. Huntingtin is found in areas of the brain that degenerate in this disease, but is also associated with pathogenic inclusions in Alzheimer disease and Pick disease. It is possible that Huntington disease has pathogenic mechanisms in common with these other neurodegenerative diseases, and that the mechanism may relate to the formation of abnormal, cytoskeletal‐associated, inclusions within cells.
Synaptic degeneration has been reported as one of the best pathological correlate of cognitive deficit in Alzheimer’s Disease (AD). However, the location of these synaptic alterations within hippocampal sub-regions, the vulnerability of the presynaptic versus postsynaptic compartments, and the biological mechanisms for these impairments remain unknown. Here, we performed immunofluorescence labeling of different synaptic proteins in fixed and paraffin embedded human hippocampal sections and report reduced levels of several presynaptic proteins of the neurotransmitter release machinery (complexin-1, syntaxin-1A, synaptotagmin-1 and synaptogyrin-1) in AD cases. The deficit was restricted to the outer molecular layer (OML) of the dentate gyrus whereas other hippocampal sub-fields were preserved. Interestingly, standard markers of postsynaptic densities (SHANK2) and dendrites (MAP2) were unaltered, as well as the relative number of granule cells in the dentate gyrus, indicating that the deficit is preferentially presynaptic. Notably, staining for the axonal components, myelin basic protein, SMI-312 and Tau, was unaffected, suggesting that the local presynaptic impairment does not result from axonal loss or alterations of structural proteins of axons. There was no correlation between the reduction in presynaptic proteins in OML and the extent of the amyloid load or of the dystrophic neurites expressing phosphorylated forms of Tau. Altogether, this study highlights the distinctive vulnerability of the OML of dentate gyrus and supports the notion of presynaptic failure in AD.
Synaptic degeneration has been reported as one of the best pathological correlate of cognitive deficit in Alzheimer's Disease (AD). However, the location of these synaptic alterations within hippocampal sub-regions, the vulnerability of the presynaptic versus postsynaptic compartments, and the biological mechanisms for these impairments remain unknown. Here, we performed immunofluorescence labeling of different synaptic proteins in fixed and paraffin embedded human hippocampal sections and report reduced levels of several presynaptic proteins of the neurotransmitter release machinery (complexin-1, syntaxin-1A, synaptotagmin-1 and synaptogyrin-1) in AD cases. The deficit was restricted to the outer molecular layer (OML) of the dentate gyrus whereas other hippocampal sub-fields were preserved. Interestingly, standard markers of postsynaptic densities (SHANK2) and dendrites (MAP2) were unaltered, as well as the relative number of granule cells in the dentate gyrus, indicating that the deficit is preferentially presynaptic. Notably, staining for the axonal components, myelin basic protein, SMI-312 and Tau, was unaffected, suggesting that the local presynaptic impairment does not result from axonal loss or alterations of structural proteins of axons. There was no correlation between the reduction in presynaptic proteins in OML and the extent of the amyloid load or of the dystrophic neurites expressing phosphorylated forms of Tau. Altogether, this study highlights the distinctive vulnerability of the OML of dentate gyrus and supports the notion of presynaptic failure in AD.
Fluoxetine (FLX) has broad neurobiological functions and neuroprotective effects; however, the preventive effects of FLX on cognitive impairments in Alzheimer's disease (AD) have not been reported. Here, we studied whether adolescent administration of fluoxetine can prevent memory deficits in AD transgenic mice that harbour PS1m146v, APPswe and TauP301L mutations (3 × TgAD). FLX was applied through peritoneal injection to the mice at postnatal day 35 (p35) for 15 consecutive days, and the effects of FLX were observed at 6-month. We found that adolescent administration of FLX improved learning and memory abilities in 6-month-old 3 × TgAD mice. FLX exposure also increased the sizes of the hippocampal CA1, dentate gyrus (DG) and extensive cortex regions, with increased numbers of neurons and higher dendritic spine density. Meanwhile, the synaptic plasticity of neurons in the hippocampus was remodelled, and the expression levels of synaptic-related proteins were increased along with activation of the cyclic AMP response element-binding (CREB) protein/brain-derived neurotrophic factor (BDNF) signalling pathway. Finally, we found that FLX effectively prevented the increase of beta-amyloid (Aβ) levels. These data suggest that adolescent administration of the antidepressant drug FLX can efficiently preserve cognitive functions and improve pathologies in 3XTg AD mice.
Alzheimer disease (AD) is the commonest cause of dementia in the elderly (for recent reviews, see Chiu, 1989; Henderson & Finch, 1989; Katzman et al., 1988). As a consequence of the increasing number of people living beyond the seventh decade, AD has become the fourth leading cause of death in the United States. Despite the recognition of AD and the brain pathology associated with it more than 80 years ago (Alzheimer, 1907), the pathogenesis and etiology of AD remain enigmatic. Several reasons account for this such as (1) the complexity and limited understanding of the central nervous system (CNS); (2) an incomplete definition of AD and variants thereof; (3) the lack of animal models for hypothesis testing; (4) overlap between early AD and normal aging as well as between AD and other neurodegenerative diseases. For example, patients with idiopathic Parkinson disease (PD) frequently develop cognitive impairments, AD patients often exhibit extrapyramidal signs, and diminished olfaction is observed in AD and PD subjects (Chiu, 1989; Doty et al., 1987, 1989).
The β-amyloid precursor protein (βAPP) is widely expressed in neurons and glial cells throughout the mammalian nervous system. APP is a large transmembrane glycoprotein having a single transmembrane domain, a large extracelluar N-terminus, and a short cytoplasmic C-terminus (Kang et al., 1987). This protein is very important as a source of amyloid β peptide (Aβ), a 40–42 amino acid peptide that forms senile plaques in Alzheimer’s disease (AD) patient brains. Senile plaques, along with neurofibrillary tangles, are the most characteristic pathological change in AD brains (Alzheimer, 1907). In normal physiological conditions, α-secretase cleaves βAPP between amino acids 16 and 17 of A. The α-secretase cleavage prevents release of amyloidogenic Aβ and yields secreted forms of APP (sAPPα), which are released into the extracellular fluid (Weidemann et al., 1989; Esch et al., 1990; Sisodia et al., 1990). An alternative cleavage of βAPP within the endosomal—lysosomal pathway at the N-terminus of Aβ results in release of intact Aβ and secreted forms of APP without the Aβ1–16 domain (sAPPβ) (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). Previous studies paid great attention to the production and the functions of Aβ, but a regulated release of sAPPα and sAPPβ seems to be equally important (Furukawa et al., 1996b).
Die Alzheimer-Krankheit ist gekennzeichnet durch einen Verlust der Merk-fähigkeit und anderer kognitiver Funktionen. Sie führt zu schwerer Demenz und letztlich zum Tode [1]. Es handelt sich um eine verbreitete Krankheit, die 0,5–1% der Bevölkerung der westlichen Welt betrifft. Ihre Ursachen sind unbekannt, und bislang konnte noch keine allgemein wirksame Behandlung entwickelt werden. Man nimmt an, daß die Alzheimer-Krankheit in den meisten Fällen sporadisch auftritt, es existieren daneben auch familiäre Krankheitsformen mit autosomal-dominantem Erbgang.
Alterations in the neuronal cytoskeleton characterize a number of human neurological disorders, including Alzheimer’s and Parkinson’s Diseases (AD and PD) (Iqbal et al., 1987). The hypothesis that environmental aluminum neurotoxicity causes Alzheimer’s disease in humans has been the subject of considerable debate (Graves et al., 1990; Crapper-McLachlan et al., 1991; Guy et al., 1991; McDermott et al., 1979; Bonhaus et al., 1980; Kruck 1993). Aluminum can readily cross the blood brain barrier (Wen and Wisniewski, 1985), is found in neurofibrillary tangle (NFT)-containing neurons (Perl & Brody, 1980), and forms the core of the senile plaque (Candy et al., 1986; Crapper et al., 1973). Aluminum is prevalent in the drinking water (Martyn et al., 1989), food preservatives (French et al., 1989) and, in fact, it is the third most prevalent element on the earth’s surface (Graves et al., 1990; King et al., 1981). High levels of aluminum in humans in dialysis dementia have been associated with degeneration of cortical cells, however, neurofibrillary changes were not present (Alfrey et al., 1976). Although the neural concentration of aluminum increases with age, there have been conflicting results concerning the correlation between aluminum deposits and NFT (McDermott et al., 1979; Perl & Brody 1980; Wen and Wisniewski, 1985). There is no correlation between the amount of aluminum in NFT in AD or demented patients as compared to age matched controls (Wen and Wisniewski, 1985).
The presence of dystrophic neurites either free in the neuropil or around senile plaques is the critical neuropathological correlate of dementia in Alzheimer’s disease. Amyloid deposition alone is nonspecific, since it is commonly present (and can be abundant) in normal aging as well as in several other conditions. There is as yet no clear understanding of what cell biological process gives rise to the dystrophic neurites. Immunocytochemically they react with nearly the same constellation of antibodies as do neurofibrillary tangles. An aberrant or chaotic regenerative origin is suggested by their appearance as microspike-studded filopodial outgrowths from the lateral aspect of pyramidal cells. Paradoxically, however, the dystrophic neurites also have features, such as ubiquitin-immunoreactivity, filamentous inclusions, and a swollen appearance, that are more likely associated with neurite degeneration or retraction. Neither do the dystrophic neurites react with the growth cone marker, GAP-43. An unusual feature of the dystrophic neurites is their violation of neuronal polarity. While these neurites contain proteins normally found in the axon, such as tau and phosphorylated neurofilament, dystrophic neurites are commonly associated with dendrites. This important pathobiological process can be modeled in some vertebrate systems that disrupt the axon close to the cell body. Under the conditions of close axotomy, there is ectopic axonal sprouting from dentritic tips. In neuronal cell culture the administration of tau antisense oligonucleotides can induce axonal retraction with concomitant elaboration of multiple short gnarled sprouts from the cell body. Concomitant regeneration and degeneration is a unique pathobiological entity that obeys rules distinct from either regeneration or degeneration alone.
This chapter provides a clear overview of the current knowledge about Alzheimer's disease (AD) related lesion structures, as well as a critical examination of the concepts that underlie the neuropathological diagnosis of AD. The understanding of lesions has progressively changed in order to encompass complex molecular, biochemical, and structural issues. Along with these, other structural abnormalities have been described in the chapter. Recent advances in neuron counting technology have provided adequate information on the extent of synaptic and neuronal loss in normal aging and AD. The historical description of senile plaques (SP) is standardized and there are substantial interlaboratory differences in the vocabulary used. It had been widely accepted that neuronal death is associated with normal brain aging. The chapter also discusses that extensive research and studies have estimated neuron densities effectively but not the total number of neutrons in a given structure. The neuropathological assessment of an AD pathologic changes renders an easy and unequivocal way to confirm the clinical diagnosis of AD.
Predominant pathological hallmarks of Alzheimer’s (AD) include the region-specific deposition of β amyloid (Aβ) plaques, vascular amyloidosis, and a number of distinct neurodegenerative changes. These involve the formation of dystrophic neurites and neuritic plaques, cytoskeletal alterations, and synaptic and neuronal loss. Astrocytosis and microgliosis are also evident in affected brain regions. Transgenic (tg) mice overexpressing a mutant form of the β-amyloid precursor protein (APP 717 V→ F) develop several of these pathologies in an age- and region-dependent manner similar to AD. Aβ plaques in the transgenic mouse share many of the tinctorial and immunohistochemical properties of AD plaques, including the relative distribution of AβX-40 and AβX-42 isoforms and the presence of other plaque-associated proteins. Initial findings using immunoassays specific to unique forms of APP and Aβ suggest that APP levels do not dramatically change with increasing age in the mouse brains, and that region-specific variations in APP metabolism, local factors or deficits in distinct populations of neurons account for the deposition of brain Aβ. The PDAPP mouse is a relevant and efficient model system to identify mechanistic properties of the disease process and offers novel opportunities to test potential therapeutics.
This chapter discusses tau proteins and their significance in the pathobiology of Alzheimer's disease (AD). Paired helical filaments are found aggregated together into neurofibrillary tangles (NFTs) in the perikarya of selectively vulnerable populations of neurons in AD. It is suggested that the accumulation of neurofibrillary lesions in AD may be caused by the aberrant reactivation of fetal protein kinases and the inactivation of fetal protein phosphatases in the AD brain. The absence of any AD lesions in the fetal central nervous system suggests that these kinases and phosphatases, which are normally present and activated in the fetal nervous system, determine the phosphorylation state of fetal τ but do not generate derivatized forms of that are fully equivalent to paired helical filaments Tau (PHFτ) in the AD brain. It is suggested that some of the differences previously noted between normal adult CNSτ and PHFτ that were obtained from studies of postmortem brain samples may reflect the more rapid dephosphorylation of normal τ versus PHFτ during similar intervals between the death of the patient and the extraction of each of these species of τ protein.
The chapter discusses the Alzheimer disease. It is described as a fatal degenerative dementing disorder with initial mild memory impairment that progresses unrelentingly to a total debilitating loss of mental and physical faculties. Following symptom onset, the course of the disease varies considerably from a few years to over 20 years, with a mean survival of approximately 8 years. Alzheimer disease affects 10-15% of individuals over 65 years and up to 47% of individuals over the age of 80. The most common and distinctive lesions present within the diseased brain are the neuritic senile plaques and neurofibrillary tangles described by Alois Alzheimer. Neuronal and dendritic loss, neuropil threads, dystrophic neurites, granulovacuolar degeneration, Hirano bodies, and cerebrovascular amyloid, as well as generalized atrophy of the brain, are also prominent pathological features. The paired helical filaments of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with Alzheimer disease. However, despite intense efforts to understand the molecular composition of paired helical filaments, biochemical studies are severely hampered by the extreme insolubility of paired helical filaments comprising neurofibrillary tangles and the consequent difficulty of purifying a homogeneous paired helical filaments fraction. Neurotransmitter alterations are one of the best-studied biochemical features of Alzheimer disease, for which, at present, all currently licensed therapeutic molecules are targeted.
ALZHEIMER'S disease (AD) is the most common cause of progressive intellectual failure in aged humans. AD brains contain numerous amyloid plaques surrounded by dystrophic neurites, and show profound synaptic loss, neurofibrillary tangle formation and gliosis. The amyloid plaques are composed of amyloid beta-peptide (A beta), a 40-42-amino-acid fragment of the beta-amyloid precursor protein (APP)(1). A primary pathogenic role for APP/A beta is suggested by missense mutations in APP that are tightly linked to autosomal dominant forms of AD(2,3). A major obstacle to elucidating and treating AD has been the lack of an animal model. Animals transgenic for APP have previously failed to show extensive AD-type neuropathology(4-10), but we now report the production of transgenic mice that express high levels of human mutant APP (with valine at residue 717 substituted by phenylalanine) and which progressively develop many of the pathological hallmarks of AD, including numerous extracellular thioflavin S-positive AP deposits, neuritic plaques, synaptic loss, astrocytosis and microgliosis. These mice support a primary role for APP/A beta in the genesis of AD and could provide a preclinical model for testing therapeutic drugs.
Sporadic inclusion body myositis (SIBM) is characterized by vacuolar degeneration of muscle fibers and intrafiber clusters
of paired helical filaments with abnormal amyloid deposition. Because of their potential involvement in other degenerative
disorders, we have examined the expression of transglutaminases (TGases) in normal and SIBM tissues. We report that at least
two different enzymes, the ubiquitous TGase 2 as well as the TGase 1 enzyme, are present in muscle tissues. However, in comparison
with normal tissue, the expression of TGases 1 and 2 was increased 2.5- and 4-fold in SIBM, accompanied by about a 20-fold
higher total TGase activity. By immunohistochemical staining, in normal muscle, TGase 2 expression was restricted to some
endomysial connective tissue elements, whereas TGase 1 and β-amyloid proteins were not detectable. In SIBM muscle, both TGases
1 and 2 as well as amyloid proteins were brightly expressed and co-localized in the vacuolated muscle fibers, but none of
these proteins colocalized with inflammatory cell markers. Next, we isolated high molecular weight insoluble proteins from
SIBM muscle tissue and showed that they were cross-linked by about 6 residues/1000 residues of the isopeptide bond. Furthermore,
by amino acid sequencing of solubilized tryptic peptides, they contain amyloid and skeletal muscle proteins. Together, these
findings suggest that elevated expression of TGases 1 and 2 participate in the formation of insoluble amyloid deposits in
SIBM tissue and in this way may contribute to progressive and debilitating muscle disease.
The gradual development of Alzheimer-related neurofibrillary changes and β-amyloid deposits have been investigated in the amygdala and cerebral cortex with advanced silver methods. Neurofibrillary tangles occur within the somata of nerve cells, while neuropil threads are located in nerve cell processes. In neuritic plaques, there are both neuronal processes containing neurofibrillary changes and extracellular deposits of β-amyloid protein. The amygdala early displays Alzheimer-type pathology and is typically rich in neuritic plaques. Neuritic plaques do not occur in the amygdala in the absence of neuropil threads and neurofibrillary tangles in the entorhinal region and amygdala. In contrast to the gradual increase in the number of neurofibrillary tangles and neuropil threads, neuritic plaques grow in size and number along with advancement in the neurofibrillary and β-amyloid pathology. The findings indicate that neuropil threads and neurofibrillary tangles in the entorhinal region and amygdala precede neuritic plaques in the amygdala, and the density of neuritic plaques is related both to the degree of neurofibrillary and β-amyloid pathology in the cerebral cortex.
Autophagy is a main pathway that clears the dysfunction organelles, misfolding proteins and oxidative lipids. It's important for maintaining life activity and conserved from yeast to mammalian. In the AD neurons the misfolding proteins were not efficiently cleared then accumulated. These caused neurons loss of function even neuron death. This review focuses on the recent progresses on regulation of autophagy and the role of autophagy in Alzheimer's diseases. Autophagy is protective in early stage of AD, although it induces autophagic cell death in late stage of AD. Autophagosome may be the main site for Aβ production and clearance. Presenilin 1 which is the key proteinase in γ-secrectase also plays a role in lysosomal acidification which is a key step for autophagic degradation. Tau may be involved in autophagosome trafficking and autophagosome-lysosome fusion. mTOR and AMPK sensing nutrients and energy in cells also regulate autophagy.
Amyloid beta peptide accumulation in the brain poses a serious threat during Alzheimer’s disease. Various strategies to remove or disrupt these plaques with nanoparticles is an emerging area for the treatment for neurodegenerative diseases. The present work attempts to develop a novel strategy to remove the plaques using magnetic field by employing magnetic nanoparticles conjugated with a plaque–targeting ligand. Thioflavin–S (TF–S) was covalently linked to magnetic iron oxide incorporated mesoporous silica, SBA–15 and the material was characterized using microscopic and spectroscopic techniques. This magnetic conjugate (IO–SBA–TF–S) was found to display the ability to remove in vitro, KLVFF peptide, a recognition motif in β–Amyloid that has been implicated in plaque formation. This system represents a potential smart system that could herald in the next generation therapeutic strategy for Alzheimer’s disease and related disorders.
This chapter shows that PDAPP mice over expressing a mutation associated with some cases of familial early onset AD express several of the major pathological hallmarks associated with AD. Amyloid plaques in PDAPP mice appear quite similiar to A/3 deposits in AD as shown by a variety of different antibodies and stains, and are of both the diffuse and compacted varieties. Additionally, a subset of these amyloid plaques appears to be neuritic plaques. Neurodegenerative changes, including the loss of synaptic and dendritic proteins, abnormal phosphorylation of cytoskeletal elements, subcellular degenerative changes, and the deposition of lysosoma1 and acute phase proteins has also been seen in PDAPP mouse brains. Reactive astrocytosis and microgliosis have also been observed in association with the amyloid plaques in the PDAPP mice. No neurofibrillary tangles or paired helical filaments have been found in the mice to date. It remains unknown whether mice are capable of generating these in a manner comparable to AD in less than two years. Extensive behavioral analyses are currently being performed in these mice, and preliminary results indicate that the PDAPP mice are significantly impaired on a variety of different learning and memory tests. In conclusion, the PDAPP mouse model doesn't display all the pathological hallmarks of AD, but it does display most of them in a robust manner that increases with age and gene dosage. Therefore, this transgenic model provides evidence that alterations in APP processing and AB production can result in AD-like neuropathology, can contribute to a mechanistic understanding of AD, as well as providing a useful animal model for the testing of various therapeutic interventions directed toward specific aspects of the neurodegenerative process.
The amyloid beta-protein (beta/A4) that is deposited in senile plaques and in cerebral vessels in Alzheimer's disease (AD) is derived from a larger membrane-associated glycoprotein, the amyloid beta-protein precursor (APP). The gene encoding APP produces at least four major transcripts. Three of the four transcripts contain an alternatively-spliced exon encoding a Kunitz protease inhibitor domain (KPI). We now report the results of a series of experiments using novel immunohistochemical reagents to anatomically localize beta/A4, APP, and KPI-containing forms of APP (APP-KPI) in the hippocampal formation and temporal neocortex. A new monoclonal antibody against beta/A4 recognized senile plaques and vascular amyloid, but no cellular elements. Anti-APP and anti-KPI monoclonal antibodies stained neurons, including proximal axons and dendrites. The neuritic component of some plaques in patients with AD and in elderly control individuals were also immunoreactive for both APP and APP-KPI. Quantitative assessment of senile plaques in temporal neocortex showed that, on average, about one-third of beta/A4 immunoreactive plaques stained with either anti-APP or anti-KPI. Amyloid beta-protein precursor and APP-KPI immunoreactivity were also found in the white and grey matter vessels of both AD patients and control individuals. These results suggest that KPI-containing forms of APP are present in dystrophic neurites of senile plaques, and normally in neurons, neuronal processes, and in the vascular compartment in the brain. Thus, APP-KPI is in a position to be intimately associated with beta/A4 deposition in the neuropil, in plaques and in amyloid angiopathy.
Using differential affinity chromatography, antibodies were prepared to the carboxy-terminal and amino-terminal portions of murine SAA. Immunohistochemical examinations of spleen AA amyloid with these antibodies, at both the light and electron microscopic levels, indicated the presence of epitopes from both regions of SAA in these fibrillary deposits. Whole spleen homogenates.from animals at varying periods of time following the induction of amyloidosis, were used in conjunction with electrophoresis and autoradiographic Western blotting to determine the SAA/AA ratio in spleen as a function of AA amyloid induction time. Intact SAA was a consistent constituent of splenic homogenates regardless of the time following induction. Furthermore, over a three to four week period the SAA/AA ratio gradually decreased with time. These morphologic and time course data are more consistent with SAA undergoing proteolytic cleavage after, rather than before, it is incorporated into an AA fibril.
Diese Übersichtsarbeit beabsichtigt, die Ergebnisse molekularbiologischer Untersuchungen zur Amyloidpathologie der Alzheimer-Demenz in einen Zusammenhang mit anderen, in den letzten Jahren weniger beachteten Aspekten der Erkrankung zu stellen. Besonderes Augenmerk soll dabei den zahlreichen Hinweisen auf einen immunologischen Prozeß als Teil der Alzheimer-Pathologie gelten. Die Frage der Spezifität von Plaques und neurofibrillärer Degeneration, die als klassische neuropathologische Marker der Alzheimer-Demenz angesehen werden, wird anhand vorliegender Befunde zur Neuropathologie bei nichtdementen und dementen älteren Personen einer kritischen Überprüfung unterzogen. Das traditionelle unidirektionale Konzept, welches die bei der Alzheimer- Demenz beeinträchtigten neuropsychologischen Funktionen nur als Folge beschädigter morphologischer Strukturen versteht, wird in Frage gestellt. Experimentelle Befunde alteren und jüngeren Datums, daß dysfunktionaler Gebrauch und fehlende Bedienung neuropsychologischer Funktionen die neuronale Plastizität beeinträchtigen und selbst zum Ausgangspunkt morphologischer Veränderungen werden können, werden in Beziehung gesetzt zu zahlreichen Hinweisen auf nichtkognitive, psychische Auffälligkeiten im Vorfeld einer Alzheimer-Erkrankung. Befunde zur Synapsenpathologie, die bei der Alzheimer-Demenz mit den neuropsychologischen Einbußen am besten korreliert ist, werden als möglicher Ausdruck eines durch psychobiologische Mechanismen mitbedingten Rückgangs neuronaler Plastizität diskutiert.
The mechanism by which the A4 (β-amyloid) domain of the Alzheimer amyloid precursor protein (APP) is deposited in plaques is unknown, and limited information is available concerning the extent to which other APP sites are associated with plaques. To address these issues, we prepared antiserum to a peptide adjacent to the N-terminus of the APP (referred to as N1) and examined its distribution in brain relative to A4 by double-immunostaining techniques. Anti-N1 localized to both neurons and glia in control and Alzheimer patients. In the Alzheimer brain, anti-N1 detected plaques. Quantitation revealed that 85% of thioflavin-positive plaques, and 91% of A4-positive plaques were also N1 positive. Double-staining methods directly demonstrated colocalization of distant APP sites. The data suggest that proposed mechanisms for amyloid deposition during plaque formation must take into account the extracytoplasmic domain, in addition to the A4 region, rather than be confined exclusively to the A4 site. (J Geriatr Psychiatry Neurol 1990;3:139-145).
The blood-brain barrier (BBB) is impermeable to IgG. Therefore, a delivery strategy has to be applied in order to use monoclonal antibodies (mAb) as diagnostic or therapeutic agents in the brain. It has been demonstrated that cationization of IgG allows for the BBB penetration following peripheral administration. A cationized mAb against betaA4-amyloid could be a sensitive and specific diagnostic tool for Alzheimer's disease (AD). The site-protected cationization and radiolabeling with In-111 of the specific anti beta-amyloid mAb, AMY33, is described. The binding affinity of the antibody was retained after these procedures (K(d) = 3.1 +/- 0.5 nM), as determined by solid-phase immunoradiometric assay and immunocytochemistry on AD brain sections. The in vitro binding by isolated brain capillaries indicated that the cationized antibody may be delivered to the brain in vivo. The ability of the modified antibody to detect cerebral beta-amyloid deposits in vivo can now be evaluated using single photon emission computed tomography (SPECT) and a suitable animal model for cerebral amyloidosis, such as non-human primates or aged canines.
It has come to our attention that the review by Suh and Checler [Suh Y-H and Checler F (2002) Amyloid precursor protein, presenilins, and α-synuclein: molecular pathogenesis and pharmacological applications to Alzheimer's disease. Pharmacol Rev 54: [469][1]–525] contains several paragraphs that
New molecular information about Alzheimer disease (AD) is appearing at an unprecedented rate. Much interest centers on the beta-A4 amyloid protein, which is progressively deposited in senile plaques and blood vessels in AD brain tissue. The discovery that some kindreds with familial AD have a mutation in the gene coding for the beta-A4 amyloid precursor protein (APP) suggests that this mutation alone may be sufficient to cause the full spectrum of clinical and pathological changes that characterize AD. Although APP point mutations may turn out to be relatively rare causes of AD, the idea that accelerated beta-A4 deposition is an early and critical event in many patients continues to gain support from studies in humans, animals, and cultured cells. Identification of the biochemical steps leading to production of the beta-A4 peptide from APP is now a critical issue. Recent reports indicate that normal lysosomal processing pathways can produce carboxyl-terminal fragments of APP that contain the entire beta-A4 sequence, and are therefore potentially amyloidogenic. The mechanisms by which such intermediate forms are further processed and released, resulting in extracellular beta-A4 deposits in plaques and vessels, are yet to be determined. It is likely that full elucidation of the beta-A4-producing pathways will ultimately yield new therapeutic approaches to this complex and tragic disorder.
Normal aging may be considered aging associated with minimal declines in physiological variables, approaching those of a healthy young adult. Certain physiological and structural changes are inevitable with aging. Aging is associated with mild decreases in attention span, short-term memory, and recall speed. Normal aging is readily contrasted with moderately advanced dementia, where memory deficits are more severe, and accompanied by the loss of a general fund of knowledge, poor orientation, naming problems, and inappropriate social behavior. The distinction between very early dementia and age-related cognitive impairments is a much more controversial matter, and an area of active research. Brains of normal elderly humans are heterogeneous, due in part to presence of subclinical disease processes that are increasingly common with advanced age. Brains of all clinically normal elderly have certain structural changes that may contribute to age-associated cognitive and motor slowing. Among these inevitable age-related changes are accumulation of pigment in neurons and glia, neuroaxonal dystrophy and granular degeneration of myelin. Neurofibrillary tangles are also found in virtually all elderly brains in an anatomically restricted distribution. In normal elderly persons, neurofibrillary tangles are usually limited to neurons of the entorhinal cortex, hippocampus, and basal forebrain, which are most vulnerable to this type of structural pathology. In contrast, neurofibrillary tangles are more numerous and widespread in Alzheimer's disease (AD).
Alzheimer's disease (AD) is a progressive degenerative encephalopathy associated with behavioral disorders, loss of memory and personality, physical debility, and, ultimately, death. It is the most common cause of dementia. While AD is an amyloidosis of the brain, it has been shown that neurotransmitter deficits more closely mirror the degree of dementia than the level of amyloidotic deposits in the AD brains. There is a case-to-case heterogeneity of the disease process, differences in diagnostic reliability among clinical centers, and complex interactions among various neurotransmitter systems within the brain. Because neuronal death is cumulative in AD, neurotic loss and neurotransmitter deficits are expected to increase progressively. Measures of plaque and tangle density reflect net formation and removal, as suggested in the only published pseudotime course study of AD. Therefore, the rate of amyloid plaque and neurofibrillary tangle (NFT) formation should plateau as the neurons responsible for the synthesis of their precursors die. Amyloid deposition is likely a participant in the progression of AD-associated neural degeneration. Amelioration of amyloid-related pathology may prove therapeutically promising, because this approach addresses all the various transmitter deficits and the neuronal degeneration responsible for dementia. This chapter discusses Alzheimer amyloid, amino acid permease (AAP) gene and transcripts, AAP synthesis and distribution, the processing and function of AAP protein, AD amyloidogenesis, and its therapeutic approaches.
Hirano bodies (HB) are cytoplasmic inclusions predominantly found in the CA, sector of the hippocampus. In Alzheimer's disease they are dislocated to the stratum pyramidale from their normal position in the stratum lacunosum. Hirano bodies are known to contain epitopes related to microfilaments (actin), neurofilaments, and microtubules (tau). In cryostat sections of the hippocampus from both Alzheimer's disease and normal patients, HB were decorated by two antisera raised against different sequences of the cytoplasmic domain of [latin sharp s]-amyloid precursor protein ([latin sharp s]-APP), and two antisera against the [latin sharp s]/A4 sequence of [latin sharp s]-APP, but not by two antisera directed against ectodomain (N-terminal) sequences of [latin sharp s]-APP. Thus, in contrast to dystrophic neurites in plaques, which are decorated by antibodies to either terminus of [latin sharp s]-APP, HB appear to be a site of preferential accumulation of C-terminal fragments of [latin sharp s]-APP, extending to include at least part of [latin sharp s]/A4.
(C) 1993 American Association of Neuropathologists, Inc
The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.
The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.
The senile plaque is one of the histopathologic changes that characterizes Alzheimer's disease and the aging brain. The histopathology of senile plaques was studied using double-labeling immunohistochemistry and lectin histochemistry with thioflavin S fluorescent microscopy in 9 cases of Alzheimer's disease, 2 nondemented elderly individuals, and 3 individuals with non-Alzheimer primary degenerative dementias. Every plaque that was visualized with thioflavin also had amyloid, but not all thioflavin-positive plaques contained neurites that could be recognized with specific monoclonal antibodies to paired helical filament, tau, or neurofilament epitopes. Some neurofilament-positive neurites were not visualized with thioflavin, but almost all tau-positive neurites were colabeled with thioflavin. Microglia were associated with most plaques. Most plaques were also surrounded by fibrous astrocytes. These results suggest that amyloid may be the common feature that defines senile plaques, but that other elements may be more specific for Alzheimer's disease, because extensive neuritic degeneration was seen only in Alzheimer brains and not in either nondemented elderly individuals with senile plaques or in non-Alzheimer dementia cases.
To clarify the distribution, morphology, and density of amyloid deposits in patients with Alzheimer's disease (AD), tissue sections from various areas of the central nervous system of 14 patients with AD and from 20 nondemented aged controls were investigated immunohistochemically using anti-beta protein antiserum. beta-protein amyloid deposits were present not only in the cores of the senile plaques and in the vascular wall (amyloid angiopathy), but also in various sized plaque-shaped fibrillary, perivascular, subpial, and subependymal deposits. Amyloid deposits were found mainly in the cerebral cortex in nondemented controls, while in AD they were distributed widely in the regions that were not affected in nondemented controls. The positivity of amyloid deposits in AD was 100% in the cerebral cortex, hippocampus, amygdala, thalamus, caudate nucleus, claustrum, hypothalamus, nucleus basalis of Meynert, and cerebellar cortex. Putamen and brain-stem nuclei were affected frequently, and the spinal cord, dentate nucleus, and globus pallidus were sometimes (less than 50%) affected. This result provides an evidence that Alzheimer's disease is a beta-protein amyloidosis of the central nervous system. An assessment of the distribution of amyloid deposits should prove to be useful for the histopathologic diagnosis of AD.
To identify the tau component in senile or kuru plaques, the authors examined brain sections from 12 patients with Alzheimer's disease (AD), 6 with Creutzfeldt-Jakob disease (CJD), and 20 nondemented aged controls using anti-beta protein, anti-buman prion protein, and affinity-purified tau-specific antibody. The tau component was identified both in senile and kuru plaques. In AD, tau-positive senile plaques were found in all cerebral cortices of almost all cases, and the tau-positivity of plaques in cerebral cortices was 5.1 to 27.5%. In CJD, tau-positive senile and kuru plaques were restricted to the hippocampus, and the tau-positivity was 4.3 and 1.2%, respectively. In nondemented aged controls, tau-positive senile plaques also were restricted mostly to the hippocampus, and the tau-positivity was 1.3%. Significant differences in the tau-positivity of senile plaques were found between AD and CJD and nondemented aged controls, and no significant differences were found between CJD and nondemented aged controls. These observations are important because increased tau accumulation in senile plaques can be a hallmark of AD.
Diffuse senile plaques are characterized by the presence of beta protein (beta P), also called A4 protein, in a dispersed form and the apparent lack of associated dystrophic neurites or reactive glial cells. They are the most common type of senile plaque found in the cerebral cortex in Alzheimer's disease (AD), Down's syndrome (DS), and normal aging. Here is reported the frequent presence of diffuse senile plaques in the molecular layer of cerebellar cortex in AD. Typical neuritic plaques were never detected in this location, making the cerebellar molecular cortex a useful site for the study of diffuse plaques because diffuse plaques in the cerebral cortex are intermingled with neuritic plaques. Diffuse cerebellar plaques were detected by modified Bielschowsky silver stain in 47 of 100 cases of clinically and pathologically diagnosed AD and in none of 40 aged demented and nondemented controls. They were immunolabeled by antibodies to purified AD meningeal or cortical beta P, and to a synthetic beta P but not by two antibodies to the carboxyl- and amino-termini of the beta protein precursor (beta PP), which label a subgroup of cerebral cortical plaques. This latter result suggests that the beta P deposited in the cerebellar molecular layer may be derived from a form of the beta PP from which the carboxyl and amino terminal regions of the precursor have already been cleaved. Diffuse cerebellar plaques were not recognized by antibodies to neurofilaments, tau, and PHF, all of which detect dystrophic neurites in cerebral cortical neuritic plaques. Also, no association of reactive astrocytes or microglial cells with diffuse cerebellar plaques was observed. Thus, diffuse cerebellar plaques represent multifocal deposits of noncompacted beta P that cause little or no morphologic reaction in their microenvironment.
The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.
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.
Progressive cerebral deposition of extracellular filaments composed of the beta-amyloid protein (beta AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the beta AP precursor (beta APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated beta AP deposition. Using two antibodies to the predicted carboxyl terminus of beta APP, we have identified the native beta APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundant in fetal brain, and detected in nonneural tissues that contain beta APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. Confirmation that the immunodetected tissue proteins are forms of beta APP was obtained when mammalian cells transfected with a full-length beta APP cDNA showed selectively augmented expression of 110- to 135-kDa proteins and specific immunocytochemical staining. Unexpectedly, the antibodies to the carboxyl terminus of beta APP labeled amyloid-containing senile plaques in AD brain. We conclude that the highly conserved beta APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of approximately 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the beta APP into insoluble filaments occurs locally in cortical regions that develop beta-amyloid deposits with age.
Early and accurate diagnosis of Alzheimer's disease (AD) has a major impact on the progress of research on dementia. To address the problems involved in diagnosing AD in its earliest stages, the National Institute on Aging, the American Association of Retired Persons, the National Institute of Neurological and Communicative Disorders and Stroke, and the National Institute of Mental Health jointly sponsored a workshop for planning research.
The purpose of the meeting was to identify the most important scientific research opportunities and the crucial clinical and technical issues that influence the progress of research on the diagnosis of AD. The 37 participants included some of the most knowledgeable and eminent scientists and physicians actively involved in the study of AD. The participants were divided among six panels representing the disciplines of neurochemistry, neuropathology, neuroradiology, neurology, neuropsychology, and psychiatry. Within each of the panels, participants discussed specific areas of research requiring further
Monoclonal antibodies generated against a synthetic peptide corresponding to amino acids 1 to 24 of cerebrovascular amyloid β-protein do not only stain amyloidotic blood vessels and the amyloid deposits of the (senile) neuritic plaques, but also the neuronal pigment lipofuscin. Staining of lipofuscin is observed in both cerebral and cerebellar cortices, subcortical nuclei as well as the brain stem, and is identical in Alzheimer and normal control brain. Western blots of a lipofuscin enriched fraction show an anti-β-protein reactive polypeptide migrating at approximately 31 kDa position on SDS-polyacrylamide gel electrophoresis. These results suggest that this polypeptide is associated with lipofuscin and is most likely derived from the predicted amyloid precursor protein. This implicates that, unlike in Alzheimer's disease where this protein is also processed extraneuronally in a manner to release an amyloid fiber forming fragment, the end point of its processing in the nerve cell seems to accumulate on a lipopigment characteristic for normal aging.
The purpose of this study was to design a method by which immunoperoxidase staining can be applied to formalin-fixed, paraffin-embedded tissue sections to demonstrate amyloid deposits in cerebral and systemic amyloidotic tissues. We used anti-prion protein, anti-beta-protein, anti-amyloid A, and anti-prealbumin antisera. The tissue sections were first treated with 100% formic acid for 5, 20, or 60 minutes and the unlabeled immunoperoxidase method (biotin-streptavidin system reagents) was used. This formic acid pretreatment enhanced immunoreactivity of the amyloid deposits which reacted positively with specific antiserum. The specificity of the immunostainings was well preserved. This method can also be used to demonstrate interspecies cross-reactivity, by using anti-human amyloid A and anti-scrapie hamster prion protein antisera, which stained negatively or faintly with amyloid deposits of heterogenous species. The technique is expected to reveal the buried epitopes of amyloid deposits in tissue sections.
Immunological and structural analyses of neurofilament (NF) proteins with greater than 500 anti-NF monoclonal antibodies (mAbs) enumerated epitopes shared by NF proteins and Alzheimer neurofibrillary tangles. We identified the multiphosphorylation domain of the rat heaviest NF subunit--tandem repeats of Lys-Ser-Pro-Xaa (where Xaa is a small uncharged amino acid and serine is phosphorylated)--as the determinant recognized by 15 of the 16 mAbs from this collection of greater than 500 mAbs that detected neurofibrillary tangles. Most (11) of these 16 mAbs also recognized the previously characterized multiphosphorylation repeat in the human middle sized NF subunit. However, although these mAbs shared the ability to recognize NFTs, the antigen-binding domains of these 16 mAbs represented 13 separate classes based on their differential recognition of 12 synthetic peptides derived from the rat heaviest NF subunit and the human middle-sized NF subunit multiphosphorylation sites, NF subunits of 10 diverse species, and normal human tau protein. We conclude that NFTs share highly specific immunological and structural properties with specific rat heaviest NF subunit and human middle-sized NF subunit multiphosphorylation repeats.
Neurofibrillary tangles (NFT) in Alzheimer's disease (AD) and in progressive supranuclear palsy (PSP) differ in their location and morphology, but both appear to express the same or similar epitopes. These immunologic data may signify that both types of NFTs contain the same components and arise as a result of the same mechanisms. To explore this hypothesis, we probed PSP and AD samples of brain stem, where both PSP and AD NFTs occur, using a large panel of monoclonal antibodies (MAbs). The epitopes expressed in brain stem PSP and AD NFTs were compared with those in the NFTs of AD hippocampus. NFTs in PSP hippocampus were too infrequent for comparative analysis. The MAbs were raised to neurofilament and tau proteins, or to AD paired helical filaments. All MAbs raised to tau (three) and paired helical filaments (two) recognized brain stem PSP NFTs and AD NFTs in brain stem and hippocampus. However, 12 anti-NF MAbs specific for multiphosphorylation repeat domains or other phosphate-dependent and independent epitopes did not bind PSP NFTs, but they did detect AD NFTs in hippocampus, and 5 of these MAbs also recognized brain stem AD NFTs. We conclude that some populations of AD NFTs contain antigenic determinants that are not found in PSP NFTs. This may reflect the effect of different pathologic events specific to PSP and AD, or the selective formation of NFTs in different groups of neurons in each of these disorders.
Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.
In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.
The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.
Progress in the study of Alzheimer's disease (AD) has been spurred by the recent application of molecular approaches in many laboratories. Attention has centered on the nature of the proteinaceous deposits that accumulate progressively both within and outside of cerebral neurons. Evidence reviewed herein suggests that intraneuronal paired helical filaments are distinct from extracellular amyloid filaments and contain altered forms of the microtubule-associated phosphoprotein, tau. Antibodies to tau detect an extensive neuritic dystrophy in AD cerebral cortex that includes aberrant somatodendritic sprouting, suggesting a role for local growth-promoting molecules in the pathogenesis of AD. Perhaps preceding these neuronal changes, deposits of the beta-amyloid protein (beta AP) occur in a diffuse, nonfibrillar form in AD and Down's syndrome brains in the absence of surrounding neuritic or glial response. Such deposits may represent the earliest structural abnormality yet detected in AD brain. Since the gene encoding the beta AP precursor appears to be distinct from a putative familial AD gene defect also localized to chromosome 21 in some families, changes in transcriptional and posttranslational processing of the precursor in aging and AD are being sought. The central and unresolved question of the origin of the beta AP molecules deposited progressively in brain is reviewed in detail. In concert with other human amyloidoses, growing evidence points to a blood-borne or vascular source for beta AP, although rigorous proof is not at hand. Advances in the molecular analysis of AD brain lesions point to new experimental strategies that should bear directly on unsolved diagnostic and therapeutic issues in the disease.
Peptides composed of variants of a B-cell epitope followed by a T-cell determinant of rabies virus ribonucleoprotein (RNP) have been synthesized to elicit antibody production in mice. Conformation of the peptides was characterized by secondary structural prediction and circular dichroism measurements. It was found that only synthetic peptides with disrupted helical structure in the antigenic region were active on immunoblot assay, performed against a natural anti-protein monoclonal antibody (MAb) and provoked virus-neutralizing antibody production.
Studies were conducted to identify neural cells that synthesize and/or process cerebral amyloid using antisera and monoclonal antibodies (MAbs) raised to synthetic peptides based on the first 28 amino acids of the amyloid beta-protein. Using rabbit and mouse antisera, and 7 MAbs, sections of neocortex, hippocampus, cerebellum, and spinal cord from Alzheimer's disease (AD), Down's syndrome (DS), and control cases were probed. The antibodies produced 3 distinct immunohistochemical patterns: 1) staining restricted to neuritic plaque and blood vessel amyloid only (antisera, 1 of 7 MAbs); 2) immunoreactivity confined to cytoplasmic granules in diverse neuronal, glial (astrocytes, ependyma) and other (leptomeningeal, perivascular, choroid plexus) cells (1 of 7 MAbs); 3) a summation of these 2 patterns (5 of 7 MAbs). Controls resembled the AD and DS cases, except for a paucity of immunoreactive plaques and blood vessels in the controls. Immunoreactivity was reduced or removed by the peptides used to produce these antibodies. Formalin- and Bouins-fixed tissues reacted weakly or not at all with these antibodies while microwave denatured tissues reacted very intensely with them. Specific staining was enhanced by treatment of the tissue sections with Triton X-100, NaDodSO4, or trypsin. These studies significantly extend earlier studies that localized amyloid beta-protein precursor mRNA to human brain cells, and they suggest that the beta-protein, its precursor, and/or fragments thereof may exist in diverse neural cell types in AD, DS, and control brains.
Information concerning the distribution of various subdomains of the amyloid precursor protein (APP) in brain may illuminate aspects of the normal metabolism of this membrane-associated protein, as well as putative abnormal processing that may occur in Alzheimer disease (AD). We prepared affinity-purified antibody, P2, against an extracytoplasmic APP site and applied it, along with monoclonal antibodies to the beta-peptide, or A4 region, in conjunction with selective cytochemical staining methods, to control and AD tissues. The following was noted: (i) in contrast to A4 epitopes, which are easily demonstrable primarily in extracellular senile plaques of AD patients, the extracytoplasmic P2 antigen was found in association with neurons, glia, and blood vessels in both normal and AD prefrontal cortex; (ii) a subset of senile plaques contained both A4 and P2 antigens; (iii) in some instances, P2 antigen occurred as an extracellular deposit in the absence of A4; (iv) the P2 antigen, but not A4, was also associated with corpora amylacea. In addition to identifying the unique cellular distribution of the APP extracytoplasmic antigen, the results support the view that a segment of this domain undergoes processing and deposition at extracellular sites, including a subset of senile plaques.
A monoclonal antibody (4D12/2/6) to a synthetic peptide consisting of residues 8-17 of the amyloid beta-protein of Alzheimer's disease was used in an immunohistochemical study to investigate the localization of beta-protein immunoreactivity in neuritic plaques in the brains of 20 cases with Alzheimer's disease and a similar number of nonAlzheimer controls. The morphology and distribution of immunoreactive plaque-like lesions and the sensitivity of immunostaining were assessed both with and without formic acid pretreatment of the sections, and these results were compared with those obtained using conventional Congo red and silver impregnation staining methods. Congo red and immunostaining without formic acid pretreatment mainly stained the core deposits of amyloid in compact plaques, whereas the silver stain could also detect numerous diffuse plaques. Immunostaining with formic acid pretreatment was the most sensitive technique, and this revealed many additional immunoreactive lesions which were impossible or difficult to detect with the other staining methods. These additional lesions included variable sized areas of faint granular staining with little evidence of amyloid deposition or degenerating neurites that are presumed to be very early stages in plaque development. Far fewer immunoreactive lesions were observed in the nonAlzheimer controls. It is concluded that an abundant presence of anti-beta-protein immunoreactive plaque lesions throughout the cortex and subcortical gray matter structures is typical of Alzheimer's disease even when only moderate numbers of plaques can be detected by Congo red or silver stain. This immunostaining procedure with a specific monoclonal antibody for beta-protein may be very useful for the postmortem diagnosis of Alzheimer's disease.
Alzheimer's disease results from the degeneration of neurons. Degenerating nerve cells express atypical proteins, and amyloid is deposited. We suggest that some of these events are strongly influenced by genetic factors and age. Animal models should be useful in investigating the pathogenic mechanisms that lead to the brain abnormalities seen in this disease.
The actual presence of the predicted precursor of Alzheimer's disease amyloid A4 protein, reported by Kang et al. (1987) in the Alzheimer brain, has yet to be verified. To identify the various regions of this precursor, antibodies were raised against three synthetic polypeptides, R35 (residues 274-286), R36 (residues 527-540), and R37 (residues 681-695), subsequences of the precursor protein; the specificity of these antibodies was ascertained by ELISA. Upon immunohistochemical examination, the antibody to R35 failed to react, but the antibody to R36 (the extracellular part) stained the amyloid of senile plaques and the staining pattern was identical to that of anti-A4 antibody. The antibody to R37 (the C-terminal intracellular part) stained what may be degenerating neurites in senile plaques whereas the amyloid remained unstained. An anti-neurofilament (NF) antibody reacted with some of the R37-positive grains, but R37-negative grains also were seen. Further, some R37-positive grains were not stained by the anti-NF antibody. The anti-GFAP antibody and the anti-macrophage antibody did not stain the R37-positive grains. These findings indicate that the amyloid protein in senile plaques actually contains a larger polypeptide than the A4 protein, and suggest that the intracellular C-terminal part of the precursor may exist in the degenerated neurites seen in senile plaques.
Monoclonal antibodies generated against a synthetic peptide corresponding to amino acids 1 to 24 of cerebrovascular amyloid beta-protein do not only stain amyloidotic blood vessels and the amyloid deposits of the (senile) neuritic plaques, but also the neuronal pigment lipofuscin. Staining of lipofuscin is observed in both cerebral and cerebellar cortices, subcortical nuclei as well as the brain stem, and is identical in Alzheimer and normal control brain. Western blots of a lipofuscin enriched fraction show an anti-beta-protein reactive polypeptide migrating at approximately 31 kDa position on SDS-polyacrylamide gel electrophoresis. These results suggest that this polypeptide is associated with lipofuscin and is most likely derived from the predicted amyloid precursor protein. This implicates that, unlike in Alzheimer's disease where this protein is also processed extraneuronally in a manner to release an amyloid fiber forming fragment, the end point of its processing in the nerve cell seems to accumulate on a lipopigment characteristic for normal aging.
Immunocytochemistry with monoclonal antibodies to the beta-protein and to antigens associated with paired helical filaments (PHF) allows us to selectively stain two major components of neuritic (senile) plaques (NP): PHF and amyloid deposits. Using this method, the structure of NP in the brains of Alzheimer disease victims was compared to their structure in the brains of non-demented aged individuals selected for high numbers of NP. It is demonstrated that the dystrophic neurites participating in the plaque formation contain PHF only when cortical nerve cells in the same brain area form neurofibrillary tangles (NFT). People with many NP and many NFT were always demented, whereas people with many NP but few, if any NFT were not. It is speculated that there is individual susceptibility to the formation of PHF and that their appearance may represent a nonspecific response of the neuronal network to different kinds of injuries, like the deposition of amyloid in Alzheimer disease, or other pathogenic factors associated with various dementive neurodegenerative diseases. It is hypothesized that the deposition of brain amyloid in people resistant to neurofibrillary pathology may induce too little dysfunction for the development of dementia.
Neurofibrillary tangles and neuritic plaques are the neuropathological hallmarks of Alzheimer's disease. The latter consist of a core of A4 amyloid protein. We now report that some neurofibrillary tangles ('tombstone tangles') are also A4 immunoreactive. This observation is consistent with the hypothesis that A4 amyloid accumulation is a component of both neurofibrillary tangles and neuritic plaques.
Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.
Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.
Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.
The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.
An alternate form of the Alzheimer amyloid protein precursor mRNA that encodes a protease inhibitor domain has recently been reported. Oligonucleotide probes that differentiate between the two mRNAs are used to describe the expression of each amyloid precursor transcript in the human brain. RNA blot analyses show that one of the mRNAs is expressed selectively in the nervous system, that the two messages display different regional distributions in the adult human brain, and that the expression of the two mRNAs is differentially affected in Down's syndrome brain and in Alzheimer's disease frontal cortex. In situ hybridization shows that the two transcripts display the same laminar distribution in the adult cortex but that the transcripts differ significantly in their levels of expression in pyramidal cells of the hippocampus.
We studied cerebral amyloid deposits in the hippocampal area immunohistochemically, using antiserum to synthetic beta peptide (1-28) in 66 patients with or without dementia and aged 17 to 91 years old. Senile plaques (SP) and amyloid angiopathy (AA) were detected in 36 (55%) and 19 (29%) patients, respectively. Also, cerebral amyloid deposits from the brains of seven patients with dementia and five patients without were studied in serial sections stained with Bodian, modified Bielschowsky, Congo red, and beta protein immunostain. In the patients with Alzheimer-type dementia (ATD) diffuse plaques, typical of this group, were stained with beta protein antiserum but not with Bodian stain, because the plaques were devoid of abnormally swollen neuritic processes. The diffuse plaques often contained one or more neuronal cell bodies. As well as primitive and classic plaques and AA, the beta protein immunostain demonstrated small deposits among the SP, small stellate deposits of layer 1, subpial fibrillar deposits, and focal cribriform deposits of parasubiculum, which may be new types of amyloid deposits. Amyloid plaques within the subcortical white matter were only found in ATD brains. In the non-demented patients various kinds of SP, including diffuse and compact ones, were immunostained. They tended to be small and few. beta protein immunostain with formic acid pretreatment is a useful method for the identification of a variety of senile cerebral amyloid deposits.
A monoclonal antibody was prepared against pooled homogenates of brain tissue from patients with Alzheimer's disease. This
antibody recognizes an antigen present in much higher concentration in certain brain regions of Alzheimer patients than in
normal brain. The antigen appears to be a protein present in neurons involved in the formation of neuritic plaques and neurofibrillary
tangles, and in some morphologically normal neurons in sections from Alzheimer brains. Partial purification and Western blot
analysis revealed the antigen from Alzheimer brain to be a single protein with a molecular weight of 68,000. Application of
the same purification procedure to normal brain tissue results in the detection of small amounts of a protein of lower molecular
weight.
A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.
We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.
The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.
During aging of the human brain, and particularly in Alzheimer's disease, progressive neuronal loss is accompanied by the formation of highly stable intra- and extraneuronal protein fibers. Using fluorescence-activated particle sorting, a method has been developed for purifying essentially to homogeneity the extracellular amyloid fibers that form the cores of senile plaques. The purified plaque cores each contain 60-130 pg of protein. Their amino acid composition shows abundant glycine, trace proline, and approximately 50% hydrophobic residues; it resembles that of enriched fractions of the paired helical filaments (PHF) that accumulate intraneuronally in Alzheimer's disease. Senile plaque amyloid fibers share with PHF insolubility in numerous protein denaturants and resistance to proteinases. However, treatment of either fiber preparation with concentrated (88%) formic acid or saturated (6.8 M) guanidine thiocyanate followed by sodium dodecyl sulfate causes disappearance of the fibers and releases proteins migrating at 5-7,000 and 11-15,000 Mr which appear to be dimerically related. Following their separation by size-exclusion HPLC, the proteins solubilized from plaque amyloid and PHF-enriched fractions have highly similar compositions and, on dialysis, readily aggregate into higher Mr polymers. Antibodies raised to the major low-Mr protein selectively label both plaque cores and vascular amyloid deposits in Alzheimer brain but do not stain neurofibrillary tangles, senile plaque neurites, or any other neuronal structure. Thus, extraneuronal amyloid plaque filaments in Alzheimer's disease are composed of hydrophobic low-Mr protein(s) which are also present in vascular amyloid deposits. Current evidence suggests that such protein(s) found in PHF-enriched fractions may derive from copurifying amyloid filaments rather than from PHF.
A purified protein derived from the twisted beta-pleated sheet fibrils in cerebrovascular amyloidosis associated with Alzheimer's disease has been isolated by Sephadex G-100 column chromatography with 5 M guanidine-HC1 in 1 N acetic acid and by high performance liquid chromatography. Amino acid sequence analysis and a computer search reveals this protein to have no homology with any protein sequenced thus far. This protein may be derived from a unique serum precursor which may provide a diagnostic test for Alzheimer's disease and a means to understand its pathogenesis.
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