Amyloid β protein 1–42/43 (Aβ 1–42/43) in cerebellar diffuse plaques: enzyme-linked immunosorbent assay and immunocytochemical study

Diffuse plaques are immature and amorphous senile plaques and believed to be in the initial phase of plaque formation. In contrast to amyloid angiopathy and the plaque core amyloid, diffuse plaques failed to be purified in preserved forms from the brain. Here, we studied the diffuse plaques in the cerebellar region of the Alzheimer's disease brain based on immunocytochemistry and ELISA using two different monoclonal antibodies specifically recognizing the car?yl termini of Aβ molecules (BA27 for Aβ 1–40 and BC05 for Aβ 1–42/43). We found that the amount of Aβ 1–40 was in proportion to the staining degree on amyloid angiopathy by immunohistochemistry. We found that Aβ 1–42/43 comprised diffuse plaques as the major component in the cerebella of AD brains. Taking these findings into consideration, diffuse plaques, the earliest pathological change in the brain with AD, are concluded to be composed mainly of Aβ 1–42/43, implicating the critical importance of this kind of Aβ species deposition in the pathogenesis of AD.     

Amyloid β protein (Aβ) deposition in chromosome 14–linked Alzheimer's disease: Predominance of Aβ42(43)

Amyloid β protein (Aβ) deposition was investigated in the frontal cortex of 8 cases of (genetically confirmed) chromosome 14–linked Alzheimer's disease (AD) using the end-specific monoclonal antibodies BA27 and BC05 to detect the presnce of Aβ₄₀ and Aβ₄₂₍₄₃₎, respectively. IN all patients, Aβ₄₂₍₄₃₎ was the predominant peptide species present. The total amount of Aβ₄₀ and Aβ₄₂₍₄₃₎ deposited was more than twice the amount deposited in cases of sporadic AD of similar disease duration, although the ratio between the extent of Aβ₄₀ and Aβ₄₂₍₄₃₎ deposition was unaltered, compared with sporadic AD. Therefore, (one of) the effects of the mutations in the presenilin 1:PS-1 (S182) gene may be to cause or at least promote an early and excessive deposition of Aβ₄₂₍₄₃₎ within the brain, a property shared with other inherited forms of AD, such as those due to amyloid precursor protein mutations, and Down's syndrome (trisomy 21).     

Characteristics of cerebral β amyloid deposition in four non-demented patients in their forties with a high apolipoprotein E ε4 allele frequency

Four autopsied brains from mentally normal patients aged 43–49 who had cerebral β amyloid deposition were examined. Three patients had breast cancer, and in one of these cases it was associated with brain metastasis and brain radiation therapy. One other case had pulmonary small cell carcinoma. In two patients, small β amyloid deposits were only found in the frontal cortex. In another two patients, β amyloid deposits were found in many cortical areas and the density of plaques was higher than in the former two patients. No β amyloid deposition was found in the cerebella, basal ganglia, or brain stem in any of the four patients. When examined with end-specific antisera for the C-terminal of amyloid β protein (Aβ), Aβ42 was predominant in the diffuse plaques and immunoreactions for Aβ40 varied among the patients. The N-terminal of Aβ was truncated in a subset of plaques. Tau- and phosphorylated tau-reactive fine neurites were only found in the entorhinal cortex of Case 3. The apoE genotype of these four patients were 3/4, 3/4, 4/4 and 3/3, and therefore, the ε4 allele frequency (50%) was as high as that in AD. Three out of four patients had at least one ε4 allele, a risk factor of AD. There is a possibility that these subjects might develop AD when in their seventies. It may take 30 years from the beginning of Aβ deposition to the clinical manifestation of dementia.     

Plasma Levels of amyloid β proteins Aβ1–40 and Aβ1–42(43) are elevated in Down's syndrome

To investigate the effect of the overexpression of β-amyloid precursor protein (APP) on the production of two major amyloid β protein (Aβ) species, Aβ40 and Aβ42(43), we measured amounts of Aβ1–40 and Aβ1–42(43) in the plasma from 44 patients with Down's syndrome (DS) (age, 19–61 years) and 66 age-matched normal controls using enzyme-linked immunosorbent assays. Plasma concentrations of both Aβ1–40 and Aβ1–42(43) were increased about 3-fold and 2-fold, respectively, in DS patients compared with normal controls. Especially, the increases in plasma Aβ1–40 in DS Patients were statistically higher than the 1.5-fold increase one might predict based on the gene dose of APP in DS. These findings showed that both Aβ1–40 and Aβ1–42(43) are increased in plasma in DS patients, the former more than the latter, suggesting that overexpression of APP and/or other genes may have different effects on the production of these two Aβ species in DS.     

Characteristics of aquaporin expression surrounding senile plaques and cerebral amyloid angiopathy in Alzheimer disease

Senile plaques (SPs) containing amyloid β peptide (Aβ) 1-42 are the major species present in Alzheimer disease (AD), whereas Aβ1-40 is the major constituent of arteriolar walls affected by cerebral amyloid angiopathy. The water channel proteins astrocytic aquaporin 1 (AQP1) and aquaporin 4 (AQP4) are known to be abnormally expressed in AD brains, but the expression of AQPs surrounding SPs and cerebral amyloid angiopathy has not been described in detail. Here, we investigated whether AQP expression is associated with each species of Aβ deposited in human brains affected by either sporadic or familial AD. Immunohistochemical analysis demonstrated more numerous AQP1-positive reactive astrocytes in the AD cerebral cortex than in controls, located close to Aβ42- or Aβ40-positive SPs. In AD cases, however, AQP1-positive astrocytes were not often observed in Aβ-rich areas, and there was a significant negative correlation between the levels of AQP1 and Aβ42 assessed semiquantitatively. We also found that Aβ plaque-like AQP4 was distributed in association with Aβ42- or Aβ40-positive SPs and that the degree of AQP4 expression around Aβ40-positive vessels was variable. These findings suggest that a defined population of AQP1-positive reactive astrocytes may modify Aβ deposition in the AD brain, whereas the Aβ deposition process might alter astrocytic expression of AQP4.     

Aβ-peptide length aid apolipoprotein E genotype in Alzheimer's disease

Apolipoprotein ∈ (ApoE)ε4 allele, a risk factor for the development of Alzheimer's disease (AD), is associated with increased amyloid deposition. We examined cerebral cortex in 68 AD cases using antibodies to βbeta;-amyloid (Aβbeta;) peptides of different length (Aβbeta;_1?40 and Aβbeta;_l?42) and found that the increased plaque frequency observed with ε4 genotypes may be largely attributed to an increase in Aβbeta;_1?40-positive plaques. Indeed, both the number of Aβbeta;_1?40-positive plaques, as well as the ratio of Aβbeta;_I?40/ Aβbeta;_1?42-positive plaques, increased with ε4 dosage. In contrast, the frequency of Aβbeta;_1?42-immunoreactive plaques was similar for ε3/ε3,ε3/ε4 and ε4/ε4 genotypes. ApoE may influence Aβbeta; length by facilitating Aβbeta; _1?40 deposition onto Aβbeta;_1?42-seeded plaques or by modulating the activity of a putative carboxypeptidase that forms Aβbeta;_1?40 from Aβbeta;_1?42 in situ.     

Deposition of amyloid β protein (Aβ) subtypes [Aβ40 and Aβ42(43)] in canine senile plaques and cerebral amyoloid angiopathy

To clarify the immunohistochemical features of canine senile plaques (SPs) and cerebral amyloid angiopathy (CAA), the distribution of the amyloid β protein (Aβ) subtypes Aβ40 and Aβ42(43), Aβ precursor protein (APP), and glial cell reaction were examined in the brains of seven aged dogs (12–18 years). Aβ42(43) was found to be deposited in all types of SPs, whereas Aβ40 was deposited only in mature (classical and primitive) plaques. CAA, which was located along parenchymal and meningeal arterioles and capillaries, consisted of both subtypes of Aβ. APP was exhibited in normal and degenerative neurons and swollen neurites of mature plaques. It was, therefore, considered that Aβ42(43) in diffuse plaques might be derived from APP in neurons, while Aβ40 and Aβ42(43) in mature plaques might be generated from APP in swollen neurites in the plaque. In contrast to the case in humans, in whom deposition of Aβ40 and Aβ42(43) in the mature plaques is predominantly associated with microglial reaction, in dogs we found that it was closely associated with astroglial reaction. The present findings showed characteristics of canine SPs which are different from those of humans. Received: 11 October 1996 / Revised: 27 March 1997, 10 April 1997 / Accepted: 10 April 1997     

Conversion of Aβ43 to Aβ40 by the successive action of angiotensin‐converting enzyme 2 and angiotensin‐converting enzyme

The longer and neurotoxic species of amyloid‐β protein (Aβ), Aβ42 and Aβ43, contribute to Aβ accumulation in Alzheimer's disease (AD) pathogenesis and are considered to be the primary cause of the disease. In contrast, the predominant secreted form of Aβ, Aβ40, inhibits amyloid deposition and may have neuroprotective effects. We have reported that angiotensin‐converting enzyme (ACE) converts Aβ42 to Aβ40 and that Aβ43 is the earliest‐depositing Aβ species in the amyloid precursor protein transgenic mouse brain. Here we found that Aβ43 can be converted to Aβ42 and to Aβ40 in mouse brain lysate. We further identified the brain Aβ43‐to‐Aβ42‐converting enzyme as ACE2. The purified human ACE2 converted Aβ43 to Aβ42, and this activity was inhibited by a specific ACE2 inhibitor, DX600. Notably, the combination of ACE2 and ACE could convert Aβ43 to Aβ40. Our results indicate that the longer, neurotoxic forms of Aβ can be converted to the shorter, less toxic or neuroprotective forms of Aβ by ACE2 and ACE. Moreover, we found that ACE2 activity showed a tendency to decrease in the serum of AD patients compared with normal controls, suggesting an association between lower ACE2 activity and AD. Thus, maintaining brain ACE2 and ACE activities may be important for preventing brain amyloid neurotoxicity and deposition in Alzheimer's disease. © 2014 Wiley Periodicals, Inc.     

Subpopulation of dogs with severe brain parenchymal β amyloidosis distinguished with cluster analysis

A study of the brains of 30 dogs, mongrels from 6.5 to 26.5 years of age, revealed that all dogs older than 13 years of age develop amyloid-β-positive plaques. Cluster analysis based on the age of the dogs and the numerical density of amyloid-positive plaques stained with monoclonal antibody 4G8 (17–24aa) revealed that the population of old dogs consists of two subpopulations: one with a very low (0.8/mm² on average) and other with a high (19.2/mm² on average) numerical density of plaques. These two groups (19.5 and 19.1 years of age, respectively) appear to emerge from the younger group (12.2 years of age on average), with moderate (2.2/mm² on average) numerical density of 4G8-positive plaques. These data may indicate that only a portion of the mongrel population (43%) is susceptible to amyloidosis β or that only this severely affected subpopulation was exposed to a factor or factors inducing this pathology and developed severe cortical amyloidosis that correlates with age. Dog plaques are only of the diffuse type, with nonfibrillar, thioflavin S-, and Congo red-negative amyloid in all groups distinguished by cluster analysis. Only from 10% of 4G8-positive plaques in the mildly affected group to 29% in the severely and 37% in the moderately affected group are Bielschowsky positive. In the younger, moderately affected group, 6E10 (1–17aa)-positive plaques prevail. In the two old groups with severe and weak changes, almost all 4G8-positive plaques are also 6E10-positive. Carboxy-terminal region immunocytochemistry reveals that BC42-positive plaques are numerous, whereas BC40-positive plaques are few or absent. The differences in the silver-positivity of plaques and their immunoreactivity in both the amino- and carboxy-terminal regions may reflect differences in amyloid-β deposition and resolution. Dog parenchymal amyloidosis β appears to be a model for the study of diffuse plaques.     

Amyloid (Aβ) deposition in chromosome 1–linked Alzheimer's disease: The volga german families

Amyloid β protein (Aβ) deposition was investigated in the frontal cortex of 6 cases of (genetically confirmed) chromosome 1–linked Alzheimer's disease (AD) (PS-2 gene mutation) among the Volga German families using the end-specific monoclonal antibodies BA27 and BC05 to detect the presence of Aβ₄₀ and Aβ₄₂₍₄₃₎, respectively. In all patients, Aβ₄₂₍₄₃₎ was the predominant peptide species present, although the total amount of Aβ₄₀ and Aβ₄₂₍₄₃₎ deposited in plaques did not differ from that seen in sporadic AD and was significantly lower than that occurring in AD due to PS-1 gene mutations. Therefore, mutations in the PS-2 gene, like those in the presenilin-1 (PS-1) and amyloid precursor protein (APP) genes, are associated with an initial and preferential deposition of Aβ₄₂₍₄₃₎ within the brain. Although the mechanisms(s) whereby the PS-1 and PS-2 gene mutations operate remains unclear, it seems from the present study that the effect of the PS-2 gene mutation on the brain is muuch less severe, at least as far as Aβ deposition is concerned, than that of the PS-1 mutation, which seems to confer a much earlier and a much more aggressive development of AD.     

Longitudinal study of cerebrospinal fluid levels of tau,Aβ1–40, and Aβ1–42(43) in Alzheimer's disease: A study in Japan

To clarify the alterations of tau, amyloid β protein (Aβ) 1–40 and Aβ1–42(43) in the cerebrospinal fluid (CSF) that accompany normal aging and the progression of Alzheimer's disease (AD), CSF samples of 93 AD patients, 32 longitudinal subjects among these 93 AD patients, 33 patients with non-AD dementia, 56 with other neurological diseases, and 54 normal control subjects from three independent institutes were analyzed by sensitive enzyme-linked immunosorbent assays. Although the tau levels increased with aging, a significant elevation of tau and a correlation between the tau levels and the clinical progression were observed in the AD patients. A significant decrease of the Aβ1–42(43) levels and a significant increase of the ratio of Aβ1–40 to Aβ1–42(43) were observed in the AD patients. The longitudinal AD study showed continuous low Aβ1–42(43) levels and an increase of the ratio of Aβ1–40 to Aβ1–42(43) before the onset of AD. These findings suggest that CSF tau may increase with the clinical progression of dementia and that the alteration of the CSF level of Aβ1–42(43) and the ratio of Aβ1–40 to Aβ1–42(43) may start at early stages in AD. The assays of CSF tau, Aβ1–40, and Aβ1–42(43) provided efficient diagnostic sensitivity (71%) and specificity (83%) by using the production of tau levels and the ratio of Aβ1–40 to Aβ1–42(43), and an improvement in sensitivity (to 91%) was obtained in the longitudinal evaluation.     

Amyloid β-proteins 1—40 and 1—42(43) in the soluble fraction of extra- and intracranial blood vessels

To investigate the process of amyloid β-protein (Aβ) accumulation in cerebral amyloid angiopathy (CAA), the levels of Aβ were determined in the soluble fraction of extra- and intracranial blood vessels and leptomeninges obtained at autopsy. Two enzyme immunoassays were employed that are known to sensitively and specifically quantify two Aβ species, Aβ1–40 and 1–42(43). Aβ was detectable in the intracranial blood vessels and leptomeninges with the latter containing the highest levels, while it was undetectable in the extracranial blood vessels. Thus the levels of soluble Aβ correlated well with the prediiection sites for CAA. Among individuals aged 20 to 90, the Aβ levels in the leptomeninges increased sharply in those aged 50 to 70 and thereafter tended to decline. However, only slight degrees of CAA were detected by immunocytochemistry, even when those leptomeninges contained high levels of Aβ comparable with those in Alzheimer's disease. The level of Aβ1–42 was almost always severalfold that of Aβ1–40 in the soluble fraction of leptomeninges. This is in good agreement with the immunocytochemical result showing the presence of Aβ40-negative, Aβ42(43)-positive meningeal vessels. These results indicate that Aβ1–42 is the initially deposited species in CAA and that the disruption of Aβ homeostasis precedes Aβ deposition in the meningeal vessels.     

Senile plaques in very aged cats

Senile plaques were found in the cerebral cortices of three very aged cats (more than 18 years old). The plaques consisted of a coarse assembly of silver staining-positive materials, and was morphologically different from the well-known classical, primitive, and diffuse plaques. Congophilic amyloid angiopathy was observed in a few cortical arterioles of the oldest cat (20 years old). The senile plaques and a few cortical blood vessels were immunopositive for amyloid β-protein (Aβ). Aβ-positive materials were also sparsely distributed in the cortical neuropil but did not form senile plaques there. These findings should help to clarify the development of senile plaques and the early stage of Aβ deposition. Received: 29 May 1995 / Revised, accepted: 28 September 1995     

Biochemical heterogeneity of Alzheimer's disease

Amyloid β protein (Aβ) amyloid deposition is an important early event in the evolution of Alzheimer's disease (AD) pathology. Serial extraction studies of AD brains showed that a large amount of modified Aβ (4, 3.7 and 3 kDa Aβ species) was accumulated as forms with differing solubilities. Immunostaining using specific antibodies to the N and C terminus of Aβ showed that N-terminally modified Aβ ending at 42A were predominant in senile plaques. Quantitative assay of Aβ in AD brain revealed that Aβ ending at 40 V were correlated with vascular amyloids and that Aβ ending at 42(43) were related with senile plaque amyloids. The site of Aβ deposits may be determined by the heterogeneous length of the C terminus Aβ. The amount of Aβ amyloids may be correlated with N-terminal modification of Aβ. Thus, heterogeneity of Aβ species is a crucial biochemical factor of AD pathogenesis.     

Elimination of amyloid precursor protein in senile plaques in the brain of a patient with Alzheimer-type dementia and Down syndrome

The average lifespan of individuals with Down syndrome has approximately doubled over the past three decades to 55–60?years. To reveal the pathogenic process of Alzheimer-type dementia in individuals with Down syndrome, we immunohistochemically examined senile plaque formation in the cerebral cortex in the autopsy brain and compared findings with our previous studies. We described a 52-year-old female with Down syndrome who developed progressively more frequent myoclonus following cognitive decline and died at the age of 59?years. Her karyotype [46XX, inv(9)(p12q13), i(21)(q10)] included triplication of the gene for amyloid precursor protein and the Down syndrome critical region. On microscopy, very few gamma-aminobutyric acid-ergic (GABAergic) neurons, in the form of small granular cells, in the cortex and Purkinje cells in the cerebellum were visible. In our previous study, amyloid precursor protein immunoreactivity was first noted in senile plaques at the age of 32?years. In this patient, even though amyloid β immunoreactivity was detected in the cores of senile plaques and diffuse plaques, amyloid precursor protein immunoreactivity was not noted in senile plaques in the frontal cortex. Amyloid precursor protein and its derivative amyloid-β play an important role in the formation of senile plaques and the time course of immunoreactive expression may be related to the pathogenic process of Alzheimer-type dementia.     

Characteristic developmental expression of amyloid beta40, 42 and 43 in patients with Down syndrome

We immunohistochemically studied the expression of beta-amyloid precursor protein (APP), Abeta40, Abeta42, and Abeta43 in the frontal lobes of 20 Down syndrome (DS) patients and 13 controls. The immunoreactivity for each antibody was different in the degree of intensity and the chronological pattern of expression. APP and Abeta43 immunoreactivity was increased in neurons initially, and then Abeta43 and 42 immunoreactivity appeared in diffuse plaques from 32 years of age. APP and Abeta43 were characteristically observed in axons around senile plaques. Finally, Abeta40 immunoreactivity was detected in the cores of senile plaques. This time course of immunoreactive expression may be related to the pathogenetic process of Alzheimer-type dementia in DS, and the axonal damage in senile plaques may lead to the formation of neurofibrillary tangles (NFT) or neuronal death through axonal flow disturbance and accumulation of Abeta43 in cortical neurons.     

APP mutations in the Aβ coding region are associated with abundant cerebral deposition of Aβ38

A?? is the main component of amyloid deposits in Alzheimer disease (AD) and its aggregation into oligomers, protofibrils and fibrils is considered a seminal event in the pathogenesis of AD. A?? with C-terminus at residue 42 is the most abundant species in parenchymal deposits, whereas A?? with C-terminus at residue 40 predominates in the amyloid of the walls of large vessels. A?? peptides with other C-termini have not yet been thoroughly investigated. We analysed A??38 in the brains of patients with A?? deposition linked to sporadic and familial AD, hereditary cerebral haemorrhage with amyloidosis, or Down syndrome. Immunohistochemistry, confocal microscopy, immunoelectron microscopy, immunoprecipitation and the electrophoresis separation of low molecular weight aggregates revealed that A??38 accumulates consistently in the brains of patients carrying APP mutations in the A?? coding region, but was not detected in the patients with APP mutations outside the A?? domain, in the patients with presenilin mutations or in subjects with Down syndrome. In the patients with sporadic AD, A??38 was absent in the senile plaques, but it was detected only in the vessel walls of a small subset of patients with severe cerebral amyloid angiopathy. Our results suggest that APP mutations in the A?? coding region favour A??38 accumulation in the brain and that the molecular mechanisms of A?? deposition in these patients may be different from those active in patients with familial AD associated with other genetic defects and sporadic AD.     

Amyloid β-protein deposition in the leptomeninges and cerebral cortex

To further investigate the process of amyloid β-protein (Aß) deposition, we determined, using sensitive enzyme immunoassays, the levels of Aβ40 and Aβ42 (Aβs) in the soluble and insoluble fractions of the leptomeninges (containing arachnoid mater and leptomeningeal vessels) and cerebral cortices from elderly control subjects showing various stages of Aβ deposition and from patients affected by Alzheimer's disease (AD). In both locations, insoluble Aβ levels were higher by ordersof magnitude than soluble Aβ levels. Soluble Aβ levels. Soluble Aβ levels in cortices were much lower than those in leptomeninges. In insoluble Aβ in the cortex, Aβ42 was by far the predominant species, and Aβ42 in AD cortices was characterized by the highest degree of modifications in the amino terminus. In contrast, this Aβ42 predominance was not observed in insoluble Aβ in the leptomenings, which were found to be able to accumulate Aβs to an extent similar to that in the cortex, on a weight basis. The levels of insoluble Aβ in the leptomeninges or cortex generally correlated with the degree of cerebral amyloid angiopathy or the abundance of senile plaque, respectively. However, the presence of plaque-free cortical samples showing significant levels of insoluble Aβ42 suggests that biochemically detectable Aβ accumulation precedes immunocytochemically detectable Aβ deposition in the cortex.     

Relative efficacies of amyloid β peptide (Aβ) binding proteins in Aβ aggregation

The aggregation of amyloid β peptide (Aβ) into its fibrillar, cross β-pleated configuration is generally viewed as a critical event in the pathophysiology of Alzheimer's disease (AD). A diverse group of molecules, the Aβ binding proteins, has been evaluated for their effects on this process. However, most of these studies have used micromolar or greater reagent concentrations, and their different methods have not permitted quantitative comparisons of the efficacy of different Aβ binding proteins in augmenting or inhibiting aggregation. In the present work we have undertaken a coherent analysis using fluorimetry of thioflavin T-stained experimental solutions. The complement protein C1q, serum amyloid P, and transthyretin significantly enhanced the formation of precipitable, cross β-pleated aggregates in solutions of 800 nM Aβ1–42. Under these same experimental conditions, α₁-antichymotrypsin had no significant effect on the aggregation process, and both the E3 and E4 isoforms of apolipoprotein E were significant inhibitors. There was a non-significant trend toward the E3 isoform exhibiting greater inhibition than the E4 isoform. Of the aggregation-facilitating molecules, C1q was substantially and significantly the most potent. © 1996 Wiley-Liss, Inc.     

Cerebrospinal fluid amyloid β40 is decreased in cerebral amyloid angiopathy

Cerebral amyloid angiopathy is caused by deposition of the amyloid β protein in the cerebral vasculature. In analogy to previous observations in Alzheimer disease, we hypothesized that analysis of amyloid β₄₀ and β₄₂ proteins in the cerebrospinal fluid might serve as a molecular biomarker. We observed strongly decreased cerebrospinal fluid amyloid β₄₀ (p < 0.01 vs controls or Alzheimer disease) and amyloid β₄₂ concentrations (p < 0.001 vs controls and p < 0.05 vs Alzheimer disease) in cerebral amyloid angiopathy patients. The combination of amyloid β₄₂ and total tau discriminated cerebral amyloid angiopathy from controls, with an area under the receiver operator curve of 0.98. Our data are consistent with neuropathological evidence that amyloid β₄₀ as well as amyloid β₄₂ protein are selectively trapped in the cerebral vasculature from interstitial fluid drainage pathways that otherwise transport amyloid β proteins toward the cerebrospinal fluid. Ann Neurol 2009;66:245–249