Immunohistochemical localization of amyloid β-protein with amino-terminal aspartate in the cerebral cortex of patients with Alzheimer's disease

We investigated immunohistochemically the localization of amyloid β-protein (Aβ) with amino-terminal aspartate (N1[D]) in brains of patients with Alzheimer's disease, diffuse Lewy body disease and Down's syndrome. A monoclonal antibody, 4G8, which recognizes the middle portion of Aβ, was used as a reference antibody to label the total Aβ deposits. Double staining with anti-Aβ(N1[D]) and 4G8 revealed that Aβ deposits in the subiculum and the neocortical deep layers often lacked N1[D] immunoreactivity, indicating N-terminal truncation of Aβ in these deposits. Aβ deposits in the neocortical superficial layers and the presubicular parvopyramidal layer always contained Aβ with N1[D]. Such regional as well as laminar differences in the distribution of Aβ beginning at N1[D] suggest that some local factors influence N-terminal processing of Aβ deposited in the brain.     

Fleecy amyloid deposits in the internal layers of the human entorhinal cortex are comprised of N-terminal truncated fragments of Abeta

The deposition of amyloid in the brain is a hallmark of Alzheimer disease (AD). Amyloid deposits consist of accumulations of beta-amyloid (Abeta), which is a 39-43 amino-acid peptide cleaved from the Abeta-protein precursor (APP). Another cleavage product of APP is the P3-peptide, which consists of the amino acids 17-42 of the Abeta-peptide. In order to study the deposition of N-terminal truncated forms of Abeta in the human entorhinal cortex, serial sections from 16 autopsy cases with AD-related pathology were immunostained with antibodies against Abeta1-40, Abeta1-42, Abeta17-23, and Abeta8-17, as well as with the Campbell-Switzer silver impregnation for amyloid. In the external entorhinal layers (pre-beta and pre-gamma), sharply delineated diffuse plaques were seen. They were labeled by silver impregnation and by all Abeta-antibodies used. By comparison, in the internal layers (pri-alpha, pri-beta, and pri-gamma) blurred, ill-defined clouds of amyloid existed, in addition to sharply delineated diffuse plaques. These clouds of amyloid were termed "fleecy amyloid." Immunohistochemically, fleecy amyloid was stained by Abeta17-23 and Abeta1-42 antibodies, but not with antibodies against Abeta8-17 and Abeta1-40. Using the Campbell-Switzer technique, the fleecy amyloid deposits were found to be fine argyrophilic amyloid fibrils. Thus, the internal entorhinal layers are susceptible to a distinct type of amyloid, namely fleecy amyloid. This fleecy amyloid obviously corresponds to N-terminal truncated fragments of Abeta1-42, probably representing the P3-peptide. These N-terminal truncated fragments of Abeta are capable of creating fine fibrillar "amyloid."     

Occurrence of the diffuse amyloid beta-protein (Abeta) deposits with numerous Abeta-containing glial cells in the cerebral cortex of patients with Alzheimer's disease

Diffuse amyloid beta-protein (Abeta) deposits with numerous glial cells containing C-terminal Abeta fragments occur in the cerebral cortex of patients with Alzheimer's disease. By using a panel of antibodies specific for various epitopes in the Abeta peptide, we have investigated the immunohistochemical nature of the diffuse Abeta deposits. The extracellular material contains Abeta with a C-terminus at residue valine40 (Abeta40) as well as residues alanine42/threonine43 (Abeta42). The N-termini include aspartate1, pyroglutamate3, and pyroglutamate11, with pyroglutamate3 being dominant. Microglia and astrocytes in and around these deposits contain intensely staining granules. Most of these granules are negative for antibodies to the N-terminally located sequences of Abeta. These include 6E10 (Abeta1-17), 6F/3D (Abeta8-17), and the N-terminal antibodies specific to aspartate1, pyroglutamate3, and pyroglutamate11. The C-termini of intraglial Abeta are comparable with those of the extracellular deposits. The microglia and astrocytes have quiescent morphology compared with those associated with senile plaques and other lesions such as ischemia. Complement activation in these deposits is not prominent and often below the sensitivity of immunohistochemical detection. Although factors which may cause this type of deposit remain unclear, lack of strong tissue responses suggests that these deposits are a very early stage of Abeta deposition. They were found only inconsistently and were absent in a number of cases examined in this study. Further analysis of these deposits might provide important clues regarding the accumulation and clearance of Abeta in Alzheimer's disease brain.     

Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta

Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.     

Imaging brain amyloid of Alzheimer disease in vivo in transgenic mice with an Abeta peptide radiopharmaceutical.

Abeta 1-40 is a potential peptide radiopharmaceutical that could be used to image the brain Abeta amyloid of Alzheimer disease in vivo, should this peptide be made transportable through the blood-brain barrier in vivo. The blood-brain barrier transport of [ 125 I]-Abeta 1-40 in a transgenic mouse model was enabled by conjugation to the rat 8D3 monoclonal antibody to the mouse transferrin receptor. The Abeta 1-40 -8D3 conjugate is a bifunctional molecule that binds the blood-brain barrier TfR and undergoes transport into brain and binds the Abeta amyloid plaques of Alzheimer disease. App SW /Psen1 double-transgenic and littermate control mice were administered either unconjugated Abeta 1-40 or the Abeta 1-40 -8D3 conjugate intravenously, and brain scans were obtained 6 hours later. Immunocytochemical analysis showed abundant Abeta immunoreactive plaques in the brains of the App SW /Psen1 transgenic mice and there was a selective retention of radioactivity in the brains of these mice at 6 hours after intravenous administration of the conjugate. In contrast, there was no selective sequestration either of the conjugate in control littermate mouse brain or of unconjugated Abeta 1-40 in transgenic mouse brain. In conclusion, the results show that it is possible to image the Abeta amyloid burden in the brain in vivo with an amyloid imaging agent, provided the molecule is conjugated to a blood-brain barrier drug-targeting system.     

Aβ species,including IsoAsp23 Aβ, in Iowa-type familial cerebral amyloid angiopathy

Mutations within the Abeta sequence of the AbetaPP gene are associated with familial forms of cerebral amyloid angiopathy (CAA). One mutation, Abeta D23N, was identified in a family in Iowa with a clinical history of early-onset dementia. We analyzed the pattern of Abeta deposition in the hippocampus of an individual with Iowa CAA. In addition to strong amyloid angiopathy, we found unusual diffuse Abeta deposits in the CA4, and in the parenchyma near amyloid-laden vessels. ELISA of cortical brain extracts showed that Abeta40 was nearly 20-fold higher than Abeta42, in both soluble and insoluble fractions. We identified an Abeta antibody that recognized wild-type Abeta but not Iowa Abeta. With this antibody, we found that wild-type Abeta was present in the Abeta deposits, but limited to the strongest deposits in the cerebrovasculature. Previous in vitro studies suggested that the presence of an asparagine at position 23 of Abeta favored formation of an isoAsp residue, which was associated with increased Abeta fibrillogenesis. Using isoAsp-specific antibodies in immunohistochemical studies, we examined the distribution of isoAsp Abeta in the Iowa brain. IsoAsp7 Abeta was present in both the parenchymal and vascular deposits, whereas isoAsp23 Abeta was present only in vascular deposits. These data suggest that alteration of Abeta Asn23 to isoAsp may be an important determinant in the deposition of Abeta in cerebral blood vessels.     

Beta-amyloid racemized at the Ser26 residue in the brains of patients with Alzheimer disease: implications in the pathogenesis of Alzheimer disease

Oligomeric and fibrillar beta-amyloid (Abeta) may be toxic in Alzheimer disease (AD), especially after post-translation modification cumulative over time. Racemization of Ser and Asp residues of Abeta in senile plaques (SPs) occurs as an age-dependent process in AD. We previously reported that Abeta1-40 racemized at Ser26 is soluble and susceptible to proteolysis yielding toxic [D-Ser26]Abeta25-35/40 fragments in vitro and in vivo. Here, we focus on the localization of racemized Ser26 residues in AD brains within the limbic system, the earliest site of AD histopathology. We developed antisera (20.1 and 22.7). each with epitopes within [D-Ser26]Abeta25-40. Two forms of truncated [D-Ser26]Abeta were detected either in SPs or within neurons in all 11 AD-affected brains, but not in age-matched controls. [D-Ser26]Abeta25/26-35 (detected by 20.1) was localized to plaque cores, extracellular neurofibrillary "ghost" tangles and vascular amyloid deposits. In contrast, [D-Ser26]Abeta25-40 (detected by 22.7) was observed in most neurons containing intracellular neurofibrillary tangles, but not in SPs. These results suggest [D-Ser26]Abeta]1-40, formed during aging, becomes soluble and diffuses from SPs. It is then proteolyzed to [D-Ser26]Abeta25-35/40, which is toxic and may contribute to the neurodegeneration. This hypothesis may explain the long lag between SP formation and neurofibrillary degeneration in AD brains.     

Commentary: Abeta N- Terminal Isoforms: Critical contributors in the course of AD pathophysiology

The assessment of protein or amino acid variations across evolution allows one to glean divergent features of disease-specific pathology. Within the Alzheimer's disease (AD) literature, extensive differences in Abeta processing across cell lines and evolution have clearly been observed. In the recent past, increased levels of amyloid beta Abeta1-42 have been heralded to be what distinguishes whether one is prone to the development of AD [59]. However, observations in naturally occurring, non-transgenic animals which display a great deal of parenchymal Abeta1-42 (Abeta found within extracellular plaque deposits) and a complete lack ofbeta1-40 within these same Abeta1-42 plaques raise the issue of whether Abetax-42 (Abeta that is truncated or modified at the N- terminus), rather than Abeta1-42, is instead the critical mediator of Abeta production and pathogenesis [47,49]. Distinct ratios of Abeta N-terminal variants (i.e. Abeta1-x, Abeta3-x, Abeta11-x, beta17-x) have been assessed in human amyloid plaques [18,21,40,41,42,47,48,49,52]. Moreover, ratios of specific Abeta N-terminal variants separate naturally occurring, non-transgenic animals which develop abundant levels of Abetax-42 and not Abetax-40 from human AD participants who harbor plaques that contain both the Abetax-42 and Abetax-40 variants [49]. Next, Teller and colleagues have demonstrated the presence of N-terminal truncated soluble 3kD (likely Abeta17-x) and 3.7kD peptides (in addition to 4kD Abeta) well before the appearance of amyloid plaques in Down Syndrome brain [51], indicating an early contribution of thebeta N-terminus to the formation of amyloid pathology. Additional critical facts concerning the major contribution of the Abeta N-terminus in AD pathogenesis include observations which support thatbeta generated by rodent neurons is predominantly truncated at Abeta11-x [13], the major form of APP C-terminal fragments in mice lacking functional PS1 is AbetaPP11-98 [9], beta11-x expression is increased as a function of BACE expression [55], and an interrelationship between presenilin-1 mutations and increased levels of N-terminally truncatedbeta [40]. This commentary highlights current understanding and potential biochemical, pathological, and cell biological contributions of Abeta N-terminal variants implicated during the course of AD pathogenesis. Although the amyloid beta protein precursor (AbetaPP) gene and Abeta are highly conserved across mammalian species, there are species-specific differences. For instance, the primate, guinea pig, canine, and polar bear share an identical Abeta sequence to that observed in human brain while the rat displays a distinct amino acid sequence with substitutions at residues 5 (Arg), 10 (Tyr), and 13 (His) [24,37]. All of these mammals generate Abeta1-42 via cleavage by at least two enzymes, beta (beta-) secretase and gamma (gamma-) secretase (Fig. 1). The enzyme that liberates the N- terminus of the Abeta peptide ('beta-secretase') is also termed BACE (beta-site AbetaPP cleaving enzyme) [55]. Cathepsin D, which accumulates within AD neurons [15], also cleaves at the N-terminal side of the first aspartate residue of amyloid beta [2].beta-secretase activity is necessary in order to initiate 4kD beta1-x formation by cleaving AbetaPP at the N-terminus and results in the release of a soluble 100kD AbetaPP N- terminal fragment and a 12kD membrane bound C-terminal fragment (C99/C100) [55]. The carboxyl-terminus of the Abetapeptide is liberated through cleavage by the enzyme termed gamma-secretase. In the past, potential AD therapeutic strategies have mainly been geared towards gamma-secretase inhibition. However, such strategies alone no longer appear sound as it is clear that the AbetaPP C99/C100 fragment itself, which requires beta-, but not gamma-, secretase cleavage for generation and includes the entire Abeta peptide, is neurotoxic when evaluated in cultured cells [12,30,34]. Thus, gamma-secretase inhibition alone would not preclude the generation of the neurotoxic C99/C100 fragment.     

The F(ab)'2 fragment of an Abeta-specific monoclonal antibody reduces Abeta deposits in the brain

This work examines whether administering the F(ab' )2 fragment of an IgG1 monoclonal antibody (mAb) targeting the N-terminal 1-13 amino acids of the beta-amyloid peptide (Abeta mAb) reduces amyloid deposition in Alzheimer's disease (AD). The F(ab')2 fragment was injected intraperitoneally or intracranially into Tg2576 mice, a murine model of human AD. Both routes of administration significantly reduced Abeta plaque formation in the brain, as determined immunohistochemically and by monitoring levels of Abeta1-40 and Abeta1-42 peptide. Use of the F(ab')2 fragment significantly reduced phagocytic infiltration in the CNS when compared to intact mAb. Since IgG1 Abs do not fix complement, these findings suggest that effective in vivo clearance of amyloid deposits can be achieved without stimulation of FcR-reactive phagocytes or activation of the complement cascade.     

Absence of beta-amyloid deposits after immunization in Alzheimer disease with Lewy body dementia

OBJECTIVE: To describe the neuropathological and biochemical findings of the brain examination of a patient enrolled in the AN-1792(QS-21) trial with an initial clinical diagnosis of Alzheimer disease (AD), in whom Lewy body variant was thereafter clinically diagnosed. DESIGN: A case report. SETTING: University memory clinic. Patient A 74-year-old woman with clinical features of probable AD. Intervention The patient received 2 injections of 225 mug of AN-1792 (beta-amyloid [Abeta]) plus 50 mug of the adjuvant QS-21 at an interval of 4 weeks. The patient was an antibody responder with an IgG anti-AN-1792 antibody titer exceeding 10 000 and an IgM titer exceeding 3500. Maximum serum anti-Abeta titers were reached in 4.7 months. During the 3 following years, while the Mini-Mental State Examination score remained globally stable despite several confusional episodes, she developed clinical features of dementia with Lewy bodies. The patient died 34 months postimmunization. An autopsy was performed. MAIN OUTCOME MEASURES: Neuropathological and biochemical examination of the brain using standardized evaluation for tau, beta-amyloid, and synuclein deposits. RESULTS: Neither neuropathological nor biochemical examinations showed amyloid deposit in the brain of this immunized patient. For tau deposition, Braak stage was IV/VI, and the Western blot analysis score was 9c/10. The neuropathological semiquantitative score for alpha-synuclein aggregation was 4. There was no inflammation. These results were compared with those of an age-matched patient with AD and a control devoid of any neurological disease. CONCLUSION: In this Lewy body variant case, with globally stable functional and cognitive features, Abeta immunization resulted in a significant clearance of amyloid deposits, with remaining tau and synuclein pathological features in the brain. Patients with a Lewy body variant of AD should not be excluded from enrollment in Abeta-immunization trials.     

Immunization with amyloid-beta using GM-CSF and IL-4 reduces amyloid burden and alters plaque morphology

Alzheimer's disease is a neurodegenerative disease characterized by the formation of plaques composed of amyloid-beta (Abeta) peptide. Vaccination of transgenic models reduced Abeta deposition and protected these mice from memory deficits. However, Phase IIa clinical trials were halted prematurely. Since several investigators have suggested that the adjuvant QS-21 may have promoted the inflammatory response we investigated alternate adjuvants. Our results suggest that GM-CSF and IL-4 drive an attenuated Th2 response to immunization with A, including moderate antibody titers. These antibodies decreased plaque load in transgenic mice by as much as 43%. Total Abeta(40) and Abeta(42) levels were reduced in Abeta/GM-CSF/IL-4 animals, while plasma Abeta(40) and Abeta(42) were increased. Reductions in Abeta resulted in altered plaque morphology. Immunohistochemical analyses show fewer compact deposits composed primarily of Abeta(40) in treated mice, with a concomitant reduction in plaque-associated microgliosis. Thus, GM-CSF and IL-4 are effective adjuvants for Abeta immunotherapy.     

Imaging of amyloid beta in Alzheimer's disease with 18F-BAY94-9172, a novel PET tracer: proof of mechanism

BACKGROUND: Amyloid-beta (Abeta) plaque formation is a hallmark of Alzheimer's disease (AD) and precedes the onset of dementia. Abeta imaging should allow earlier diagnosis, but clinical application is hindered by the short decay half-life of current Abeta-specific ligands. (18)F-BAY94-9172 is an Abeta ligand that, due to the half-life of (18)F, is suitable for clinical use. We thus studied the effectiveness of this ligand in identifying patients with AD. METHODS: 15 patients with mild AD, 15 healthy elderly controls, and five individuals with frontotemporal lobar degeneration (FTLD) were studied. (18)F-BAY94-9172 binding was quantified by use of the standardised uptake value ratio (SUVR), which was calculated for the neocortex by use of the cerebellum as reference region. SUVR images were visually rated as normal or AD. FINDINGS: (18)F-BAY94-9172 binding matched the reported post-mortem distribution of Abeta plaques. All AD patients showed widespread neocortical binding, which was greater in the precuneus/posterior cingulate and frontal cortex than in the lateral temporal and parietal cortex. There was relative sparing of sensorimotor, occipital, and medial temporal cortex. Healthy controls and FTLD patients showed only white-matter binding, although three controls and one FTLD patient had mild uptake in frontal and precuneus cortex. At 90-120 min after injection, higher neocortical SUVR was observed in AD patients (2.0 [SD 0.3]) than in healthy controls (1.3 [SD 0.2]; p<0.0001) or FTLD patients (1.2 [SD 0.2]; p=0.009). Visual interpretation was 100% sensitive and 90% specific for detection of AD. INTERPRETATION: (18)F-BAY94-9172 PET discriminates between AD and FTLD or healthy controls and might facilitate integration of Abeta imaging into clinical practice.     

Alzheimer disease: mouse models pave the way for therapeutic opportunities

Research into the molecular mechanisms of Alzheimer disease (AD) continues to clarify important issues in aberrant protein processing while seeking to identify therapeutic targets. Mutations of genes on chromosomes 1, 14 (presenilins 1 and 2), and 21 (the amyloid-beta [Abeta] amyloid precursor protein [APP]) cause the familial forms of AD that often begin before age 65. An allelic polymorphism on chromosome 19 (apolipoprotein E ) affects the age of onset of the more common forms of sporadic AD. Multiple studies in transgenic mice provide strong evidence to support the view that Abeta amyloid formation is an early and critical pathogenic event: mice expressing pathogenic human APP mutations develop Abeta deposits; coexpression of mutant presenilin genes accelerates the rate of Abeta deposition; and apolipoprotein E plays a role in this process. Thus, the 3 established genetic causes or risk factors for AD affect Abeta deposition. The fact that elevation of the Abeta42/Abeta40 ratio (differing only in 2 amino acids in length) is also linked to amyloid deposition in the APP mice and is temporally linked to cognitive impairment suggests that Abeta42 may be a principal inducing factor of AD. The exact sequence of events is still unknown, but the transgenic models generated so far have shown their usefulness in clarifying this complex part of the pathology. The continuing progress in elucidation of the molecular pathogenesis of AD suggests a range of rational pharmacological interventions for this disorder. The most promising strategy involves the development of approaches to retard, halt, or prevent Abeta-mediated disease progression, and these can now be tested in transgenic animals.     

The metallobiology of Alzheimer's disease

The cause of Alzheimer's disease (AD) is closely related to the aggregation of a normal protein, beta-amyloid (Abeta), within the neocortex. Recently, evidence has been gathered to suggest that Abeta precipitation and toxicity in AD are caused by abnormal interactions with neocortical metal ions, especially Zn, Cu and Fe. However, Abeta might also participate in normal metal-ion homeostasis. An inevitable, age-dependent rise in brain Cu and Fe might hypermetallate the Abeta peptide, causing the catalysis of H(2)O(2) production that mediates the toxicity and auto-oxidation of Abeta. The greater incidence of AD in females could be due to greater constitutive activity of the synaptic Zn transporter ZnT3, and attenuated binding of metal ions to the rodent homologue of Abeta might explain why these animals are spared Alzheimer's pathology. Compounds that interdict metal-ion binding to Abeta dissolve brain deposits in vitro and one such compound, clioquinol, inhibits Abeta deposition in the Tg2576 mouse model for AD and could be useful clinically. These insights could also apply to other degenerative disorders in which metal-ion-protein interactions have been implicated.     

Native human neocortex release-regulating dopamine D2 type autoreceptors are dopamine D2 subtype.

Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 nM-10 microM) inhibited tritium release with a calculated EC50 of 17 nM and a maximal inhibition of approximately 75% reached at 1 microM. In the presence of the D2/D3 receptor antagonist (-)-sulpiride (0.1 and 1 microM), the concentration-response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14-8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor-selective antagonist [7-N,N-dipropylamino-5,6,7, 8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane] (S 14297) and the D4 receptor-selective antagonist 3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine (L-745,870) (0.01-1 microM in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype.     

Niemann-Pick type C disease: accelerated neurofibrillary tangle formation and amyloid beta deposition associated with apolipoprotein E epsilon 4 homozygosity

Niemann-Pick type C disease is a neurovisceral storage disorder. Neurofibrillary tangles similar to those in Alzheimer's disease have been reported in most juvenile/adult patients without amyloid beta protein (Abeta) deposits. Recently, we found deposits of Abeta in the form of diffuse plaques in three (31- and 32-year-old sisters and a 37-year-old man) of nine Niemann-Pick type C disease patients, who presented with most severe tauopathy and with numerous neurofibrillary tangles. Abeta deposits were not detected in any of the control brains of patients younger than age 42 years. These three patients with Abeta deposit all were homozygotes of apolipoprotein E epsilon 4. Our study suggested that NPC1 gene mutations combined with homozygosity of apolipoprotein E epsilon 4 alleles could manifest neuropathology similar to that of Alzheimer's disease. Investigation of these patients may provide an important clue for understanding the pathogenesis of Alzheimer's disease.     

A novel monoclonal antibody specific for the amino-truncated beta-amyloid Abeta5-40/42 produced from caspase-cleaved amyloid precursor protein

Deposition of beta-amyloid peptide (Abeta) as senile plaques and amyloid angiopathy are the major neuropathological features of Alzheimer's disease (AD). Heterogeneity is observed in the N- and C-termini of the deposited Abeta species. Recent evidence implicates caspase activation and apoptosis in AD neurodegeneration. We previously reported that a distinct N-terminally truncated Abeta species, Abeta5-40/42 is preferentially produced from the caspase-cleaved form of amyloid precursor protein (APP) lacking its C-terminal 31 amino acids and that it is deposited in AD brain tissues. Here, we generated a novel monoclonal antibody specific to the N-terminal end of Abeta5-40/42. Western blotting confirmed that this antibody recognizes Abeta5-40 but not Abeta1-40. We also showed that the antibody is able to immunoprecipitate Abeta5-40 but not Abeta1-40. Immunoprecipitation with the antibody followed by mass spectrometric analysis further detected Abeta5-40 in the conditioned media from neuroblastoma cells expressing the caspase-cleaved APP. The antibody reacted weakly with Abeta derived from AD brains. These results suggest that our novel monoclonal antibody is useful for detecting the N-terminally truncated Abeta produced in conjunction with caspase activation.     

Novel presenilin-1 mutation with widespread cortical amyloid deposition but limited cerebral amyloid angiopathy

OBJECTIVE: To clarify the phenotypic heterogeneity in deposition of amyloid beta (Abeta) in the parenchyma and in cerebral vessels of the brains of the patients having presenilin-1 (PS1) mutations. Mutations in PS1 induce increased production of Abeta42(43), resulting in an enhanced overall deposition of Abeta protein within the cerebral cortex. METHODS: Sequence analysis of the PS1 gene of DNA from patients with early onset Alzheimer's disease, and immunostaining of brain tissues by end specific monoclonal antibodies against Abeta. RESULTS: Sequence analysis disclosed a novel mutation (N405S) in the PS1 gene in a Japanese patient with early-onset Alzheimer's disease. Postmortem examination of one patient with N405S showed limited cerebral amyloid angiopathy, whereas postmortem examination of another Japanese patient with Alzheimer's disease with the E184D mutation disclosed severe cerebral amyloid angiopathy. The brains of both patients showed widespread neuritic plaques, neurofibrillary tangles, and neuronal loss. Immunostaining showed that Abeta42 was predominant over Abeta40 in neuritic plaques in both patients, whereas Abeta40 was found to be predominant over Abeta42 in cerebral amyloid angiopathy in the patient with E184D. However, most cortical vessels of the patient with N405S were not reactive with either of the antibodies. CONCLUSION: The N405S mutation of PS1 is a major determinant of cortical Abeta deposition but not cerebral amyloid angiopathy in Alzheimer's disease.     

Aggregation and catabolism of disease-associated intra-Abeta mutations: reduced proteolysis of AbetaA21G by neprilysin

Five point mutations within the amyloid beta-protein (Abeta) sequence of the APP gene are associated with hereditary diseases which are similar or identical to Alzheimer's disease and encode: the A21G (Flemish), E22G (Arctic), E22K (Italian), E22Q (Dutch) and the D23N (Iowa) amino acid substitutions. Although a substantial body of data exists on the effects of these mutations on Abeta production, whether or not intra-Abeta mutations alter degradation and how this relates to their aggregation state remain unclear. Here we report that the E22G, E22Q and the D23N substitutions significantly increase fibril nucleation and extension, whereas the E22K substitution exhibits only an increased rate of extension and the A21G substitution actually causes a decrease in the extension rate. These substantial differences in aggregation together with our observation that aggregated wild type Abeta(1-40) was much less well degraded than monomeric wild type Abeta(1-40), prompted us to assess whether or not disease-associated intra-Abeta mutations alter proteolysis independent of their effects on aggregation. Neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin play a major role in Abeta catabolism, therefore we compared the ability of these enzymes to degrade wild type and mutant monomeric Abeta peptides. Experiments investigating proteolysis revealed that all monomeric peptides are degraded similarly by IDE and plasmin, but that the Flemish peptide was degraded significantly more slowly by NEP than wild type Abeta or any of the other mutant peptides. This finding suggests that resistance to NEP-mediated proteolysis may underlie the pathogenicity associated with the A21G mutation.     

Laminar specific loss of isocortical presenilin 1 immunoreactivity in Alzheimer's disease. Correlations with the amyloid load and the density of tau-positive neurofibrillary tangles

Presenilin 1 has been shown to be mutated in a high proportion of cases of familial Alzheimer's disease. Immunoreactive epitopes of the protein have been found mainly in neurones devoid of neurofibrillary tangles - an observation that has led to the conclusion that presenilin 1 could have a protective role. In this study, the relationship between deposits of Abeta peptide (both the 40 and 42 isoforms), tau positive neurofibrillary tangles and presenilin 1-positive neuronal profiles were analysed in three cases of presenilin 1 mutation, four cases of sporadic Alzheimer's disease and five controls. Immunohistochemistry was performed in a sample from the supramarginal gyrus. The proportion of volume occupied by the Abeta1-40 and Abeta1-42 deposits (amyloid load) was evaluated by a point-counting technique. Tau-positive neurofibrillary tangles, and presenilin 1-positive neuronal profiles were directly counted. The location of the lesions in the thickness of the cortex was recorded. The density of PS1-positive neuronal profiles in Alzheimer's disease cases was lower than in the controls. The deficit was significant only in the upper layers of the cortex. The density of presenilin 1 neuronal profiles was negatively correlated with Abeta1-40 and Abeta1-42 loads, and with the density of tau-positive neurofibrillary tangles. Multivariate analysis showed that the Abeta1-42 load was the best determinant of the decrease in presenilin 1-positive neuronal profiles. Presenilin 1-positive neurones appear to be lost rather than protected in the course of Alzheimer disease.