LRP and senile plaques in Alzheimer's disease: colocalization with apolipoprotein E and with activated astrocytes

The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer's disease (AD). It is suggested to play an important role in the balance between amyloid beta (Abeta) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Abeta. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Abeta deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Abeta. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Abeta/ApoE complexes.     

Increased senile plaques without microglia in Alzheimer's disease

Summary To clarify the association of microglia with senile plaques, the brains from 13 patients with Alzheimer's disease (AD) and 23 nondemented aged controls were investigated immunohistochemically by a double-labeling method using anti--protein antiserum and anti-ferritin antibody, which is a recently reported microglia marker. In addition, a quantitative analysis was performed. The senile plaques which appeared initially in the nondemented aged controls consisted of a diffuse type without any amyloid cores and these were found in the group aged 50–59 years. The great majority of them were found to contain no ferritin-positive microglia. The number and proportion (percentage) of microglia-containing diffuse plaques increased with age. Classical and compact plaques began to appear in the brains of the group aged 70 years and over, and practically all of them contained microglia. These results suggest that microglia are not associated with initial plaque formation, but correlate with amyloid core formation. In AD, the most prominent feature was that the diffuse plaques, which contained either no or only a few ferritin-positive microglia, increased markedly.Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease

Abstract It has been shown that human monocytes express monocyte chemoattractant protein-1 (MCP-1), an inflammatry factor, in response to non-fibrillar β-amyloid protein. Reactive microglia and inflammatory factors were reported to be present in β-amyloid deposits (senile plaques) in Alzheimer's disease, suggesting the presence of MCP-1 in senile plaques. To address this issue, we examined MCP-1-immunoreactivity in senile plaques using a mouse monoclonal anti-MCP-1 antibody. Monocyte chemoattractant protein-1 was found immunohistochemically in mature senile plaques and reactive microglia but not in immature senile plaques of brain tissues from five patients with Alzheimer's disease. These findings suggest that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques.     

Morphological characteristics of senile plaques in familial Alzheimer's disease

Summary The distribution and morphology of senile plaques (SPs) in the cerebral cortices and subcortical nuclei of six cases of familial Alzheimer's disease (AD) were examined using the Methenamine-Bodian method and compared with those of sporadic AD cases. SPs were grouped into three types according to their morphology. SP types were generally constant at each anatomical site in all of the cases. The SPs of familial cases, however, had a greater tendency to fuse together than those of sporadic cases, especially in the cingulate cortex, presubiculum and striatum. This tendency was more evident in cases with severe amyloid angiopathy. Here it appeared that a SP type corresponding to diffuse plaques at least in part, might be formed by transformation from another type. In the globus pallidus, all the familial cases had many compact-like plaques which appeared to be derived from drusige Entartung of the capillaries. Furthermore, the regional proportion of two types of SPs occuring in this nucleus varied along its anteroposterior axis. These findings may be the histological hallmarks of atypical AD rather than familial AD.     

Prion protein expression in senile plaques in Alzheimer's disease

Prion protein (PrPC) is a glycolipid-anchored cell membrane sialoglycoprotein that localises in presynaptic membranes. Since synapses are vulnerable to Alzheimer's disease (AD), the present study examines PrPC expression in senile plaques, one of the major structural abnormalities in AD, by single- and double-labelling immunohistochemistry. Punctate PrPC immunoreactivity is found in diffuse plaques, whereas isolated large coarse PrPC-positive granules reminiscent of dystrophic neurites are observed in neuritic plaques. Finally, PrPC deposition also occurs as dense filamentous and amorphous precipitates in amyloid cores of senile plaques, but not in the walls of blood vessels with amyloid angiopathy. In contrast to PrPC, betaA4-amyloid immunoreactivity is preserved and even enhanced following incubation of the tissue sections with proteinase K prior to immunohistochemistry, thus indicating no PrPC and betaA4-amyloid cross-reactivity in dense amyloid cores of senile plaques. Punctate PrPC deposition in diffuse plaques is similar to that of synaptophysin, a synaptic vesicle-associated protein, as already reported in other studies. Immunoprecipitation, electrophoresis and Western blot studies have shown that synaptophysin, amyloid precursor protein (APP) and betaA4 do not co-precipitate with PrP. These results suggest that synaptophysin, APP and betaA4 are likely not bound to PrP. PrPC accumulation in betaA4-amyloid dense cores may be the consequence of the release of PrP into the extracellular space. Whether PrPC accumulation in the extracellular space is the result of impaired endocytosis and subsequent hydrolysis in the endosomal compartment, in contrast to normal degradation of PrPC, resulting from or occurring in parallel to abnormal APP degradation, deserves further study.     

Fibrous astrocytes in Alzheimer's disease and senile dementia of Alzheimer's type

In four patients with Alzheimer's disease (AD), on patient with senile dementia of Alzheimer's type (SDAT) and five age-matched controls, occipital cortex, frontal cortex, and hippocampus were evaluated for the distribution of fibrous astrocytes (FA), using peroxidase-anti-peroxidase for glial fibrillary acidic protein (GFAP). FA, neuronal cells, neurofibrillary tangles (NFT), and senile plaques (SP) have been quantified in the occipital cortex. In AD and SDAT there was a significant increase in the number of FA in the molecular layer as well as in the other layers of the cortex. No correlation was found between the increase in FA and the number of neurons, NFT or SP.The GFAP positivity was most pronounced around small blood vessels. Electron-microscopic studies of four cortical biopsies of AD revealed dense perivascular gliosis in 48.8% of the capillaries examined as opposed to 17.8% of capillaries in three controls without dementia. The significance of increased perivascular gliosis in AD and SDAT is unknown. It may be related to a defect in the blood-brain barrier.     

Electron-microscopical study on senile plaques in Alzheimer's disease.

The cerebral cortex taken post mortem from a case of Alzheimer's disease was examined especially with regard to the relation between blood vessels and senile plaques. Many senile plaques had central cores, composed of such matter as degenerated blood vessels and basement membranes with abundant amyloid fibrils. The components of senile plaques seemed to be degenerated neuronal and glial tissue compressed by developed basement membranes and amyloid fibrils. From this we would like to emphasize that senile plaques seem to be caused by amyloid fibrils and degenerated capillaries.     

Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques

The sequestration of RNA in Alzheimer's disease (AD) senile plaques (SPs) and the production of intraneuronal amyloid-β peptides (Aβ) prompted analysis of the mRNA profile in single immunocytochemically identified SPs in sections of AD hippocampus. By using amplified RNA expression profiling, polymerase chain reaction, and in situ hybridization, we assessed the presence and abundance of 51 mRNAs that encode proteins implicated in the pathogenesis of AD. The mRNAs in SPs were compared with those in individual CA1 neurons and the surrounding neuropil of control subjects. The remarkable demonstration here, that neuronal mRNAs predominate in SPs, implies that these mRNAs are nonproteinaceous components of SPs, and, moreover, that mRNAs may interact with Aβ protein and that SPs form at sites where neurons degenerate in the AD brain. Ann Neurol 1999;45:174–181     

Sequestration of RNA in Alzheimer's disease neurofibrillary tangles and senile plaques

The polypeptide composition of neurofibrillary tangles (NFTs) and senile plaques (SPs) has been characterized extensively within the Alzheimer's disease (AD) brain. Because few data exist on the nonproteinaceous components of these lesions, we sought to determine if NFTs, neuropil threads (NTs), and SPs contain RNA species. To acoomplish this, acridine orange (AO) histofluorescence was employed, alone or in combination with thioflavine S (TS) staining and immunohistochemistry to identify RNAs in paraffin-embedded tissue sections of hippocampus and entorhinal cortex. Postmortem brain samples came from 32 subjets including AD and elderly Down's syndrome (DS) patints, age-matched normal controls, and non-AD diseased controls. AO stained the cytoplasm of normal hippocampal and entorhinal neurons in all of the cases, while NFTs, NTs, and SPs were AO-positive in the same regions of AD and DS brains. Cytoplasmic AO histofluorescence was abolished with RNase, but not DNase or proteinase K, indicating the relative specificity of AO for RNA species. Quantitative analysis of double-labeled sections demonstrated that approximately 80% of TS-positive NFTs also were AO-positive, whereas approximately 55% of TS-stained SPs contained AO labeling. These novel observations demosntrate the presence of RNAs in NFTs, NTs, and SPs.     

Regression stage senile plaques in the natural course of Alzheimer's disease

Resolution process of cerebroparenchymal amyloid beta-protein (Abeta) deposition has become of increasing interest in the light of recent advance in the Abeta-vaccination therapy for Alzheimer's disease (AD). However, the neuropathological features of degraded and disappearing senile plaque remain poorly characterized, especially in the natural course of the disease. To clarify the natural removal processes of Abeta burden in the brain with AD, we devised a triple-step staining method: Bodian for dystrophic neurites, anti-glial fibrillary acidic protein for astrocytes, and anti-Abeta. We thus examined 24 autopsied AD brains. A novel form of senile plaques, termed 'remnant plaques', was identified. Remnant plaques were characterized by mesh-like astroglial fibrils within the entire plaque part, Abeta deposit debris exhibiting weak Abeta immunoreactivity, and only a few slender dystrophic neurites. In remnant plaques, amyloid burden was apparently decreased. The density of remnant plaques increased significantly with disease duration. Dual-labelling immunohistochemistry revealed many Abeta-immunoreactive granules in astrocytes and a modest number in microglia, both of which accumulated in senile plaques. We consider amyloid deposits of diffuse and neuritic plaques to be shredded by astrocytic processes from the marginal zone of plaques, and to gradually disintegrate into smaller compartments. Cerebroparenchymal Abeta deposits undergo degradation. After a long-standing resolution process, diffuse and neuritic plaques may finally proceed to remnant plaques. Astrocytes are actively engaged in the natural Abeta clearance mechanism in advanced stage AD brains, which may provide clues for developing new therapeutic strategies for AD.     

Presence of calpain II immunoreactivity in senile plaques in Alzheimer's disease.

Calpains are calcium-dependent neutral cysteine proteinases. We utilized a specific anti-calpain II antibody to examine immunohistochemically whether calpain II is associated with pathological changes in Alzheimer's disease (AD). Calpain II was mainly expressed in the neurons in control human brains. In AD brains, intense immunoreactivity was present in the dystrophic neurites of senile plaques. These results suggest that calpain II is involved in the pathogenesis of AD.     

Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease

Reactive astrogliosis, a complex process characterized by cell hypertrophy and upregulation of components of intermediate filaments, is a common feature in brains of Alzheimer's patients. Reactive astrocytes are found in close association with neuritic plaques; however, the precise role of these glial cells in disease pathogenesis is unknown. In this study, using immunohistochemical techniques and light and electron microscopy, we report that plaque‐associated reactive astrocytes enwrap, engulf and may digest presynaptic dystrophies in the hippocampus of amyloid precursor protein/presenilin‐1 (APP/PS1) mice. Microglia, the brain phagocytic population, was apparently not engaged in this clearance. Phagocytic reactive astrocytes were present in 35% and 67% of amyloid plaques at 6 and 12 months of age, respectively. The proportion of engulfed dystrophic neurites was low, around 7% of total dystrophies around plaques at both ages. This fact, along with the accumulation of dystrophic neurites during disease course, suggests that the efficiency of the astrocyte phagocytic process might be limited or impaired. Reactive astrocytes surrounding and engulfing dystrophic neurites were also detected in the hippocampus of Alzheimer's patients by confocal and ultrastructural analysis. We posit that the phagocytic activity of reactive astrocytes might contribute to clear dysfunctional synapses or synaptic debris, thereby restoring impaired neural circuits and reducing the inflammatory impact of damaged neuronal parts and/or limiting the amyloid pathology. Therefore, potentiation of the phagocytic properties of reactive astrocytes may represent a potential therapy in Alzheimer's disease.     

Ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of Alzheimer's disease

Summary Senile plaques are present in the cerebellum of most Alzheimer patients. They are composed of beta-amyloid deposits lacking neurites detectable with immunocytochemistry for neurofilament, tau and paired helical filament proteins. Recent studies, however, have shown that cerebellar plaques usually contain round structures that are reactive with ubiquitin antibodies. In this immunoelectron microscopic study, the nature of these structures is explored. Ubiquitin-positive structures in cerebellar senile plaques were composed of degenerating neurites that contained membranous and vesicular dense bodies, but no paired helical filaments. A minority of the neurites contained finely granular material. Thus, cerebellar plaques are associated with neuritic degeneration, and the neurites in cerebellar plaques resemble dystrophic neurites in senile plaques of non-demented elderly subjects and subjects with non-Alzheimer dementias. They differ from some of the neurites in senile plaques in the neocortex in Alzheimer's disease by the absence of paired helical filaments. These results suggest that the same mechanisms involved in the generation of dystrophic neurites in pathological aging are involved in generating dystrophic neurites in the cerebellum in Alzheimer's discase.Supported by NIH grant: AG06803 and AG1136     

Untangling Alzheimer's disease from fibrous lesions of neurofibrillary tangles and senile plaques

Neuropathological evidence suggests that the two fibril lesions of neurofibrillary tangles (NFT) and senile plaques are the major findings in brain tissue of Alzheimer's disease (AD) and that their occurrence is strongly associated with the symptoms of dementia. Genetic findings have indicated that the pathological molecules from the lesions function as causal agents. There is little evidence, however, to directly indicate that fibril lesions themselves kill neuronal cells in vivo. In spite of such limitations it is important to consider the molecular events involved in AD etiology. In this review of the contribution of Japanese neuropathologists to studies of AD, I will introduce briefly their work and highlight some current topics for consideration on the etiology of AD, and the basis of cell death, and will offer my perspective on outstanding conflicting issues.     

Microangiopathy and the colocalization of heparan sulfate proteoglycan with amyloid in senile plaques of Alzheimer's disease

While the pathogenetic mechanisms responsible for Alzheimer's Disease (AD) remain unknown, blood vessel deformities, thickened vascular basement membrane (VBM), and amyloid fibrils emanating from the VBM all suggest vascular involvement. The present study immunocytochemically localized the VBM constituent heparan sulfate proteoglycan (HSPG), which is said to play a role in filtration of anionic and neutral proteins. In addition, thioflavine S was used to double-label each tissue section for the presence of amyloid. Samples were taken from frontal, temporal and parietal lobes of 8 patients who exhibited the neuropathologic lesions of AD and 6 patients who did not. HSPG immunolabeled the capillary bed in all samples. Tissue from patients with AD, however, exhibited severe microangiopathic changes: ragged and irregular outer capillary walls, both thickened and attenuated capillary diameters, and regionally increased capillary density. In addition, plaque-like extravascular accumulations of HSPG were seen in all patients with AD. These accumulations were found in the vicinity of capillaries, and were commonly colocalized with amyloid. Neither extravascular clouds of HSPG immunoreactivity nor fluorescing accumulations of amyloid were found in non-AD patients. The pattern of HSPG immunostaining confirms: (1) the high incidence of microangiopathy in AD; (2) the close anatomic relationship between plaque constituents and capillaries; and (3) the colocalization of HSPG with extravascular amyloid. The cerebral vasculature, and specifically the VBM, may thus be actively involved in the pathogenesis of AD.     

Melanotransferrin is produced by senile plaque-associated reactive microglia in Alzheimer's disease

Melanotransferrin (MTf), also known as p97, has been localized in capillary endothelial cells of human brain. In Alzheimer's-diseased (AD) brain tissues, reactive microglial cells located in senile plaques exhibit elevated levels of MTf. The localization of the p97 protein may reflect its site of synthesis or could reflect a paracrine site of action. We examined the expression of MTf mRNA by in situ hybridization histochemistry using AD and healthy brain tissues. We also examined normal liver tissues by immunohistochemistry and in situ hybridization. In all the brain tissues examined, capillaries had positive signals for MTf mRNA. In AD tissues, expression of MTf mRNA appeared in reactive microglial cells in the grey matter specifically associated with dense plaques. In liver tissues, immunohistochemistry using anti-p97 antibody demonstrated that sinusoids were positively stained. In addition, in situ hybridization histochemistry revealed that hepatocytes had positive signals. These results suggest that p97 expression in reactive microglial cells are closely related to AD pathology. These results also support the notion that p97, which appears elevated in the cerebral spinal fluid and serum of AD patients, originates in the reactive microglia associated with dense senile plaques. Thus, p97 is a unique cellular hallmark of AD and further suggests that metal transport mechanisms play a role in this disease.     

Expression of monoamine oxidase B activity in astrocytes of senile plaques

Summary Monoamine oxidase (MAO) histochemistry has been performed in brains from patients with dementia of Alzheimer type (DAT) and aged controls. Conspicuous MAO-positive cell clusters were frequently observed in the amygdala, hippocampus, and insular cortex in the brains of DAT. Double staining with glial fibrillary acidic protein immunohistochemistry revealed that the clusterforming MAO-positive cells were astrocytes. Using Bielschowsky's method, Congo red and thioflavin S counterstaining, this astrocytic mass was shown to be associated with senile plaques. By the enzyme inhibition experiment, MAO activity in senile plaques was revealed to be of type B. The present results clearly indicate that MAO-B activity is expressed in fibrillary astrocytes in or around senile plaques, suggesting that these astrocytes metabolize exogenous amines in senile plaques.     

Selective PrP-like protein, doppel immunoreactivity in dystrophic neurites of senile plaques in Alzheimer's disease

Doppel (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene PRNP in several species. The present study examines by immunohistochemistry Dpl expression in brain samples from 10 patients with Alzheimer's disease (AD), three patients with Pick's disease, four patients with Parkinson's disease, eight patients with diffuse Lewy body disease (DLBD), six patients with sporadic Creutzfeldt-Jakob disease (CJD) methionine/methionine at the codon 129, two patients with sporadic CJD methionine/valine at the codon 129 and numerous kuru plaques in the cerebellum, one patient with fatal familial insomnia (FFI), and 10 age-matched controls. In the adult human brain, Dpl immunoreactivity was restricted to scattered granule cells of the cerebellum and scattered small granules in the cerebral cortex. Dpl immunoreactivity was seen around betaA4 amyloid deposits in neuritic plaques, but not in diffuse plaques, AD and the common form of DLBD. Neurofibrillary tangles, Pick bodies and Lewy bodies were not stained with anti-Dpl antibodies. No modifications in Dpl immunoreactivity were observed in CJD excepting those associated with accompanying senile plaques. No Dpl-positive deposits were seen in FFI. Whether Dpl in neuritic plaques may attenuate amyloid-induced oxidative stress and participate in the glial response around amyloid cores is discussed in light of the few available data on Dpl functions.