An immunohistochemical study of β-protein in Alzheimer-type dementia brains

Summary An immunohistochemical study with a polyclonal anti--protein antiserum was performed in order to understand the mechanism of deposition of amyloid fibrils in senile plaques in Alzheimer-type dementia (ATD). Serial cortical sections cut from ATD brains were necessary to investigate the structural correlation between senile plaques and blood vessels. The senile plaques were stained well and a blood vessel or capillary-like structure was found in most of -protein-immunoreactive deposits. These findings may suggest an involvement of blood vessels in the formation of at least some of the amyloid deposits in ATD brains.     

Expression of intercellular adhesion molecule (ICAM)-1 by a subset of astrocytes in Alzheimer disease and some other degenerative neurological disorders

Summary Intercellular adhesion molecule-1 (ICAM-1) was localized immunohistochemically in postmortem brain tissue of Alzheimer's disease (AD), progressive supranuclear palsy, amyotrophic lateral sclerosis, Pick's disease, and controls. In controls, only capillaries were stained for ICAM-1. In affected areas of neurologically disease brains, a subset of reactive astrocytes was also strongly stained. In addition, there were irregular, diffuse patches of positive staining in the tissue matrix. In AD, many of these patches had dense cores which corresponded with senile plaques. Double immunostaining for glial fibrillary acidic protein and ICAM-1 indicated that some reactive astrocytes at the periphery of senile plaques were positive for ICAM-1. Within such plaques, microglial aggregates were stained intensely for leukocyte function-associated antigen-1 (LFA-1), the adhesion molecule for ICAM-1. The LFA-1/ICAM-1 system appears to play an important role in the interaction of astrocytes and microglia in several neurological diseases.Supported by grants from the Foundation for Total Health Promotion (HA), the Sasakawa Research Foundation (HA), the Alzheimer Society of B.C. and the MRC of Canada, as well as donations from individual British Columbians     

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques

CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-μm-thick serial sections were stained with H & E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection. Received: 4 March 1999 / Revised: 9 June 1999 / Accepted: 28 June 1999     

Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis

In this study, we demonstrate that grossly unaffected white matter from secondary progressive multiple sclerosis (SP-MS) patients is heavily citrullinated, as compared to normal white matter from control patients. Citrullination was most pronounced at plaque interfaces and was shown to colocalize with glial fibrillary acidic protein (GFAP)-immunoreactivity using dual color immunofluorescence. In contrast, the plaques themselves weakly stained for citrullinated proteins compared to control white matter and usually contained a blood vessel with surrounding astrocytes that were positive both for citrullinated proteins and GFAP. In SP-MS brain samples, but not in normal brains, long fibers of colocalized GFAP- and citrullinated proteins extended into the gray matter. Increased numbers of astrocytes containing citrullinated proteins and GFAP were also present at the junction between the gray and white matter in SP-MS brains. Western blot analysis of acidic brain proteins from nonplaque-containing white matter showed upregulation of multiple citrullinated GFAP proteins in SP-MS brains as compared to controls. Our results demonstrate that increased amounts of citrullinated GFAP are present in SP-MS brains, but also shows that these proteins are present in areas of MS brains that were grossly normal appearing. These data raise the possibility that citrullination of GFAP contributes to the pathophysiology of MS.     

Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin

To investigate the role of proteolysis in amyloid formation, we studied the localization of the proteolytic enzymes, cathepsin D and cathepsin B, in the prefrontal cerebral cortex and hippocampus of human postmortem brains from patients with Alzheimer's disease and from individuals free of neurological disease. In control and Alzheimer brains, cathepsin immunoreactivity within cells was localized to lysosome-related structures, which were particularly abundant in neuronal perikarya. In Alzheimer brain, cathepsin immunoreactivity was also heavily concentrated extracellularly within senile plaques. Cathepsin immunoreactivity associated with plaques was not confined to lysosomes and was distributed throughout the plaque. Isolated amyloid cores, however, were not immunostained. Cathepsin-laden perikarya of degenerating neurons were frequently seen within senile plaques and, in the more advanced stages of degeneration, cathepsin immunoreactivity was present throughout the cytoplasm. Other identified constituents of senile plaques appeared to be less significant sources of cathepsin immunoreactivity, including astrocytes, degenerating neurites, microglia and macrophages. These results demonstrate that lysosomal proteinases are major constituents of the senile plaque and that degenerating neuronal perikarya are a principal source of the cathepsin immunoreactivity. We propose that the unregulated action of extracellular cathepsins liberated from degenerating neurons may lead to abnormal processing of the amyloid precursor protein and to the formation of amyloid locally within senile plaques in Alzheimer's 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.     

Amyloid-P-component-like immunoreactivity in β/A4-immunoreactive deposits in Alzheimer-type dementia brains

An immunohistochemical study using the mirror-image technique was performed in order to establish whether amyloid P component is involved in the mechanism of deposition of amyloid fibrils in senile plaques (SPs) in Alzheimer-type dementia (ATD). Ninety percent of /A4 protein-immunoreactive SPs were also stained by the anti-amyloid P component immunchistochemistry, and this applied to all of the diffuse, primitive and classical types of /A4 deposits. These findings may suggest an involvement of amyloid P component in the formation of amyloid fibrils in senile plaques in ATD brains.     

The identity of cells expressing MHC class II antigens in normal and pathological human brain

Major histocompatibility complex class II antigen (Ag) expression in human brain was investigated in autopsied human brain tissues, using anti-human class II monoclonal antibodies. In normal brains, class II Ag was usually absent or was low in positivity. When it was found immunohistochemically, it appeared more frequently in the meninges (meningeal macrophages) and the neurohypophysis (pituicytes) than in the cerebral cortex (microglia and perivascular cells). The identity of the latter cell types was confirmed by immunoelectron microscopy. Class II-positive microglial cells were usually present in the cerebral white matter, but in senile brains showing numerous senile plaques, their numbers were increased in the grey matter. In diseased brains, numerous reactive microglia and macrophages containing class II Ag were observed in the affected lesions of neural tissue destruction, neuronal degeneration, and inflammation. Astrocytes, which were identified with an antibody to glial fibrillary acidic protein, did not contain class II Ag, although a small number of reactive astrocytes showed an equivocal class II staining. Staining for class II Ag on cerebral endothelial cells was mostly negative; however, class II Ag was microscopically identified in a case of secondary CNS T-cell lymphoma.     

Does damage of perivascular astrocytes in multiple sclerosis plaques participate in blood-brain barrier permeability?

In six young and two senile MS cases perivascular astrocytes within demyelination lesions were evaluated immunocytochemically. The peroxidase-antiperoxidase method of Sternberger et al. (1970) was used for visualization of glial fibrillary acidic protein (GFAP). In all cases very weak immunoreactivity of perivascular astrocytes was noted. Accumulation of perivascular glial fibers, and infrequently their fragmentation were observed both within active and old demyelination plaques. Clasmatodendrosis, Rosenthal's fibers and prominent regressive changes of astrocyte perikarya were found only in old plaques. A lack of immunoreactivity of perivascular astrocytes was noted within old demyelination lesions. The background of the latter was often immunonegative to GFAP. It is suggested that secondary damage of perivascular astrocytes influences vascular permeability within demyelination lesions including old plaques.     

A variety of cerebral amyloid deposits in the brains of the Alzheimer-type dementia demonstrated byβ protein immunostaining

Summary We studied cerebral amyloid deposits in the hippocampal area immunohistochemically, using antiserum to synthetic 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 protein immunostain. In the patients with Alzheimer-type dementia (ATD) diffuse plaques, typical of this group, were stained with 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 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. protein immunostain with formic acid pretreatment is a useful method for the identification of a variety of senile cerebral amyloid deposits.     

Senile plaques: staining for acetylcholinesterase and A4 protein: a comparative study in the hippocampus and entorhinal cortex

Summary In 20 unselected autopsy cases tissue blocks from the hippocampus with adjacent entorhinal cortex and neocortex were stained for acetylcholinesterase (AChE). From five brains shown to have large numbers of senile plaques tissue, adjacent to that taken for AChE tissue blocks, was embedded in paraffin and sections were immunostained for the A4 protein. The morphological aspects were compared. Equivalent types of plaques and plaque-like structures were observed in the A4- and ACHE-stained sections. On selected tissue blocks from patients with many senile plaques two immediately adjacent cryostat sections were stained, one for AChE and one for A4 protein. The same individual plaques could be identified on the two sections. These findings suggest that high AChE activity is intimately associated with the process of A4 protein formation and accumulation in plaques and that this association already occurs at a very carly stage of plaque formation.     

Down‐regulation of MET in hippocampal neurons of Alzheimer's disease brains

We found that mRNA of MET, the receptor of hepatocyte growth factor (HGF), is significantly decreased in the hippocampus of Alzheimer's disease (AD) patients. Therefore, we tried to determine the cellular component‐dependent changes of MET expressions. In this study, we examined cellular distribution of MET in the cerebral neocortices and hippocampi of 12 AD and 11 normal controls without brain diseases. In normal brains, MET immunoreactivity was observed in the neuronal perikarya and a subpopulation of astrocytes mainly in the subpial layer and white matter. In AD brains, we found marked decline of MET in hippocampal pyramidal neurons and granule cells of dentate gyrus. The decline was more obvious in the pyramidal neurons of the hippocampi than that in the neocortical neurons. In addition, we found strong MET immunostaining in reactive astrocytes, including those near senile plaques. Given the neurotrophic effects of the HGF/MET pathway, this decline may adversely affect neuronal survival in AD cases. Because it has been reported that HGF is also up‐regulated around senile plaques, β‐amyloid deposition might be associated with astrocytosis through the HGF signaling pathway.     

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.     

Alpha-1-antichymotrypsin in human glioblastoma multiforme cells and its relation to GFAP immunostaining.

Biopsies of 20 different patients with glioblastoma multiforme were treated with 0.1% protease and stained for alpha-1-ACT. Simultaneous glial fibrillary acidic protein (GFAP) staining of mirror sections was performed. In four cases no neoplastic cells were stained with alpha-1-ACT. The other 11 cases displayed minute accumulations of immunostained neoplastic cells in spongiotic foci. Some scattered immunostained cells not differentiable from neoplastic cells were noticed. Staining of neoplastic as well as branched intratumoral astrocytes for alpha-1-ACT and GFAP of equal distribution and intensity was detected in one case. The alpha-1-ACT immunostaining of peritumoral reactive astrocytes has been observed in 8 cases, 7 of them, however with a smaller amount of cells, entirely corresponding to the distribution and intensity of GFAP. This might represent the absorption of antigen by degenerating cells.     

Cdk5 and munc-18/p67 co-localization in early stage neurofibrillary tangles-bearing neurons in Alzheimer type dementia brains

Hyperphosphorylation of tau protein occurs during the formation of paired helical filament (PHF) in the brain with Alzheimer's disease. As previously reported, cyclin-dependent kinase (cdk) 5 can phosphorylate tau at the site of abnormally phosphorylated in PHF. To characterize the relationship between cdk5 and PHF-tau, we investigated the localization of cdk5 and its regulator, p67 (munc 18), in the hippocampus and temporal lobes from 12 Alzheimer type dementia (ATD) patients and 5 controls using immunohistochemical procedures. The specificity of antibodies was confirmed with Western blot analysis. Anti-cdk5 antibody diffusely stained the perikarya of some tau2-positive or neurofibrillary tangle (NFT)-bearing neurons in ATD brains, while cdk5-positive staining was scarcely found in control brains. Anti-p67 antibody also showed stronger immunoreactivity of pyramidal neurons in ATD brains than in control brains. Double immunostaining with anti-cdk5 and anti-p67 antibodies revealed co-localization of both molecules in some pyramidal neurons. These findings suggest that cdk5 is activated by p67 at the early stage of NFT formation and accelerates NFT formation. In cdk5-positive and p67-negative neurons, cdk5 may be activated by other regulator molecules such as p35. In addition, cdk5-positive reactive astrocytes were found close to cdk5-positive NFT-bearing neurons m ATD brains but not in control brains, suggesting a correlation between NFT and reactive astrocytes.     

Hematopoietic prostaglandin D synthase and DP1 receptor are selectively upregulated in microglia and astrocytes within senile plaques from human patients and in a mouse model of Alzheimer disease

Prostaglandin (PG) D2 is produced in activated microglia by the action of hematopoietic PGD synthase (HPGDS) and plays important roles in neuroinflammation. Because the fact that neuroinflammation accelerates progression of Alzheimer disease (AD) has been documented, we investigated whether PGD2 is also involved in the pathology of AD. Here, we report that the level of the mRNA of the receptor for PGD2 (DP1) was increased in AD brains compared with the level in non-AD brains. Immunocytochemical analysis showed HPGDS expression to be localized in the microglia surrounding senile plaques. In situ hybridization studies revealed that DP1 mRNA was specifically localized in microglia and reactive astrocytes within senile plaques of AD brains. In the brain of Tg2576 mice, a model of AD, HPGDS and DP1 proteins were mainly localized immunocytochemically in microglia and astrocytes in the plaques, and the levels of their mRNAs increased in parallel with amyloid beta deposition. These results indicate that PGD2 may act as a mediator of plaque-associated inflammation in AD brain and may explain the pharmacologic mechanisms underlying the favorable response of patients with AD to nonsteroidal anti-inflammatory drugs.     

Selective distribution of matrix metalloproteinase-3 (MMP-3) in Alzheimer’s disease brain

A growing amount of evidence indicates that matrix metalloproteinases (MMPs) may play an important role in the pathogenesis of Alzheimer’s disease (AD). Stromelysin-1 (MMP-3) plays a central role in activating latent-type MMPs, which are originally secreted as proenzymes. We examined MMP-3 immunoreactivity in the brains of patients who had suffered from Alzheimer’s disease and in those of neurologically normal persons. The interstitium between myelinated axons and astrocytes in the white matter of all brain tissues, and senile plaques in the gray matter of the patients with AD were stained with a monoclonal antibody to MMP-3. Comparison of the number of senile plaques stained with the antibody against MMP-3 in the parietal cortex with that in the hippocampus showed that fewer plaques were stained in the hippocampus. The selective distribution of MMP-3 in the human brain suggests that MMP-3 might play an important role in the pathogenesis of AD, especially in the degradation of β-amyloid protein. Received: 16 November 1998 / Revised: 6 April / 24 June 1999 / Accepted: 24 June 1999     

Examination of β-Protein Deposits in Alzheimer's Cerebral Cortices by Three-Dimensional Observation

Abstract: We examined the location of β-protein deposits (small diffuse plaques) in the cerebral cortices of Alzheimer's patients and aged nondemented controls by observing the methenamine silver stained serial sections. Some vessels, astrocytes or neuronal elements existed near or within the diffuse plaques, and the preamyloids in the plaques were frequently detected in direct contact with vessels, astrocytes or neuronal elements. Although these findings do not demonstrate that the β-protein within the senile plaques is produced in various cellular elements of the brain, they suggest some associations between β-protein deposition and vessels, astrocytes or neuronal elements.     

Ultrastructural localization of Alzheimer amyloid β/A4 protein precursor in the cytoplasm of neurons and senile plaque-associated astrocytes

Summary The ultrastructural localization of amyloid /A4 protein precursor (APP) in the brains of control and Alzheimer's disease patients was examined immunohistochemically using antisera against the N and C termini of APP. In both control and Alzheimer brains, immunoreaction for APP was seen in the cytoplasm of most neurons, on plasma membranes, outer membrane of mitochondria, granular substance and neurofilaments. Cell bodies and foot processes of astrocytes, containing glial filaments, were also labeled. In primitive and classic type senile plaques, APP immunoreaction products were localized in the astroglial processes that surrounded the amyloid mass of the senile plaques. Swollen degenerating neurites in the senile plaques were also labeled. Amyloid fibrils were negative with APP antisera.Supported by Grant-in-Aid for Scientific Research on Priority Area 02240105 from the Ministry of Education, Science, and Culture, Japan