Astroglial localization of cholesteryl ester transfer protein in normal and Alzheimer's disease brain tissues

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of cholesteryl esters, phospholipids and triglycerides between the lipoproteins, and regulates plasma high-density lipoprotein levels. We examined CETP-like immunolabeling in non-neurological and Alzheimer's disease (AD) liver and brain tissues. The anti-CETP antibodies showed positive staining in round cells in the liver sinusoid and in brain astrocytes. In the brains of non-neurological cases, positively stained astrocytes were preferentially distributed in the white matter. In AD tissue, many reactive astrocytes in the gray matter as well as the white matter astrocytes had CETP-like immunoreactivity. CETP-positive astrocytes may play a role for AD pathology such as tissue repair.     

Pick's disease immunohistochemistry: new alterations and Alzheimer's disease comparisons

Pick's disease (PD) brains were examined immunohistochemically for the expression of antigens known to be associated with Alzheimer's disease (AD) lesions. Most antibodies which label intracellular neurofibrillary tangles (NFTs) in AD were found to stain Pick bodies (PBs). Among them was the monoclonal antibody A2B5, which is known to recognize neuronal surface gangliosides. This result indicates that membrane proteins are probably incorporated into PBs as into NFTs. However, PBs, in contrast to NFTs, showed a paucity of staining for heparan sulfate glycosaminoglycan and basic fibroblast growth factor (bFGF). Staining for midline, seen in senile plaques in AD, was not seen in PD. The relative lack of staining for these two neurotrophic factors in PD brain may reflect underlying mechanisms which are distinct from those in AD. We also describe two glial abnormalities in PD: glial fibrillary tangles and clusters of granules positive for the complement protein C4d in the hippocampal dentate fascia. These are presumably related to complement-activated oligodendroglia, and both pathological structures are more abundant in advanced cases, suggesting that they may be hallmarks of the disease progression.     

Expression of glia maturation factor in neuropathological lesions of Alzheimer's disease

R. Thangavel, D. Stolmeier, X. Yang, P. Anantharam and A. Zaheer (2012) Neuropathology and Applied Neurobiology 38, 572–581 Expression of glia maturation factor in neuropathological lesions of Alzheimer's disease Aims: The pathology of Alzheimer's disease (AD) is characterized by the presence of amyloid plaques (APs), neurofibrillary tangles (NFTs), degenerating neurones, and an abundance of reactive astrocytes and microglia. We aim to examine the association between glia maturation factor (GMF) expression, activated astrocytes/microglia, APs and NFTs in AD‐affected brain regions. Methods: Brain sections were stained with Thioflavin‐S to study AD pathology and sequentially immunolabeled with antibodies against GMF, glial fibrillary acidic protein (marker for reactive astrocytes), and Ionized calcium binding adaptor molecule 1 (Iba‐1, marker for activated microglia) followed by visualization with avidin‐biotin peroxidase complex. Results: Our double immunofluorescence labelling with cell‐specific markers demonstrated the glial localization of GMF. The immunohistochemical data showed that APs and NFTs are associated with increased expression of GMF in reactive glia of AD brains compared with non‐AD controls. Conclusions: This is the first report that shows GMF, a mediator of central nervous system inflammation, is expressed in the brain regions affected in AD and that GMF is mainly localized in reactive astrocytes surrounding APs/NFTs. The distribution of GMF‐immunoreactive cells in and around Thioflavin‐S stained APs and NFTs suggests involvement of GMF in inflammatory responses through reactive glia and a role of GMF in AD pathology.     

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.     

Up-regulation of cell division cycle (cdc) 2 kinase in neurons with early stage Alzheimer's disease neurofibrillary degeneration

The major component of Alzheimer's disease (AD) neurofibrillary tangles (NFTs) is abnormally hyperphosphorylated tau aggregated as paired helical filaments (PHFs). Cell division cycle (cdc) 2 kinase is one of the main candidate kinases that phosphorylates normal tau in vitro at several sites seen in PHF-tau. Using brains staged according to Braak and Braak criteria, we investigated the role of cdc2 in neurofibrillary changes in the hippocampal formation, and the entorhinal and temporal cortices. Neurons with tangle-like inclusions positive for active cdc2 were found to appear first in the Pre-alpha layer of the entorhinal cortex, and then extend to other brain regions co-incident with the progressive sequence of neurofibrillary changes. This predictable progressive pattern is not associated with amyloid. The intraneuronal accumulation of active cdc2 appeared to precede the deposition of PHF-tau phosphorylated at Ser 202/Thr 205 sites. These data are consistent with the notion that cdc2 might be involved in the abnormal hyperphosphorylation of tau and consequently aggregation of tau into PHF at an early stage and that increased cdc2 activity is not consequent to the deposition of beta-amyloid in AD brain.     

Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain

Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so through a mitochondrial pathway employing caspase-9 or through an alternative, receptor-mediated pathway involving caspase-8. Considering the role of mitochondrial dysfunction in AD, we examined the possible activation of caspase-9 in the AD brain using an antibody that recognizes the active fragments of caspase-9, but not the full-length proform of the enzyme. In vivo immunohistochemical analysis demonstrated little caspase-9 activation in the majority of hippocampal brain sections from control brains. However, labeling of neurons as well as dystrophic neurites within plaque regions was observed in all AD hippocampal brain sections examined. In addition, active caspase-9 was colocalized with active caspase-8 and the accumulation of caspase-3-cleavage products of fodrin. The activation of caspase-9 was also observed in neurons positive for oxidative damage to DNA/RNA. A quantitative analysis indicates that as the number of neurons containing neurofibrillary tangles (NFTs) increases, the extent of caspase-9 activation decreases, supporting the idea that caspase-9 activation may precede NFT formation. In addition, a site-directed caspase-cleavage antibody was designed to the amino-terminal caspase-3 consensus cleavage site located in tau, and shown to be an effective marker for caspase-cleaved fragments of tau in vitro. Analysis with this antibody using age-matched control or AD brain sections demonstrated no staining in control brains while widespread labeling of NFTs, neuropil threads, and dystrophic neurites was observed in AD sections. Taken together, these results demonstrate the activation of caspases and cleavage of tau in the AD brain, events which may precede and lead to the formation of NFTs.     

Human neurotropic JC virus early protein deregulates glial cell cycle pathway and impairs cell differentiation

Progressive multifocal leukoencephalopathy (PML), a human demyelinating disease of the central nervous system (CNS), is induced upon replication of the human neurotropic virus, JCV, in glial cells. Similar to other polyomaviruses, replication of JCV is initiated and orchestrated by the viral early protein, T-antigen, and results in the cytolytic destruction of oligodendrocytes, the subset of glial cells responsible for myelin production, and the appearance of bizarre astrocytic glial cells in affected individuals. Earlier results from studies in transgenic animals have suggested that in the absence of viral replication, expression of JCV T-antigen induces pathology consistent with hypomyelination of the brain. These observations suggest that JCV T-antigen has the ability to deregulate oligodendrocyte and perhaps astrocyte function in the CNS. Here we demonstrate that expression of JCV T-antigen in the bipotential glial cell line, CG-4, severely affects the ability of these cells to differentiate toward oligodendrocyte and astrocyte lineages as evidenced by their distinct morphological changes. Examination of the activity of cell cycle regulatory proteins including cyclins and their associated kinases reveals that in the absence of T-antigen, differentiation of CG-4 cells toward astrocytes and oligodendrocytes is accompanied by a decline in cyclin E, cdk2, cyclin A, and cyclin B activity. In contrast, cdc2 activity increased upon CG-4 differentiation. In T-antigen-producing cells, distinct variations in the activity of several cyclins was observed. For example, while the activity of cdk2 and cyclin E was enhanced in T-antigen expressing astrocytes compared to their levels in control cells, the activity of cdc2 was decreased in this cell type. In oligodendrocytes, expression of T-antigen decreased the activity of several cyclins and cdks including cyclin E and cdc2. On the other hand, the level of expression and activity of cyclin A was increased. Thus, it is evident that JCV T-antigen deregulates several important cell cycle regulators during CG-4 differentiation, and these alterations may contribute to the process of cell growth and differentiation in glial cells. The importance of our findings with regard to the neuropathogenesis of PML is discussed.     

S6 kinase phosphorylated at T229 is involved in tau and actin pathologies in Alzheimer's disease

The 70‐kDa ribosomal protein S6 kinase (S6K), a serine/threonine kinase that modulates the phosphorylation of the 40S ribosomal protein S6, regulates cell cycle progression and is known as a tau kinase in Alzheimer's disease (AD). In AD brains, neurofibrillary tangles (NFTs) have been shown to be positively stained with antibodies against S6K proteins phosphorylated at T389 (pT389‐S6K) or T421/S424 (pT421/S424‐S6K) by the mammalian target of rapamycin and mitogen‐activated protein kinase pathways, respectively. However, there is little information available about S6K proteins directly phosphorylated at T229 (pT229‐S6K) by the PI3K‐PDK1 pathway. In the present study, we investigated the distribution of pT229‐S6K in post mortem human brain tissues from elderly (control) and patients with AD using immnunoblotting and immunohistochemistry. pT229‐S6K immunoreactivity was localized to small granular structures in neurons and endothelial cells in control and AD brains. In AD brains, intense pT229‐S6K immunoreactivity was detected in 16.3% of AT8‐positive NFTs, neuropil threads, and dystrophic neurites in the hippocampus and other vulnerable brain areas. In addition, Hirano bodies were also positive for pT229‐S6K but were negative for pT389‐S6K or pT421/S424‐S6K. The present results indicate that S6K phosphorylation via the PI3K‐PD1 pathway is involved in tau pathology in NFTs and abnormal neurites as well as actin pathology in Hirano bodies.     

Accumulation of cyclin-dependent kinase 5 (cdk5) in neurons with early stages of Alzheimer''s disease neurofibrillary degeneration

Cyclin-dependent kinase 5 (cdk5) is one of the candidate kinases involved in the abnormal hyperphosphorylation of tau. To have a direct effect on tau hyperphosphorylation, cdk5 protein levels and enzyme activity should be upregulated in especially those neurons that develop neurofibrillary tangles (NFTs). We studied the distribution of cdk5 immunoreactivity in neurons with or without early- and late-stage NFTs in hippocampal, entorhinal, transentorhinal, temporal and frontal cortices, and cerebellum of Alzheimer's disease (AD) and control brain. The immunocytochemical localisation of cdk5 was compared with that obtained using antibodies to PHF-tau (tau in paired helical filaments of NFTs, mAb AT8) and ubiquitin as markers of early and late stage NFTs, respectively. Immunoreactivities of cdk5 and PHF-tau were found in neuronal perikarya and processes of hippocampal, entorhinal, transentorhinal, temporal and frontal, and cerebellar cortices. An apparent increase of cdk5 immunoreactivity was seen in pretangle neurons and in neurons bearing early stage NFTs. These findings suggest that this kinase might be involved in the formation of NFTs at a relatively early stage in the neocortex.     

Astroglial expression of hepatocyte growth factor and hepatocyte growth factor activator in human brain tissues

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and also has multifunctional effects on some other cells in various organs. A HGF activating protease, HGF activator (HGFA) has recently been identified as a key enzyme that regulates the activity of HGF in vivo. HGFA appears to be associated with the cell surface. We examined HGFA immunolabelling in the brains of neurologically normal and Alzheimer disease (AD) cases. Furthermore, we identified the expression of the mRNA for HGF and HGFA by in situ hybridization histochemistry. The HGFA antibody stained only astrocytes in the white matter in all the brain tissues. Expression of the mRNAs of HGF and HGFA was also seen in white matter astrocytes. These results suggest that, in human brain, secreted pro-HGF from astrocytes might be activated by HGFA on/or near the astrocytic cell surface.     

Vimentin immunoreactivity in normal and pathological human brain tissue

Summary Vimentin immunoreactivity was examined in brain tissues from non-neurological and various human central nervous system disease cases. In all brain tissues examined, vimentin immunoreactivity was intensely positive in ependymal cells and subpial tissues, and weakly positive in some capillaries and some white matter astrocytes. In affected areas of Alzheimer's disease (AD), Pick's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and cerebral infarction cases, numerous intensely vimentin-immunopositive astrocytes of both protoplasmic and fibrous morphology were demonstrated. A few such astrocytes were also observed in Parkinson's disease and progressive supranuclear palsy. ALS, MS and infarction brains also had numerous, strongly vimentin-positive, round and fat-laden microglia/macrophages. In AD and ALS, a few reactive microglia with irregularly enlarged shapes were vimentin positive. In AD, they were almost exclusively related to senile plaques.Supported by grants from the Medical Research Council of Canada, and the Alzheimer Society of B. C., and donations from individual British Columbians. D. G. W. is a Research Fellow of the Alzheimer Society of B. C.     

Apoptosis of astrocytes with enhanced lysosomal activity and oligodendrocytes in white matter lesions in Alzheimer's disease

Cerebral white matter lesions in Alzheimer's disease (AD) consist of subcortical degeneration and ischaemic-hypoxic changes. Glial changes are intimately associated with the white matter lesions, and regressive changes in astrocytes and loss of oligodendroglial cells have been reported. We quantitatively compared glial changes including apoptosis and enhanced lysosomal activity in the frontal and temporal white matter by using terminal dUTP nick end labelling (TUNEL) and immunohistochemistry for glial markers, lysosomes and apoptosis-regulating proteins in non-familial AD brains. The degree of myelin pallor and axonal loss varied considerably in both the frontal and temporal white matter but fibrillary gliosis in demyelinated lesions tended to be less prominent in the temporal white matter in AD cases. A morphometric study with planimetric methods for cross-sectional areas of frontal and temporal white matter revealed that the white matter of AD cases manifested atrophy with significant reduction in frontal (11.9%) and temporal (29.4%) white matter compared to normal controls. Double immunolabelling for glial fibrillary acidic protein (GFAP) and KP1 (CD68) revealed KP1-positive fragmented structures within the weakly GFAP-labelled astrocytes. These KP1-positive structures correspond to process fragmentation and cytoplasmic vacuoles, which in turn indicate enhanced lysosomal activity during regressive changes in astrocytes. The KP1-modified astrocytes were not found in Pick's disease and corticobasal degeneration. The density of apoptotic glial cells, largely oligodendroglial, was significantly higher in the temporal than in the frontal white matter, and most GFAP-positive astrocytes with regressive changes were apoptotic. GFAP-positive astrocyte density was statistically the same in the frontal and temporal white matter, but the density of KP1-modified astrocytes was higher in the temporal than in the frontal white matter. The rate of white matter shrinkage was significantly correlated with the density of apoptotic glial cells and the density of KP1-modified astrocytes in the temporal lobe in AD cases. An increase in apoptotic glial cell density was found to contribute to GFAP-positive astrocytes with regressive changes in temporal white matter, while apoptosis of vascular smooth muscle cells did not show topographical accentuation. Astrocytes labelled with beta amyloid protein were not apoptotic, and the density of apoptotic cells labelled with CD95 and caspase-3 was too low in both types of white matter to be statistically evaluated. Our results imply that regressive changes in astrocytes and glial apoptosis are, to some extent, associated with white matter lesions, particularly of the temporal lobe in AD brains. The presence of apoptotic astrocytes with evidence of regressive change could therefore be a histological hallmark for white matter degeneration in AD.     

Widespread occurrence of argyrophilic glial inclusions in Parkinson's disease.

Argyrophilic glial inclusions, which are immunohistochemically positive for alpha-synuclein but negative for tau protein, were examined in the brain of Parkinson's disease (PD) patients. Autopsied brains of 10 individuals who died from PD, of two incidental Lewy body disease cases and of five age-matched individuals whose deaths were caused by non-neurological diseases were studied, histopathologically, by Gallyas-Braak staining and, immunohistochemically, with anti-alpha-synuclein antibody, anti-ubiquitin, and anti-tyrosine hydroxylase. All postmortem PD brains showed a significant number of argyrophilic glial inclusions, but no glial inclusions were found in control brains. The inclusions were found not only in the regions showing neuronal loss and gliosis, such as the substantia nigra, locus ceruleus and dorsal vagal nucleus, but also in regions without neuronal loss and gliosis, such as the cerebral cortex, cerebral white matter, striatum, globus pallidus, thalamus, cerebellum and spinal cord. The distribution and density of glial inclusions in PD brains varied from case to case but, in the cerebral cortex, the number of glial inclusions were fairly well correlated with the number of Lewy bodies. The distribution pattern of glial inclusions also showed a striking resemblance to that of catecholaminergic neurones and fibres. The abnormal accumulation of alpha-synuclein in glial cells was more widespread than neurone loss, and appears to be an important pathological feature of PD.     

Neuronal and glial coexpression of argininosuccinate synthetase and inducible nitric oxide synthase in Alzheimer disease

The enzyme argininosuccinate synthetase (ASS) is the rate limiting enzyme in the metabolic pathway leading from L-citrulline to L-arginine, the physiological substrate of all isoforms of nitric oxide synthases (NOS). ASS and inducible NOS (iNOS) expression in neurons and glia was investigated by immunohistochemistry in brains of Alzheimer disease (AD) patients and nondemented, age-matched controls. In 3 areas examined (hippocampus, frontal, and entorhinal cortex), a marked increase in neuronal ASS and iNOS expression was observed in AD brains. GFAP-positive astrocytes expressing ASS were not increased in AD brains versus controls, whereas the number of iNOS expressing GFAP-positive astrocytes was significantly higher in AD brains. Density measurements revealed that ASS expression levels were significantly higher in glial cells of AD brains. Colocalization of ASS and iNOS immunoreactivity was detectable in neurons and glia. Occasionally, both ASS-and iNOS expression was detectable in CD 68-positive activated microglia cells in close proximity to senile plaques. These results suggest that neurons and astrocytes express ASS in human brain constitutively, whereas neuronal and glial ASS expression increases parallel to iNOS expression in AD. Because an adequate supply of L-arginine is indispensable for prolonged NO generation, coinduction of ASS enables cells to sustain NO generation during AD by replenishing necessary supply of L-arginine.     

A brain regional analysis of morphologic and cholinergic abnormalities in Alzheimer's disease

In the brains of 21 patients with Alzheimer's disease (AD) and 10 nondemented controls, senile plaques (SPs), neurofibrillary tangles (NFTs), and three indexes of cholinergic function were quantified in the middle frontal (MF) and superior temporal (ST) cortex, the entorhinal cortex (HEN), and the prosubiculum (HPR) of the hippocampus. Control brains contained few SPs without preferential distribution in any of the brain regions examined, while NFTs were found almost exclusively in the HPR. In brains from patients with AD, an inverse relationship of SPs and NFTs was found in the brain regions examined; SPs were preferentially in the neocortex and NFTs preferentially in the hippocampus. The specific activities of choline acetyltransferase and acetylcholinesterase were reduced in all regions examined, while no significant change in the density of muscarinic binding sites was observed in any region. Numerous NFTs were associated with an earlier age at onset, while the presence of SPs was related to the cholinergic deficit in AD. Earlier-onset (less than 67 years) AD was also associated with a qualitative difference in the regional distribution of NFTs compared with cases with a later onset. In the latter group, most NFTs were observed in the hippocampus, a distribution pattern similar to that observed with normal aging. In AD cases with an earlier onset, NFTs were more globally distributed in the neocortex and allocortex.     

Role of protein kinase B in Alzheimer's neurofibrillary pathology

Protein kinase B (PKB) is an important intermediate in the phosphatidylinositol-3 kinase signaling cascade that acts to phosphorylate glycogen synthase kinase-3 (GSK-3) at its serine 9 residue, thereby inactivating it. Activated GSK-3 has been previously shown to be preferentially associated with neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brain. In the present study, we performed immunohistochemistry with an antibody to the active form of PKB in brains with different stages of neurofibrillary degeneration. We found that the amount of activated PKB (p-Thr308) increased in correlation to the progressive sequence of AT8 immunoreactivity and neurofibrillary changes assessed according to Braak's criteria. By confocal microscopy, activated PKB (p-Thr308) was found to appear in particular in neurons that are known to later develop NFTs in AD. Western blotting showed that activated PKB was increased by more than 50% in the 16,000- g supernatants of AD brains as compared with normal aged and Huntington's disease controls. This increase in PKB levels corresponded with a several-fold increase in the levels of total tau and abnormally hyperphosphorylated tau at the Tau-1 site. These studies suggest the involvement of PKB/GSK-3 signaling in Alzheimer neurofibrillary degeneration.     

DJ-1 (PARK7) is associated with 3R and 4R tau neuronal and glial inclusions in neurodegenerative disorders

Mutations in the DJ-1 gene are associated with autosomal recessive Parkinson's disease (PD), but its role in disease pathogenesis is unknown. This study examines DJ-1 immunoreactivity (DJ-1 IR) in a variety of neurodegenerative disorders, Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) with Pick bodies, FTLD with MAPT mutations, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), in which hyperphosphorylated tau inclusions are the major pathological signature. DJ-1 IR was seen in a subset of neurofibrillary tangles (NFTs), neuropil threads (NTs), and neurites in extracellular plaques in AD; tau inclusions in AD contained both 3R and 4R tau. A subset of Pick bodies in FTLD showed DJ-1 IR. In PSP, DJ-1 IR was present in a few NFTs, NTs and glial cell inclusions. In CBD, DJ-1 IR was seen only in astrocytic plaques. In cases of FTLD with MAPT mutations that were 4R tau positive (i.e. N279K and exon 10+16 mutations), DJ-1 IR was present mostly in oligodendroglial coiled bodies. However, in MAPT R406W mutation cases, DJ-1 IR was associated mainly with NFTs and NTs and these were both 3R and 4R tau positive. No DJ-1 IR was present in FTLD with ubiquitin inclusions (FTLD-U). In AD and FTLD with Pick bodies, DJ-1 protein was enriched in the sarkosyl-insoluble fractions of frozen brain tissue containing insoluble hyperphosphorylated tau, thus strengthening the association of DJ-1 with tau pathology. Additionally using two-dimensional gel electrophoresis, we demonstrated accumulation of acidic pI isoforms of DJ-1 in AD brain, which may compromise its normal function. Our observations confirm previous findings that DJ-1 is present in a subpopulation of glial and neuronal tau inclusions in tau diseases and associated with both 3R and 4R tau isoforms.     

Expression of heme oxygenase-1 in the senescent and alzheimer-diseased brain

Heme oxygenase-1 is a cellular stress protein expressed in brain and other tissue in response to oxidative challenge and other noxious stimuli. Using immunohistochemistry and immunofluorescent labeling in conjunction with laser scanning confocal microscopy, we observed intense immunoreactivity of heme oxygenase-1 in neurons of the hippocampus and temporal cortex of Alzheimer-diseased (AD) brain relative to age-matched control specimens. Furthermore, we demonstrated consistent colocalization of heme oxygenase-1 to glial fibrillary acidic protein–positive astrocytes, neurofibrillary tangles, and senile plaques in the AD specimens. In AD hippocampus, approximately 86% of glial fibrillary acidic protein–positive astrocytes expressed heme oxygenase-1, whereas only 6.8% of hippocampal astrocytes in normal senescent control specimens were immunopositive for heme oxygenase-1 (p < 0.0001). In regions other than the hippocampus and neocortex, such as the substantia nigra, the proportion of astrocytes expressing heme oxygenase-1 in the experimental group (12.8%) was not significantly different from that in the controls (6.4%, p > 0.05). Robust 32-kd bands corresponding to heme oxygenase-1 were observed by Western blotting of protein extracts derived from AD temporal cortex and hippocampus after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Heme oxygenase-1 bands were very faint or absent in protein extracts prepared from control specimens. These results indicate that heme oxygenase-1 is significantly overexpressed in neurons and astrocytes of AD hippocampus and cerebral cortex relative to control brains. Upregulation of heme oxygenase-1 in AD brain supports the contention that the affected tissues are experiencing chronic oxidative stress. In addition, the excessive generation of carbon monoxide, a metabolite of heme degradation, may participate in the pathogenesis of AD.