Localization of a novel type trypsin-like serine protease, neurosin, in brain tissues of Alzheimer's disease and Parkinson's disease.

Neurosin, a novel type of trypsin-like serine protease, has been shown to be preferentially expressed in human brain by northern blotting. We examined neurosin immunolabeling in the brains of neurologically normal persons and patients with Alzheimer's disease (AD) and with Parkinson's disease. We also identified the expression of the mRNA for neurosin by in situ hybridization histochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The neurosin antibody stained all of the nuclei of various cell types. In neurons, there was also staining of neuronal cytoplasm, nucleoli and their processes. In AD, staining of neurons with processes was rare in the damaged areas. Some senile plaques, extracellular tangles and Lewy bodies were also positive for neurosin. Expression of the mRNA for neurosin was seen in neurons in the gray matter, and in microglial cells in the white matter. In AD, the intensity of the signal for neurosin mRNA in the gray matter was decreased compared with normal control brains. The relative levels of neurosin mRNA in AD brains, measured by RT-PCR, were lower than those in controls. These results suggest that in human brain neurosin plays various physiological roles, and that in AD this molecule, like other serine proteases, may have a role in the degradation of such substances as beta-amyloid protein.

Different expression of calreticulin and immunoglobulin binding protein in Alzheimer's disease brain.

Both calreticulin (CRT) and immunoglobulin binding protein (Bip) have a role in the folding and assembly of oligomeric membrane proteins in the endoplasmic reticulum (ER). Recent studies have demonstrated the generation of beta-amyloid protein (Abeta) 1-42, a key peptide for amyloid deposits, in the ER. We, therefore, examined the localization and expression of CRT, Bip and their mRNA by immunohistochemistry, Western blot, in situ hybridization and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in both neurologically normal and Alzheimer's disease (AD) brains. Two polyclonal anti-CRT antibodies gave similar positive staining of CRT in neurons and glia. In neuronal cells, the cytoplasm, nucleoli and their processes were positive for CRT. In glial cells, perinuclear staining was frequently seen and the processes of some glial cells were also stained. In AD, these antibodies stained clearly damaged neurons but the number and the intensity of positive cells were decreased compared to controls. Processes of microglial cells were markedly positive in the AD white matter. Western blots using an anti-CRT antibody showed significantly lower immunoreactive bands in AD than control brains. By in situ hybridization, the number of neurons which express the CRT mRNA was less in AD than in controls. Using RT-PCR, the relative levels of the CRT mRNA in AD brains were also found to be significantly lower than those in controls. On the other hand, the number of Bip-positive cell, the production of Bip and the expression of mRNA for Bip did not differ between control and AD brains. These results suggest that CRT may be a multifunctional protein in human brain, and that the weak expression of CRT and the positive staining of microglial processes in AD brain may be part of the pathological processes in 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.

Cross desensitizations on contractions by P2-agonists of guinea pig ileum.

The present study was designed to clarify the characteristics of contractions of guinea pig ileal longitudinal muscles evoked by alpha,beta-methylene ATP as compared with those by other P2-agonists. alpha,beta-Methylene ATP, ADP-beta-S and 2-methylthio ATP as P2-agonists produced remarkable phasic contractions of the segment in a suramin-sensitive- and reactive blue-2-insensitive manner. However, ADP-beta-S and 2-methylthio ATP, unlike alpha,beta-methylene ATP, showed a biphasic contraction accompanied by a second sustained phase. Their second sustained contractions were notably suppressed by 30 microM reactive blue-2, probably being a component mediated by P2Y-purinoceptor. The phasic contractile response to alpha,beta-methylene ATP, but not ADP-beta-S and 2-methylthio ATP, was largely reduced by tetrodotoxin and atropine, indicating that the contraction is due to acetylcholine released from the cholinergic nerves. At 100 microM, alpha,beta-methylene ATP inhibited the phasic contractions caused by a low concentration of itself, but not those induced by ADP-beta-S and 2-methylthio ATP, presumably serving as a desensitizer of the P2-receptor. Although beta,gamma-methylene ATP per se showed little contraction, it prevented the contraction evoked by alpha,beta-methylene ATP, but not those by ADP-beta-S and 2-methylthio ATP. The contraction evoked by 100 microM 2-methylthio ATP was attenuated in the presence of ADP-beta-S at 10 and 30 microM. From separate cross-interactions between two groups of P2-agonists, there seems to be different subtypes of P2X-purinoceptors in the pre- and postsynapse in producing phasic contractions, but not sustained contractions that are mediated by, presumably, the P2Y-purinoceptor of the ileum.

Glycosylation of microtubule-associated protein tau in Alzheimer's disease brain.

In the neurofibrillary pathology of Alzheimer's disease (AD), neurofibrillary tangles (NFTs) contain paired helical filaments (PHFs) as their major fibrous component. Abnormally hyperphosphorylated, microtubule-associated protein tau is the major protein subunit of PHFs. A recent in vitro study showed that PHF tangles from AD brains are highly glycosylated, whereas no glycan is detected in normal tau. Deglycosylation of PHF tangles converts them into bundles of straight filaments and restores their accessibility to microtubules. We showed that PHF tangles from AD brain tissue were associated with specific glycan molecules by double immunostaining with peroxidase and alkaline phosphatase labeling. Intracellular tangles and dystrophic neurites in a neuritic plaque with abnormally hyperphosphorylated tau, detected with the monoclonal antibodies AT-8 and anti-tau-2, were also positive with lectin Galanthus nivalis agglutinin (GNA) which recognizes both the N- and O-glycosidically linked saccharides. Colocalization was not seen in the extracellular tangles and amyloid deposition, suggesting that the glycosylation of tau might be associated with the early phase of insoluble NFT formation. Thus, although abnormal phosphorylation might promote aggregation of tau and inhibition of the assembly of microtubules, glycosylation mediated by a GNA-positive glycan appears to be responsible for the formation of the PHF structures in vivo.

Localization and expression of cdc2 and cdk4 in Alzheimer brain tissue.

Two regulators of the eukaryotic cell cycle, cell division cycle 2 (cdc2) and cyclin-dependent kinase 4 (cdk4), have been reported to be related to Alzheimer's disease (AD) pathology, and especially to hyperphosphorylated tau protein. Using well-characterized polyclonal antibodies which recognize the C termini of cdc2 kinase and cdk4, we examined by immunohistochemistry brain tissues from patients with non-neurological conditions, AD and cerebral infarction. Semiquantitative mRNA analysis by RT-PCR was also done using non-neurological and AD brains. In AD, as previously reported, the antibody to cdc2 showed positive staining of a few intracytoplasmic neurofibrillary tangles (NFTs). In addition, this antibody gave positive immunolabelling in astrocytes and capillaries in all brains studied. In both AD and cerebral infarct cases, the staining of astrocytes was more intense than in non-neurological brain tissue. In all cases, the antibodies to cdk4 showed positive immunolabelling in the nuclei of all cell types except neurons. In AD tissue, the antibody showed additional staining of neuronal nuclei and cytoplasm. In contrast to a previous report, we did not find positive labelling of NFTs with the anti-cdk4 antibody. In infarct areas, particularly strong nuclear staining in glial cells was seen. The relative levels of cdk4 mRNA in AD brains were higher than those in controls. These data suggest that cdc2 kinase appears in glial cells capable of cell division and may play a role in the regulation of amyloid precursor protein processing and NFT formation in neurons. As suggested in a report on rat brain, neuronal expression of cdk4 may reflect some pathological process in damaged cells in AD.

The differential effects of aspirin on platelets, leucocytes and vascular endothelium in an in vivo model of thrombus formation.

Unanswered questions remain with regard to the therapeutic use of aspirin and the selective inhibition of thromboxane A2 and prostacyclin in platelets and endothelial cells. In the present study, the effects of aspirin on platelets and endothelial cells in vivo were examined using a helium-neon (He-Ne) laser-induced thrombosis model. Single intravenous injections of aspirin at concentrations of more than 0.5 mg/kg body weight mediated a dose dependent inhibition of thrombus formation in arterioles but not in venules. This antithrombotic effect was optimum after 15 min and declined after 90 min. Potent antithrombotic activity in arterioles was manifest at doses of 2.5 mg/kg to 50 mg/kg, and initial inhibition of thrombogenesis in vivo was most pronounced at high doses. Oral aspirin also inhibited thrombus formation in arterioles but not in venules, although the antithrombotic effects were delayed and prolonged. Maximum inhibition of ex vivo, collagen induced platelet aggregation by aspirin was observed approximately 180 min after intravenous injection. The results demonstrated that, although aspirin might have differential effects on platelets and endothelial cells, potent antithrombotic activity was manifest in arterioles at all concentrations. The findings suggest that the concept of the aspirin dilemma might be ignored for therapeutic purposes in many clinical circumstances. The antithrombotic effects of aspirin were unchanged in granulocyte-depleted animals, indicating that leucocyte-related mechanisms including neutrophil superoxide anion production did not modulate the potency of aspirin in this model.

Immunohistochemistry and in situ hybridization of T-cell acute lymphoblastic leukemia-associated antigen 1 in human brain tissues.

One of the proteins belonging to the transmembrane 4 superfamily, T-cell acute lymphoblastic leukemia-associated antigen 1 (TALLA1), behaves like a potential tumor-associated antigen. Furthermore, its mRNA is expressed in normal brain. We examined here the histochemical localization of the protein and its mRNA in human brain tissues. Both nonneurological and Alzheimer disease (AD) brains showed astroglial staining for the TALLA1 molecule. In AD brain tissues, globular dystrophic neurites were positively stained. In damaged white matter showing leukoaraiosis by CT scan there was varicose axonal staining with the anti-TALLA1 antibody. In situ hybridization histochemistry using a RNA probe demonstrated neuronal expression of the mRNA. These results suggest that TALLA1, like amyloid precursor protein or chromogranin A, is produced in neurons and transported by axonal flow.

White matter astrocytes produce hepatocyte growth factor activator inhibitor 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 activator (HGFA) has been identified as a key enzyme that regulates the activity of HGF in vivo. Our previous studies have shown that brain astrocytes produce both proteins. Recently, HGFA inhibitor-1 (HAI-1), a novel Kunitz-type serine protease inhibitor, has been isolated. We examined HAI-1 immunolabeling in the brains of neurologically normal persons and patients with Alzheimer's disease (AD) and cerebral infarction. Furthermore, we identified the expression of the mRNA for HAI-1 by in situ hybridization histochemistry. The HAI-1 antibody stained astrocytes in the white matter of all brain tissues and was present in plasma. In AD, the intensity of HAI-1 immunolabeling was less than in the other cases. Expression of the mRNA for HAI-1 was also seen in astrocytes. The intensity of the signal for HAI-1 mRNA was similar in AD and normal control brains. These results suggest that, in human brain, secreted pro-HGF from astrocytes may be activated by HGFA and inhibited by HAI-1 on or near the astrocytic cell surface and that rapid HAI-1 consumption may occur in the white matter in AD.