Independent accumulations of tau and amyloid beta-protein in the human entorhinal cortex.

BACKGROUND: Previous studies have repeatedly described that neurofibrillary tangles arise earlier than senile plaques (SPs) in the entorhinal cortex, but one study suggested that SPs, if present, enhance the former lesions. All of these studies were performed at the histologic or immunocytochemical level, which may not accurately reflect the actual levels of amyloid beta-protein (Abeta) and tau. OBJECTIVE: To determine whether there is significant interaction between Abeta and tau in the human entorhinal cortex with regard to the Braak stage. METHODS: Biochemical studies were conducted on 50 brains from elderly people, who were mainly at Braak stages I to III. All the cases were examined neuropathologically and staged according to Braak and Braak. A small piece of brain tissue for each case was dissected from the anterior portion of the right entorhinal cortex. The amounts of tau and Abeta in the insoluble fraction of the tissue were quantified using western blotting. RESULTS: The levels of tau and possibly Abeta42 in the entorhinal cortex appeared to rise steeply at approximately age 75. The levels of insoluble tau increased as the Braak stage increased from I to II; however, it had a tendency to remain between stages II and III. The levels of Abeta42 showed a small increase, whereas those of Abeta40 increased continuously as the Braak stage advanced. In contrast, the extent of Abeta42 accumulation increased with increasing Braak stage for SPs. There was no significant correlation between the levels of insoluble tau and Abeta42 in the entorhinal cortex. Even if Abeta did not accumulate to significant extents, substantial accumulation of insoluble tau occurred. CONCLUSION: Accumulations of tau and amyloid beta-protein occur independently in the human entorhinal cortex.

Selective deposition of mutant tau in the FTDP-17 brain affected by the P301L mutation.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder exhibiting autosomal dominant inheritance. Linkage analyses have led to the identification of many exonic and intronic mutations in the tau gene in affected families. Because FTDP- 17 causes extensive neuronal loss and intracellular tau deposits in affected regions, investigation of this disease should provide an important insight into the significance of tau deposits leading to neurodegeneration. Using site-specific antibodies that distinguish between wild-type and mutant tau, we have analyzed the proportions of wild-type and mutant tau in the soluble and insoluble fractions of the P301L brain. Western blotting showed that mutant tau was selectively deposited in the Sarkosyl-insoluble fraction. Consistent with this, immunocytochemistry showed that intraneuronal tau deposits consisted exclusively of mutant tau. In one case in which abundant senile plaques occurred, in addition to mutant tau, small amounts of wild-type tau were also deposited. On the other hand, the protein levels of mutant tau in the soluble fraction were selectively decreased despite no detectable decrease in the levels of mutant tau mRNA.

Accumulation of amyloid beta-protein in the low-density membrane domain accurately reflects the extent of beta-amyloid deposition in the brain.

To learn more about the process of amyloid beta-protein (Abeta) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Abeta content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Abeta. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Abeta deposits in those cases in which Abeta accumulated to significant levels. Abeta42 levels in the low-density membrane domain were proportional to the extent of total Abeta42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Abeta in a similar manner to aging humans, Abeta42 accumulation in the low-density membrane domain also increased as Abeta deposition progressed with aging. These observations indicate that the Abeta42 associated with low-density membrane domains is tightly coupled with the process of extracellular Abeta deposition.

Molecular analysis of mutant and wild-type tau deposited in the brain affected by the FTDP-17 R406W mutation.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder, characterized genetically by autosomal dominant inheritance, clinically by behavioral abnormalities and parkinsonism, and neuropathologically by tauopathy. Linkage analyses of affected families have led to identification of several exonic and intronic mutations in the tau gene. In this study, we analyzed molecular species of tau in the soluble and insoluble fractions of brain affected by the FTDP-17 R406W mutation. Protein chemical analysis and Western blotting using site-specific antibodies indicated that almost equal amounts of wild-type and mutant tau were present in the Sarkosyl-insoluble fraction of the R406W brain. Consistent with this, wild-type and mutant tau colocalized in neurofibrillary tangles in the frontal cortex and hippocampus of the R406W brain. In contrast to soluble R406W tau, which was less phosphorylated than soluble wild-type tau, the Sarkosyl-insoluble mutant tau was highly phosphorylated as well as the insoluble wild-type tau.

[Molecular analysis of tau deposited in the FTDP-17 brain].

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder, characterized genetically by autosomal dominant inheritance, clinically by behavioral abnormalities and parkinsonism, and neuropathologically by tauopathy. Linkage analyses of affected families have led to identification of many exonic and intronic mutations in the tau gene. Using site-specific antibodies that distinguish between wild-type and mutant tau, we analyzed molecular species of tau in the soluble and insoluble fractions of the brain affected by two FTDP-17 mutations, P301L and R406W. Western blotting showed that mutant tau was preferentially deposited in the Sarkosyl-insoluble fraction of the P301L brain. Consistent with this, immunocytochemistry showed that intraneuronal tau deposits consisted exclusively of mutant tau. On the other hand, the protein levels of mutant tau in the soluble fraction were selectively decreased despite no detectable decrease in its mRNA levels. In contrast, almost equal amounts of wild-type and mutant tau were present in the Sarkosyl-insoluble fraction of the R406W brain and their levels in the soluble fraction did not differ from each other. Wild-type and mutant tau colocalized in neurofibrillary tangles in the frontotemporal cortices. In contrast to soluble R406W tau, which was less phosphorylated than soluble wild-type tau. Sarkosyl-insoluble R406W tau was highly phosphorylated to a similar extent as insoluble wild-type tau.

Effect of apolipoprotein E allele epsilon4 on the initial phase of amyloid beta-protein accumulation in the human brain.

Deposition of amyloid ss-protein (Ass), a hallmark of Alzheimer's disease, occurs to some extent in the brains of most elderly individuals. We sought to learn when Ass deposition begins and how deposition is affected by apolipoprotein E allele epsilon4, a strong risk factor for late-onset Alzheimer's disease. Using an improved extraction protocol and specific enzyme-linked immunosorbent assay, we quantified the levels of Ass40 and Ass42 in the insoluble fractions of brains from 105 autopsy cases, aged 22 to 81 years at death, who showed no signs of dementia. Ass40 and Ass42 were detected in the insoluble fractions from all of the brains examined; low levels were even found in the brains of patients as young as 20 to 30 years of age. The incidence of significant Ass accumulation increased age-dependently, with Ass42 levels beginning to rise steeply in some patients in their late 40's, accompanied by much smaller increases in Ass40 levels. The presence of the apolipoprotein E epsilon4 allele was found to significantly enhance the accumulation of Ass42 and, to a lesser extent, that of Ass40. These findings strongly suggest that the presence of epsilon4 allele results in an earlier onset of Ass42 accumulation in the brain.

Mutant presenilin 2 transgenic mice. A large increase in the levels of Abeta 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin.

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid beta protein (Abeta) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Walter, J., Grünberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313-322). Here, using a new extraction protocol, we quantitated the Abeta40 and Abeta42 levels in the Tris-saline-insoluble fraction. The insoluble Abeta levels were found to be higher than the soluble Abeta levels, and the insoluble Abeta42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble Abeta42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutant PS2 transgenic mice contained remarkably increased levels of Abeta42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of Abeta42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.

Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells.

Several lines of evidence suggest that enhanced oxidative stress is involved in the pathogenesis and/or progression of Alzheimer's disease (AD). Amyloid beta-protein (Abeta) that composes senile plaques, a major neuropathological hallmark of AD, is considered to have a causal role in AD. Thus, we have studied the effect of oxidative stress on Abeta metabolism within the cell. Here, we report that oxidative stress induced by H(2)O(2) (100-250 microM) caused an increase in the levels of intracellular Abeta in human neuroblastoma SH-SY5Y cells. Treatment with 200 microM H(2)O(2) caused significant decreases in the protein levels of full-length beta-amyloid precursor protein (APP) and its COOH-terminal fragment that is generated by beta-cleavage, while the gene expression of APP was not altered under these conditions. A pulse-chase experiment further showed a decrease in the half-life of this amyloidogenic COOH-terminal fragment but not in that of nonamyloidogenic counterpart in the H(2)O(2)-treated cells. These results suggest that oxidative stress promotes intracellular accumulation of Abeta through enhancing the amyloidogenic pathway.

Presence of sodium dodecyl sulfate-stable amyloid beta-protein dimers in the hippocampus CA1 not exhibiting neurofibrillary tangle formation.

The amyloid cascade hypothesis of Alzheimer's disease postulates that accumulation of amyloid beta-protein (Abeta) precedes neurofibrillary tangle formation or neuronal loss in the cortex. Although this temporal profile has been proved in the neocortex by silver staining and immunocytochemical methods, CA1 of the hippocampus exhibits a distinct temporal profile during normal aging: the formation of neurofibrillary tangles precedes senile plaque formation. This temporal profile has been further confirmed by two-site enzyme immunoassay (EIA) quantitation of sodium dodecyl sulfate (SDS)-dissociable Abeta42; neurofibrillary tangles are already present despite undetectable levels of SDS-dissociable Abeta42. However, when the same specimens were subjected to Western blotting, many cases with or without neurofibrillary tangles showed some accumulation of SDS-stable Abeta dimers that cannot be detected by EIA. Thus, the temporal profile prerequisite for the hypothesis is still valid in CA1, and this finding also suggests that SDS-stable Abeta dimers have some significant effects on CA1 pyramidal neurons, which are most vulnerable to neurofibrillary tangle formation.

Appearance of sodium dodecyl sulfate-stable amyloid beta-protein (Abeta) dimer in the cortex during aging.

We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid beta-protein (As) by enzyme immunoassay (EIA) provided prominent signals at 6 approximately 8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Abeta dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Abeta, capture SDS-dissociable Abeta but not SDS-stable Abeta dimer. Thus, all cortical specimens in which the levels of Abeta were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6 approximately 8 kd, but not SDS-dissociable A(beta) monomer at approximately 4 kd, as judged from the blot. This A(beta) dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the A(beta) dimer, and (ii) until 12 hours postmortem, such SDS-stable A(beta) dimer is detected only faintly in PDAPP transgenic mice. The presence of A(beta) dimer in the cortex may characterize the accumulation of A(beta) in the human brain, which takes much longer than that in PDAPP transgenic mice.

The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells.

To investigate the intracellular compartmentalization of amyloid beta-protein (Abeta), human neuroblastoma SH-SY5Y cells were fractionated and the Abeta content in each fraction was quantitated by the well-characterized two-site enzyme-linked immunosorbent assay (ELISA). Subcellular fractionation of the cell revealed two distinct pools of Abeta within the cells: a Triton-soluble and a Triton-insoluble pools with the latter being larger than the former. Because Triton insolubility points to caveolae-like domains, we prepared detergent-insoluble, low-density membrane domains from SH-SY5Y cells using two different protocols. The low-density membrane fraction prepared by either protocol was found to contain a substantial proportion of intracellular Abeta40 and Abeta42. These results indicate that the distinct membrane domains are involved in the generation and/or trafficking of Abeta.

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

Amyloid beta-protein (A beta) accumulation in the putamen and mammillary body during aging and in Alzheimer disease.

Immunocytochemical studies clearly showed that amyloid beta-protein (A beta) deposits are widely distributed in the subcortical regions as well as the cortices of normal aged and Alzheimer disease (AD) brains. To investigate the temporal profile of A beta accumulation in the subcortical region, we quantitated A beta40 and A beta42 levels, using sensitive enzyme immunoassays, in the putamen and mammillary bodies of normal individuals aged 24 to 87 years and of AD patients. In these two regions, A beta42 was the predominant species; in particular, A beta42 was the only A beta species detected in the putamen. In several cases the mammillary body contained only A beta40, but not A beta42. Although the extent of A beta accumulation in the 2 subcortical regions was much less than that in the cortex of the same subject, A beta42 appears to accumulate in both subcortical regions at the same time as in the cortex and leptomeninges. In addition, the A beta42 levels in the putamen or in the mammillary body correlated with those in the occipitotemporal cortex. This strongly suggests that the extent of A beta42 accumulation in the brain is determined not only by the duration of A beta accumulation but also by other unknown regional factors. Western blotting showed that the initial A beta species to accumulate in the putamen or mammillary body varied among individuals. In some cases, an A beta42 stable dimer was the most predominant species, while in other cases a 3 or 4 kD A beta42 monomer was more abundant, suggesting that the clearance rates of the A beta42 stable dimer and monomer are different in vivo.

Amyloid beta-protein deposition in the leptomeninges and cerebral cortex.

To further investigate the process of amyloid beta-protein (Abeta) deposition, we determined, using sensitive enzyme immunoassays, the levels of Abeta40 and Abeta42 (Abetas) in the soluble and insoluble fractions of the leptomeninges (containing arachnoid mater and leptomeningeal vessels) and cerebral cortices from elderly control subjects showing various stages of Abeta deposition and from patients affected by Alzheimer's disease (AD). In both locations, insoluble Abeta levels were higher by orders of magnitude than soluble Abeta levels. Soluble Abeta levels in cortices were much lower than those in leptomeninges. In insoluble Abeta in the cortex, Abeta42 was by far the predominant species, and Abeta42 in AD cortices was characterized by the highest degree of modifications in the amino terminus. In contrast, this Abeta42 predominance was not observed in insoluble Abeta in the leptomeninges, which were found to be able to accumulate Abetas to an extent similar to that in the cortex, on a weight basis. The levels of insoluble Abeta in the leptomeninges or cortex generally correlated with the degree of cerebral amyloid angiopathy or the abundance of senile plaque, respectively. However, the presence of plaque-free cortical samples showing significant levels of insoluble Abeta42 suggests that biochemically detectable Abeta accumulation precedes immunocytochemically detectable Abeta deposition in the cortex.

The pool of map kinase associated with microtubules is small but constitutively active.

Mitogen-activated protein kinase (MAPK) is activated by many kinds of stimuli and plays an important role in integrating signal transduction cascades. MAPK is present abundantly in brain, where we have studied its association with microtubules. Immunofluorescence of primary hippocampal neurons revealed that MAPK staining co-localized with microtubules and biochemical analyses showed that MAPK co-purified with microtubules. Approximately 4% of MAPK in cytosolic extracts was associated with microtubules, where it was associated with both tubulin and microtubule-associated proteins (MAPs) fractions. Further fractionation of MAPs suggested that a portion of MAPK is associated with MAP2. An association with MAP2 was also demonstrated by co-immunoprecipitation and in vitro binding experiments. A similar association was shown for the juvenile MAP2 isoform, MAP2C. The pool of MAPK associated with microtubules had a higher activity relative to the nonassociated pool in both brain and proliferating PC12 cells. Although MAPK was activated by nerve growth factor in PC12 cells, the activity of microtubule-associated MAPK did not further increase. These results raise the possibility that microtubule-associated MAPK operates through constitutive phosphorylation activity to regulate microtubule function in neurons.

Hyperphosphorylation of tau in PHF.

Tau in PHF is known to be highly phosphorylated and immunochemical study has indicated the similarity of the phosphorylation between PHF-tau and fetal tau. We have determined the exact phosphorylation sites in both PHF-tau and fetal rat tau by ion-spray mass spectrometry and sequencing of ethanethiol-modified peptides. In PHF-tau, 19 sites have been identified; all the phosphorylation sites except for Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Half of them are shared by fetal tau. Thus, PHF-tau is much more phosphorylated. Whereas most of the sites in fetal tau are proline-directed, half of them in PHF-tau are nonproline-directed. Overall, the hyperphosphorylation of PHF-tau can be considered to consist of fetal-type phosphorylation and additional proline-directed and nonproline-directed phosphorylation. This extraphosphorylation may provide PHF-tau with the unusual characteristics including assembly incompetence.

Proline-directed and non-proline-directed phosphorylation of PHF-tau.

To gain insight into the abnormal phosphorylation of PHF-tau, we have determined the phosphorylation sites by identifying phosphopeptides by means of ion spray mass spectrometry followed by sequencing of ethane-thiol-modified peptides. Nineteen sites have been identified; all but Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Eleven sites correspond to fetal type sites. Unexpectedly, 10 are non-proline-directed, whereas the others are proline-directed. Thus, the abnormal phosphorylation of PHF-tau can be considered to consist of fetal type phosphorylation and additional proline-directed and non-proline-directed phosphorylation. This non-fetal type phosphorylation may provide PHF-tau with the unusual characteristics.

In vivo phosphorylation sites in fetal and adult rat tau.

Fetal tau and tau in paired helical filaments show similar immunoreactivities with several phosphorylation-dependent paired helical filament-polyclonals and monoclonals, suggesting that the two molecules share several distinct phosphorylated epitopes. To make clear the similarities and differences between the two, we have undertaken work to identify the in vivo phosphorylation sites in fetal rat tau. We have approached this problem by identifying phosphopeptides by means of mass spectrometry and sequencing of those phosphopeptides after modification with ethanethiol. Although remarkable heterogeneity was present, fetal tau was found to bear at most 10 phosphates at Ser-189, Ser-190, Ser-193, Ser-226, Ser-387, Ser-395, Thr-172, Thr-222, and, presumably, Ser-391 and Thr-208 (numbering is according to the longest form of rat tau; Kosik, K. S., Orecchio, L. D., Bakalis, S., and Neve, R. L. (1989) Neuron 2, 1389-1397). In contrast, adult rat tau was much less phosphorylated; only Thr-172, Ser-190, Ser-193, Thr-222, and Ser-395 were phosphorylated to a slight-to-moderate extent. All these sites except for Ser-189 and Ser-391 were followed by Pro residues. Thus, tau is an in vivo substrate for proline-directed protein kinase(s), and its phosphorylation state is developmentally regulated.