Neuronal cytoskeletal abnormalities in human cerebral cortical dysplasia

The cytoskeletal abnormalities of cortical neurons in human cerebral cortical dysplasia were compared by immunohistochemical methods to the neurofibrillary tangles of Alzheimer's disease (AD). Surgical specimens from cortical resections performed for the treatment of intractable childhood seizures as well as autopsied samples from AD patients were analyzed with different antibodies directed against high- or medium-molecular mass neurofilament epitopes, phosphorylated or non-phosphorylated forms of neurofilaments, ubiquitin, the microtubule-associated protein tau, and paired helical filaments (PHF), a defining feature of AD tangles. A strong abnormal increase in immunoreactivity to the high and medium molecular mass neurofilament epitopes was seen in hypertrophic neurons of cortical dysplasia. These neurofilamentous accumulations of cortical dysplasia as well as AD tangles also displayed immunoreactivity with antibodies also displayed immunoreactivity with antibodies against phosphorylated and non-phosphorylated neurofilament epitopes, tau and ubiquitin. Only the AD tangles, however, were immunoreactive to the antiserum to PHF. These results replicate and extend our previous findings that the neurofibrillary accumulations in cerebral cortical dysplasia share some common antigens with the neurofibrillary tangles of AD but do not demonstrate immunoreactivity to PHF antiserum. The results also suggest that the cytoskeletal abnormalities observed in neurons of cortical dysplasia may result in part from alterations in the level of expression, in phosphorylation state or in transport of cytoskeletal components.Supported by NIH Training Grant HD 07032 (T.D.), NIH NS28383 (M.J.D., R.S.F., H.V.V.), NIH NS24596 (R.S.F.) and NIH NS26312 (H.V.V.). Ongoing work of the UCLA Pediatric Epilepsy Group is supported by the Milken Family Medical Foundations     

Microtubule-affinity regulating kinase (MARK) is tightly associated with neurofibrillary tangles in Alzheimer brain: a fluorescence resonance energy transfer study

Paired helical filaments, the main structural components of the neurofibrillary tangles in Alzheimer disease, consist of phosphorylated tau protein. Because the levels and degree of phosphorylation are significantly higher in paired helical filament (PHF)-derived tau than in normal adult tau, and because phosphorylation of tau severely disrupts microtubule stability, it is postulated that tau phosphorylation is an important step in PHF formation. The kinases and/or phosphatases that act in vivo to help induce such a pathological state of tau, however, are not yet known. In this study we implicate the non-proline directed kinase MARK in PHF-tau phosphorylation, by virtue of its close intermolecular association with the phosphorylated Ser262 epitope on PHF-tau as assessed by fluorescence resonance energy transfer. Moreover, because this tight enzyme-substrate association is observed in neurofibrillary tangles in Alzheimer tissue, we suggest that PHF-tau phosphorylation may occur to some extent on assembled PHF filaments.     

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. Received: 25 June 1998 / Revised: 3 November 1998 / Accepted: 20 November 1998     

Tau,paired helical filaments and amyloid in the neocortex: a morphometric study of 15 cases with graded intellectual status in aging and senile dementia of Alzheimer type

Summary Tau immunoreactivity was studied in temporal neocortex, area 22, in 15 cases with graded intellectual status and compared with the immunoreactivity observed with an antiserum against paired helical filaments (PHF) and with the density of amyloid revealed by thioflavin S. Samples came from women over 75 years either intellectually normal or affected by senile dementia of the alzheimer type at various degrees of severity. Mental status had been prospectively assessed by the Blessed's test score. Antitau labelled a neuropil meshwork, the density of which increased with the severity of the disease. This meshwork was denser in layers II, III and V in the most affected cases. The number and the size of the taupositive fibers within the senile plaques increased with the intellectual deficit. Senile plaques were more numerous in layers II and III and neurofibrillary tangles in layers III and V whatever the staining technique: tau or PHF immunocytochemistry, and thioflavin S. The densities of senile plaques and of neurofibrillary tangles (NFT) were correlated with the severity of the disease whatever the staining method. The three methods revealed a systematically different number of changes. This systematic difference could greatly influence the neuropathological diagnosis. It could be the consequence of various factors: different sensitivities of the staining methods or changes in the antigenic and amyloid composition of the lesion according to the stage of the disease. In line with the last hypothesis, a higher proportion of amyloid-rich plaques was noted in the less affected cases, suggesting that tau and PHF epitopes appeared secondarily. Tau epitopes seemed to be present at least as early as PHF epitopes in the NFT. The pathological changes best linked to dementia were NFT revealed by tau antiserum.Supported by a fellowship from the European Community Commission (PD)     

Rabbit IgG cross-reacts with Alzheimer neurofibrillary tangles

Biochemical studies have demonstrated that the paired helical filaments (PHF) of Alzheimer neurofibrillary tangles are mostly made up of tau and to a lesser degree of ubiquitin and other proteins. In addition, immunocytochemical labeling of tangles with antibodies to various other neuronal proteins has been shown previously. We report here the labeling of the locations of PHF, i.e., Alzheimer neurofibrillary tangles, neuropil threads and plaque neurites in tissue sections with a goat antiserum to rabbit IgG (GAR-T). The labeling is comparable in strength and distribution to that of tau and ubiquitin antibodies. The PHF-staining antibodies could be removed by absorption with native rabbit IgG but not with human IgG, IgG-depleted rabbit serum, rabbit IgG heavy chains or light chains eluted from nitrocellulose membranes. Furthermore, the PHF reactivity was obliterated by absorption with brain homogenate and a fraction enriched in soluble abnormally phosphorylated tau, but not with purified bovine tau or SDS-washed preparations of the relatively insoluble population of PHF. On immunoblots of both normal human tau and Alzheimer abnormally phosphorylated tau-enriched preparations, GAR-T labeled a set of three to five polypeptides in the tau region. Some of these polypeptides co-migrated with the tau bands. These results indicate (i) that PHF in Alzheimer's disease brain cross-react with a structural epitope/s present on native rabbit IgG, and (ii) that the cross-reactivity with PHF is probably due to tau.Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and grants NS 18105, AG04220, AG05892 and AG08076 from the National Institutes of Health     

Alzheimer's paired helical filaments: Amyloid precursor protein epitope mapping

Paired helical filaments (PHF) were electro-phoretically purified and solubilized from Alzheimer's neurofibrillary tangles and consisted of a primary 66 kDa protein on SDS-PAGE analysis. A panel of antibodies raised against restricted regions of the beta-amyloid precursor protein (APP) were employed for epitope mapping studies of this 66 kDa PHF protein. Western blot studies revealed that C-terminal APP antibodies were immunoreactive with the 66 kDa PHF protein. Further analysis revealed that only antisera raised against peptides that include the beta/A4-amyloid region within the C-terminal portion of APP were immunoreactive with PHF proteins. These data complement previous immunocytochemical studies which indicated that C-terminal APP antibodies preferentially label PHF-containing neurofibrillary tangles in Alzheimer's brain. The present data suggest a similarity of secondary or tertiary structure between beta/A4-amyloid and PHF which accounts for the cross-reactivity of beta/A4-amyloid antibodies with PHF proteins.     

Site-specific effects of tau phosphorylation on its microtubule assembly activity and self-aggregation

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172–251) and C-terminal tail (residues 368–441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3β increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (∼70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.     

Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer’s disease

Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer’s disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and β-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate the state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF. Received: 9 March 1995 / Revised, accepted: 27 November 1995     

Biochemical and ultrastructural study of neurofibrillary tangles in amyotrophic lateral sclerosis/parkinsonism-dementia complex in the Kii peninsula of Japan

Amyotrophic lateral sclerosis/parkinsonism-dementia complex of the Kii peninsula (Kii ALS/PDC) is a neurodegenerative disorder endemic to natives in the southern coast area of the Kii peninsula of Japan. The disorder closely resembles Guamanian ALS/PDC clinically and neuropathologically. The characteristic neuropathological finding is abundant neurofibrillary tangles (NFTs) without amyloid deposition. To elucidate the biochemical properties of hyperphosphorylated tau protein, the major component of the NFTs, we examined Kii ALS/PDC brains by immunoblotting and immunohistochemical analysis using well-characterized anti-tau antibodies specific to phosphorylation-dependent or -independent epitopes. Hyperphosphorylated tau in Kii ALS/PDC had phosphorylated epitopes common to tau of paired helical filaments (PHFs) in Alzheimer disease (AD): immunoblot showed triplet bands composed of 6 tau isoforms. Ultrastructurally, NFTs revealed a twisted filamentous shape similar to PHF of AD. The biochemical properties of its phosphorylated tau protein and the ultrastructural characteristics of the NFTs of Kii ALS/PDC are very similar, if not identical, to PHF tau in AD, although they are different taupopathies.     

NACP/α-synuclein and tau constitute two distinctive subsets of filaments in the same neuronal inclusions in brains from a family of parkinsonism and dementia with Lewy bodies: double-immunolabeling fluorescence and electron microscopic studies

The co-localization of NACP/α-synuclein and tau epitopes was examined in the brain stem and hippocampal formation in two patients from a family of autosomal dominant parkinsonism and dementia with Lewy bodies (LBs) without two reported missense mutations in the NACP gene. Double-labeling immunofluorescence study revealed that some brain stem LBs, cortical LBs, pale bodies, Lewy-related neurites, and neurofibrillary tangles expressed both NACP epitopes and the PHF tau AT8 epitope. Double-immunolabeling electron microscopy demonstrated that the NACP antibody selectively labeled 9- to 13-nm-thick straight filaments (LB filaments), whereas AT8 recognized twisted tubules with 80- to 100-nm-interval constrictions in the same neuronal inclusions. We show that NACP and tau aggregate into different filamentous components even if both proteins are incorporated into the same inclusions. Received: 15 March 1999 / Revised, accepted: 25 October 1999     

Young adult-form of dementia with neurofibrillary changes and lewy bodies

Summary Alzheimer's neurofibrillary tangles, Lewy bodies and chromatolytic neurons were found in the brain at autopsy of a 28-year-old male with pyramidal and extrapyramidal signs, and severe dementia of 7-year duration prior to his death. Review of histological material showed generalized changes involving both cortical and subcortical structures. These changes were characterized by the presence of neurofibrillary tangles, Lewy bodies and chromatolytic neurons. Neuritic plaques were not found. There was also loss of neurons and gliosis in the prefrontal cortex, hippocampus, amygdaloid nucleus, basal ganglia, midbrain and pons. There were spongiform changes due to loss of neurons. Myelin stain showed pallor of myelin in long tracts and in subcortical regions. The neurofibrillary tangles were mostly composed of Alzheimer's paired helical filaments (PHF). PHF were immunostained with both polyclonal and monoclonal antibodies to PHF and the microtubule-associated protein tau. Some Lewy bodies were immunolabelled with monoclonal antibodies to PHF. To the best of our knowledge it is the first reported case of a young adult-form of dementia with extensive formation of neurofibrillary changes and Lewy bodies.Supported in part by grants from the NIH NS18105 and POINS04220     

Pharmacological targets to inhibit Alzheimer neurofibrillary degeneration

Neurofibrillary degeneration appears to be required for the clinical expression of Alzheimer disease (AD) and related tauopathies. Given the polyetiology of these diseases and the pivotal involvement of neurofibrillary degeneration in their pathogenesis, inhibition of this lesion offers a promising therapeutic target. Studies from our laboratories have shown that there is a protein phosphorylation/dephosphorylation imbalance and that the microtubule associated protein tau is abnormally hyperphosphorylated in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau which is polymerized into PHF/NFT neither promotes or inhibits in vitro microtubule assembly. In contrast the cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau neither associates with tubulin nor promotes in vitro microtubule assembly but instead it sequesters normal tau, MAP1 and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments under physiological conditions of protein concentration, pH, ionic strength and reducing conditions and this self assembly requires the abnormal hyperphosphorylation of this protein. The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP)-2A which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of protein phosphatase-2A and tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion.     

Alzheimer''s disease: exposure of neurofilament immunoreactivity in SDS-insoluble paired helical filaments

In Alzheimer's disease, neurofibrillary tangles (NFT) react with both antibodies to paired helical filaments (PHF) and certain antibodies to neurofilaments, for example, monoclonal RT97 against the 200,000 mol. wt. neurofilament protein. After isolating NFT by extraction in sodium dodecyl sulfate (SDS), PHF antibodies remain reactive while RT97 weakly stains only a minority of the extracted tangles. We now show that isolated NFT initially unreactive with RT97 apparently develop exposed antigenic sites following incubation under denaturing conditions. Our results suggest that at least one neurofilament epitope is buried in the SDS-insoluble PHF fiber and that conformational changes in PHF proteins allow its exposures.     

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site

Summary Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several differnt methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration, e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.Supported by NIA AG06803, AG01136 and AG04145     

Aggregation of tau protein by aluminum

Aluminum has been detected in Alzheimer neurofibrillary tangles, but the significance of its presence is unknown. The principal component of tangles is the paired helical filament (PHF), comprised of tau protein. We investigated whether aluminum could induce tau protein to form filaments or aggregate. When 10 μM bovine tau or non-phosphorylated recombinant human tau was combined with 400 μM or more aluminum, tau protein appeared to aggregate, observed as a dose-dependent decrease in electrophoretic mobility on SDS-PAGE. Tau appeared as a smear above the region of the expected tau bands and, at higher aluminum,doses, failed to enter the gel. A tau fragment encompassing the microtubule binding domains did not show decreased mobility in the presence of aluminum, but did form aggregates that failed to electrophorese. However no fibrillar structures were observed in the aluminum-treated tau samples when observed by electron microscopy. The effect of aluminum on tau mobility was reversed by incubating with 1 mM deferoxamine. In contrast, the morphology of PHF fibrils was unaffected by deferoxamine treatment and the characteristic abnormal mobility of PHF-tau was not reduced by deferoxamine. This suggests that aluminum is not, by itself, a significant factor in maintaining the assembly of PHF-tau as fibrils or in its abnormal mobility on SDS gels. Aluminum treatment of 3T3 fibroblasts transfected with human tau resulted in toxicity, but did not change tau expression levels or induce tau aggregation. In conclusion, aluminum appears to induce isolated tau protein to aggregate in a phosphate-independent way, without the formation of fibrils. This effect was not observed when tau-transfected cells were treated with toxic doses of aluminum.     

Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments

We performed phosphate analysis of tau proteins isolated from normal human brain, tau proteins associated with paired helical filaments (PHF-tau), and Alzheimer tau not associated with PHF. These tau fractions were of high purity. Normal and Alzheimer tau were purified by heat treatment, acid extraction and calmodulin-affinity chromatography with or without HPLC. Fractions containing primarily PHF-tau polypeptides of 60, 64 and 68 kDa and their degraded fragments were purified either on a sucrose density gradient as filaments (PHF) or by heat treatment and acid extraction as amorphous proteins (PHF-tau). PHF and PHF-tau were found to contain 6-8 mol phosphate/mol protein while normal and Alzheimer tau proteins contained 1.9 and 2.6 mol phosphate/mol protein, respectively. Upon 2-h incubation with alkaline phosphatase, PHF lost two of the phosphate groups without apparent changes in the stability and morphology of PHF. The released phosphate originated from the N-terminal half of PHF-tau as determined by immunoblotting with antibodies to epitopes blocked by phosphorylation. Tau-1 and E-2, and by a prominent shift in the electrophoretic mobility of some fragments of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, which retained the epitope to Tau 46. The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region of PHF-tau and may play a role in structural stability of PHF.     

Substructure of 20 nm filaments of progressive supranuclear palsy

Summary In contrast to the ultrastructure of Alzheimer's neurofibrillary tangles (NFT), which has been well characterized as accumulations of paired helical 10-nm filaments (PHF) with 80-nm regular constrictions, the morphology of the neurofibrillary changes of PSP remains ill-defined. Until recently, the fine structure of PSP tangles was generally accepted as 15-nm straight filaments or tubules, although many reports describing different electron-microscopic findings have appeared in the literature.In this report, we present morphological data indicating a protofilamentous substructure present in straight filaments of PSP which has some points of similarity with the protofilamentous architectures which have been reported for paired helical filaments of Alzheimer's disease. The straight filaments were found to be composed of six or more helically symmetric 2–5-nm protofilaments. We conclude that despite the varied morphology of filaments in neurofibrillary tangles observed in PSP there may be some underlying identity at the molecular level with the PHF of neurofibrillary tangles of Alzheimer's disease.Supported in part by grant no. 7301400 from the Ottawa General Hospital Research Fund     

Alzheimer's neurofibrillary tangles and paired helical filaments in the pheochromocytoma cells of the adrenal medulla —Electron microscopic and immunoelectron microscopic observations

Summary Alzheimer's neurofibrillary tangles (NFT) and paired helical filaments (PHF) were found in the pheochromocytoma cells of the adrenal gland removed from a 54-year-old female. By electron microscopy they were identical to those found in the brains affected by dementia of Alzheimer type. In the tumor cells, most of the PHF were found dispersed loosely in the cytoplasm, while typical NFT were infrequent. By immunoelectron microscopy using peroxidase-antiperoxidase method, both NFT and dispersed PHF were stained positively with a polyclonal antiserum to human tau protein. This is the first observation of NFT and PHF in paraneuronal tumor cells. The patient has no obvious Alzheimer's disease.     

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.     

Elevated transglutaminase-induced bonds in PHF tau in Alzheimer's disease

Transglutaminase-induced epsilon-(gamma-glutamyl)lysine bonds covalently cross-link and polymerize peptides into insoluble high molecular weight protein aggregates resistant to degradation and proteolytic digestion. We investigated the hypothesis that excessive deposition of epsilon-(gamma-glutamyl)lysine bonds is a neuropathological mechanism which induces the polymerization of tau protein into stable aggregates leading to the formation of paired helical filaments (PHFs) which deposit into neurofibrillary tangles in Alzheimer's disease (AD) brain. We demonstrate a significant (45%) elevation in epsilon-(gamma-glutamyl)lysine cross-links in AD cortex as compared to control cortex. In vivo, PHF tau, and high and medium molecular weight neurofilament proteins have significantly greater cross-linking by epsilon-(gamma-glutamyl)lysine bonds in AD brains as compared to controls. The cross-linking of PHF tau occurs both intra-molecularly and inter-molecularly. The inter-molecular cross-linking of tau could account for the formation of high molecular weight tau polymers. These results suggest that transglutaminase-induced cross-linking of tau protein could play a role in the formation and stabilization of neurofibrillary tangles. Inhibition of transglutaminase-induced cross-linking may therefore, provide a novel strategy for the treatment of AD.