Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.     

Immunocytochemical evidence that the beta-protein precursor is an integral component of neurofibrillary tangles of Alzheimer's disease.

Amyloid beta (A beta) immunoreactivity has been demonstrated in all extracellular neurofibrillary tangles (E-NFT) and most intraneuronal neurofibrillary tangles (I-NFT). We undertook this immunocytochemical study to understand the relationship between A beta immunoreactivity localized in NFT and beta-protein precursor (beta PP). We found epitopes of amino-, mid-, and carboxyl-terminal domains of beta PP in I-NFT and the majority of E-NFT. NFT retained beta PP after ionic detergent extraction, demonstrating that beta PP is an integral component of NFT. Finding beta PP in regions of A beta immunoreactivity raises the possibility that beta PP or its fragments associate with amyloid, and that the stability of A beta is responsible for its dominance in amyloid deposits.     

Activation of the ERK and JNK signaling pathways caused by neuron-specific inhibition of PP2A in transgenic mice

A reduced activity of protein phosphatase 2A (PP2A) has been shown in brains of patients with Alzheimer's disease (AD), a neurodegenerative disorder characterized histopathologically by amyloid plaques and neurofibrillary tangles. Tau, as the principal component of neurofibrillary tangles, can be hyperphosphorylated by a reduced activity of PP2A in vitro and by pharmacological approaches, suggesting a crucial role of PP2A in tangle formation. To dissect the role of PP2A in vivo, we previously generated transgenic mice with chronically reduced PP2A activity by expressing a dominant-negative mutant form of the PP2A catalytic subunit Calpha, L199P, under the control of a neuron-specific promoter. In these mice, endogenous tau is phosphorylated at the epitopes Ser202/Thr205 and Ser422. In vitro, these tau phospho-epitopes can be phosphorylated by the kinases ERK and JNK, and the kinases themselves are negatively regulated by PP2A. In this study, we show that chronic inhibition of PP2A activity in L199P transgenic mice causes the activation of ERK and JNK as demonstrated by the phosphorylation and nuclear accumulation of the ERK and JNK substrates, Elk-1 and c-Jun. TUNEL staining revealed that activated JNK signaling was not associated with cell death. Our findings imply that PP2A is a negative regulator of the ERK and JNK signaling pathways in vivo, suggesting that in AD, tau hyperphosphorylation may be caused in part by PP2A dysfunction.     

Presenilin Binding Protein Is Associated with Neurofibrillary Alterations in Alzheimer’s Disease and Stimulates Tau Phosphorylation

A novel presenilin binding protein, PBP, has recently been identified. PBP is localized to the particulate fraction of extracts of Alzheimer's disease brain but is found in the soluble fractions of brain from age matched normal controls. It is shown here that PBP is associated with neurofibrillary tangles in Alzheimer's disease brain. In addition, the expression of PBP increases the phosphorylation of tau in cultured cells. Therefore PBP may have a regulatory role in tau phosphorylation and in the genesis of neurofibrillary tangles.     

Biochemical and anatomical redistribution of tau protein in Alzheimer's disease.

We have developed assays that distinguish tau protein incorporated into the core structure of the paired helical filament (PHF) from non-PHF tau protein in brain tissue, whether soluble or insoluble. The PHF content was 19-fold greater in Alzheimer's disease cases compared with cognitively intact controls, and in temporal cortex the difference was 40-fold. There was a threefold decrease in soluble tau protein in Alzheimer's disease cases compared with normal age-matched controls, the decrease being greatest in frontal cortex. The PHF content was closely correlated with the number of tau-immunoreactive dystrophic neurites in plaques and throughout the neuropil, whereas counts of neurofibrillary tangles were poorer predictors of PHF content. beta-Amyloid deposits correlated neither with PHF content nor with neurofibrillary pathology. These findings suggest that Alzheimer's disease is characterized by a substantial redistribution of available tau protein from free to PHF-incorporated fractions and that PHF accumulation may be important in neurites as well as tangles in predicting the extent of cognitive impairment in Alzheimer's disease.     

E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures.

Lipid peroxidation increases with age in brain and is elevated further in Alzheimer's disease. E-4-hydroxy-2-nonenal and malondialdehyde are products of lipid peroxidation that can adduct and cross-link protein. Neurofibrillary tangles, a feature of Alzheimer's disease composed mostly of tau protein, contain cross-linked and ubiquitin-conjugated protein. In P19 neuroglial cultures, E-4-hydroxy-2-nonenal was a potent cytotoxin that cross-linked cytoskeletal proteins, including tau into high molecular weight species that were conjugated with ubiquitin. Malondialdehyde formed monoadducts with cell protein but did not cross-link and was not cytotoxic. A non-crosslinking analogue of E-4-hydroxy-2-nonenal was not cytotoxic. E-4-Hydroxy-2-nonenal may contribute to neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease.     

Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats

Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer's disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer's-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.     

Co-occurrence of Alzheimer's disease ß-amyloid and τ pathologies at synapses

Although beta-amyloid (Abeta) plaques and tau neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) neuropathology, loss of synapses is considered the best correlate of cognitive decline in AD, rather than plaques or tangles. How pathological Abeta and tau aggregation relate to each other and to alterations in synapses remains unclear. Since aberrant tau phosphorylation occurs in amyloid precursor protein (APP) Swedish mutant transgenic mice, and since neurofibrillary tangles develop in triple transgenic mice harboring mutations in APP, tau and presenilin 1, we utilized these well-characterized mouse models to explore the relation between Abeta and tau pathologies. We now report that pathological accumulation of Abeta and hyperphosphorylation of tau develop concomitantly within synaptic terminals.     

A4 amyloid protein immunoreactivity is present in Alzheimer's disease neurofibrillary tangles

Neurofibrillary tangles and neuritic plaques are the neuropathological hallmarks of Alzheimer's disease. The latter consist of a core of A4 amyloid protein. We now report that some neurofibrillary tangles ('tombstone tangles') are also A4 immunoreactive. This observation is consistent with the hypothesis that A4 amyloid accumulation is a component of both neurofibrillary tangles and neuritic plaques.     

beta PP and Tau interaction. A possible link between amyloid and neurofibrillary tangles in Alzheimer's disease.

Extracellular deposition of amyloid fibrils and intraneuronal accumulation of paired helical filaments (PHFs) are the neuropathological hallmarks of Alzheimer's disease. The major constituent of amyloid fibrils is a 39- to 43-residue peptide (termed A beta), which is derived from a 695- to 770-amino-acid precursor protein (termed beta PP). The main component of PHFs identified so far is the microtubule-associated protein tau. Yet, there is no direct evidence of interconnection between these two pathological states. We report here that antibodies to an epitope located between residues 713 and 723 of beta PP770 (ie, the transmembrane region of beta PP distal to A beta) consistently labeled PHFs in the brain of Alzheimer patients. Solid phase immunoassay showed that a peptide homologous to residues 713 to 730 of beta PP770 bound tau proteins. This beta PP peptide spontaneously formed fibrils in vitro and, in the presence of tau, generated dense fibrillary assemblies containing both molecules. These data suggest that beta PP or beta PP fragments containing the tau binding site are involved in the pathogenesis of PHFs in Alzheimer's disease.     

Neurofibrillary degeneration in amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam. Immunochemical characterization of tau proteins.

Neurofibrillary tangles are observed in several neurodegenerative disorders including Alzheimer's disease, progressive supranuclear palsy, and amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam. The major components of neurofibrillary tangles are hyperphosphorylated tau proteins that can be directly detected in brain homogenates, using immunoblotting with specific immunological probes. To investigate whether tau proteins differ biochemically among various neurodegenerative disorders, we analyzed a series of brain samples from Guamanian patients in comparison with Alzheimer's disease, progressive supranuclear palsy, and normal aging. In Alzheimer's disease, these hyperphosphorylated tau proteins are composed of a triplet referred to as tau 55, 64, and 69, whereas in progressive supranuclear palsy, neurofibrillary degeneration is characterized by a tau doublet (tau 64 and 69). In the present study, characterization of tau proteins was performed by immunoblotting, on different cortical and subcortical regions of postmortem brain specimens from Guamanian natives. In all of the cases, biochemical data were always consistent with neuropathological findings. In contrast to Alzheimer's disease patients where the tau triplet is found mostly in cortical regions, a similar triplet was strongly detected in both cortical and subcortical areas in Guamanian patients. The tau profile differed quantitatively from case to case demonstrating that the Alzheimer's disease-related tau triplet had a heterogeneous regional distribution. These data suggest that the tau triplet found in amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam is similar to that observed in Alzheimer's disease, and the regional distribution of tau proteins differs in these disorders.     

Microtubule-associated protein tau epitopes are present in fiber lesions in diverse muscle disorders.

The microtubule-associated protein tau is a major cytoskeletal protein involved in the neurofibrillary tangles of Alzheimer's disease. Although tau is predominantly a neuronal protein, it has been demonstrated in glia and other nonneuronal cells. We describe the presence of microtubule-associated protein tau epitopes in various muscle fiber lesions in oculopharyngeal and Becker muscular dystrophy, dermatomyositis, central core disease, neurogenic atrophy, and in the recovery phase of an attack of malignant hyperthermia. Western blot demonstrated a 100- to 110-kd tau-immunoreactive protein probably corresponding to 'big tau' as described in peripheral nerves. Tau immunoreactivity in muscle fiber lesions usually co-localized with tubulin, although electron microscopy failed to show an increase in microtubules. Tau and tubulin reactivity also correlated with the presence of desmin and vimentin epitopes. Possible explanations for the presence of tau are briefly discussed.     

Localization of the Alz-50 epitope in recombinant human microtubule-associated protein tau.

Alz-50 is a monoclonal antibody that stains the neurofibrillary pathology of Alzheimer's disease, as well as apparently normal nerve cells that are at risk of developing neurofibrillary tangles. On immunoblots it recognizes microtubule-associated protein tau and proteins of 60-68 kDa that are associated with Alzheimer's disease. We have used recombinant tau proteins expressed in E. coli to map the Alz-50 epitope to amino-terminal residues 2-10, a region common to all known human tau isoforms. A direct correspondence between immunoblots and histological staining was established by the abolition of Alz-50 staining following adsorption with recombinant tau proteins retaining amino-terminal sequences. This suggests that tau pathology represents an early event in the development of the neurofibrillary pathology of Alzheimer's disease.     

First transgenic rat model developing progressive cortical neurofibrillary tangles

Neurofibrillary degeneration induced by misfolded protein tau is considered to be one of the key pathological hallmarks of Alzheimer's disease (AD). In the present study, we have introduced a novel transgenic rat model expressing a human truncated tau that encompasses 3 microtubule binding domains (3R) and a proline-rich region (3R tau151-391). The transgenic rats developed progressive age-dependent neurofibrillary degeneration in the cortical brain areas. Neurofibrillary tangles (NFTs) satisfied several key histological criteria used to identify neurofibrillary degeneration in human Alzheimer's disease including argyrophilia, Congo red birefringence, and Thioflavin S reactivity. Neurofibrillary tangles were also identified with antibodies used to detect pathologic tau in the human brain, including DC11, recognizing an abnormal tau conformation and antibodies that are specific for hyperphosphorylated forms of tau protein. Moreover, neurofibrillary degeneration was characterized by extensive formation of sarkosyl insoluble tau protein complexes consisting of rat endogenous and truncated tau species. Interestingly, the transgenic rats did not show neuronal loss either in the cortex or in the hippocampus. We suggest that novel transgenic rat model for human tauopathy represents a valuable tool in preclinical drug discovery targeting neurofibrillary degeneration of Alzheimer's type.     

Cytoskeletal protein pathology and the formation of beta-amyloid fibers in Alzheimer's disease

Discovery of the abnormally phosphorylated tau in paired helical filaments, its accumulation preceding the formation of the tangles and the in vitro microtubule assembly defect suggest that an abnormality in the protein phosphorylation/dephosphorylation system is involved in the pathogenesis of Alzheimer cytoskeletal pathology. The levels of mRNA for the beta-amyloid precursor protein (beta APP) in the brain suggest that only a small deficiency in the processing of the precursor would be sufficient to account for the accumulation of beta-amyloid in Alzheimer brain. Identification of reticuloendothelial system cells responsible for the production/processing of beta-amyloid will help to elucidate the pathogenesis of the brain amyloidosis. The disproportionate accumulation of paired helical filaments and amyloid within the same affected brain and from disease to disease raises the possibility of different etiologies for each of these lesions coexisting in Alzheimer's disease.     

Mitogen-activated protein kinases and the evolution of Alzheimer's: a revolutionary neurogenetic axis for therapeutic intervention?

Alzheimer's disease (AD) is a neurogenetic condition that affects the processes via which the brain functions. Major observable hallmarks of AD are accumulated clusters of proteins in the brain. These clusters, termed neurofibrillary tangles (NFT), resemble pairs of threads wound around each other in a helix fashion accumulating within neurons. These tangles consist of a protein called Tau, which binds to tubulin, thus forming microtubules. Unlike NFTs, deposits of amyloid precursor protein (beta-APP) gather in the spaces between nerve cells. The nearby neurons often look swollen and deformed, and the clusters of protein are usually accompanied by reactive inflammatory cells, microglia, which are part of the brain's immune system responsible for degrading and removing damaged neurons or plaques. Since phosphorylation/dephosphorylation mechanisms are crucial in the regulation of Tau and beta-APP, a superfamily of mitogen-activated protein kinases (MAPKs) has recently emerged as key regulators of the formation of plagues, eventually leading to dementia and AD. The complex molecular interactions between MAPKs and proteins (plagues) associated with the evolution of AD form a cornerstone in the knowledge of a still burgeoning field of neurodegenerative diseases and ageing. This review overviews current understanding of the molecular pathways related to MAPKs and their role in the development of AD and, possibly, dementia. MAPKs, therefore, may constitute a neurogenetic, therapeutic target for the diagnosis and evolution of a preventative medical strategy for early detection, and likely treatment, of Alzheimer's.     

Induction of tau pathology by intracerebral infusion of amyloid-beta -containing brain extract and by amyloid-beta deposition in APP x Tau transgenic mice

Alzheimer's disease presents morphologically with senile plaques, primarily made of extracellular amyloid-beta (A beta) deposits, and neurofibrillary lesions, which consist of intracellular aggregates of hyperphosphorylated tau protein. To study the in vivo induction of tau pathology, dilute brain extracts from aged A beta-depositing APP23 transgenic mice were intracerebrally infused in young B6/P301L tau transgenic mice. Six months after the infusion, tau pathology was induced in the injected hippocampus but also in brain regions well beyond the injection sites such as the entorhinal cortex and amygdala, areas with neuronal projection to the injection site. No or only modest tau induction was observed when brain extracts from aged nontransgenic control mice and aged tau-depositing B6/P301L transgenic mice were infused. To further study A beta-induced tau lesions B6/P301L tau transgenic mice were crossed with APP23 mice. Although A beta deposition in double-transgenic mice did not differ from single APP23 transgenic mice, double-transgenic mice revealed increased tau pathology compared to single B6/P301L tau transgenic mice predominately in areas with high A beta plaque load. The present results suggest that both extract-derived A beta species and deposited fibrillary A beta can induce the formation of tau neurofibrillary pathology. The observation that infused A beta can trigger the tau pathology in the absence of A beta deposits provides an explanation for the discrepancy between the neuroanatomical location of A beta deposits and the development and spreading of tau lesions in Alzheimer's disease brain.     

The Alzheimer's disease-associated gamma-secretase complex: functional domains in the presenilin 1 protein

Alzheimer's disease is neuropathologically characterized by the presence of neurofibrillary tangles and amyloid plaques in the brain. Amyloid plaques are extracellular deposits primarily composed of the amyloid beta-peptide, which is derived from the amyloid beta-precursor protein (APP) by sequential cleavages at the beta-secretase and gamma-secretase sites. gamma-Secretase cleavage is performed by a high molecular weight protein complex containing presenilin (PS), nicastrin, Aph-1 and Pen-2. The gamma-secretase complex is an unusual transmembrane aspartyl protease that cleaves APP within the transmembrane domain. In addition to APP, a large number of other single membrane-spanning proteins have been shown to be cleaved within their transmembrane domains by the gamma-secretase complex in a process referred to as regulated intramembrane proteolysis. Here we review recent research leading to the identification and understanding of the gamma-secretase complex components with emphasis on PS, which harbors the catalytic site. In addition, we summarize our own work focused on identifying and studying domains in PS1 that are critical for mediating gamma-secretase activity. Biochemical understanding of the gamma-secretase complex is important from a basic biological and physiological point of view, and could help in the development of small molecules that modulate gamma-secretase processing in an APP-specific manner.     

Biomarkers and evolution in Alzheimer disease

Brain regions and their highly neuroplastic long axonal connections that expanded rapidly during hominid evolution are preferentially affected by Alzheimer disease. There is no natural animal model with full disease pathology (neurofibrillary tangles and neuritic amyloid plaques of a severity seen in Alzheimer's disease brains). Biomarkers such as reduced glucose metabolism in association neocortex, defects in long white matter tracts, RNA neurochemical changes, and high CSF levels of total and phosphorylated tau protein, which are helpful to identify MCI and preclinical Alzheimer disease patients, may also provide insights into what brain changes led to this disease being introduced during hominid evolution.