Non-phosphorylated Tau Research Articles

The Synaptic Accumulation of Hyperphosphorylated Tau Oligomers in Alzheimer Disease Is Associated With Dysfunction of the Ubiquitin-Proteasome System

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys(245)-Ala(277). p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1-42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Crowded Cell-like Environment Accelerates the Nucleation Step of Amyloidogenic Protein Misfolding

To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244-441), when hyperphosphorylated by glycogen synthase kinase-3beta, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244-441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human alpha-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human alpha-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.

Fluorescent BODIPY-Based Zn(II) Complex as a Molecular Probe for Selective Detection of Neurofibrillary Tangles in the Brains of Alzheimer’s Disease Patients

We have developed a new fluorescent binuclear Zn(II) complex for the detection of neurofibrillary tangles (NFTs) of hyperphosphorylated tau proteins, a representative hallmark of Alzheimer's disease (AD). The probe 1 incorporates a fluorescent BODIPY unit and two Zn(II)-2,2'-dipicolylamine (Dpa) complexes as a binding site for phosphorylated amino acid residues. Using fluorescence titration to evaluate the binding and sensing properties of 1 toward several phosphorylated peptide segments derived from hyperphosphorylated tau protein, we found that 1 binds preferentially to peptides presenting phosphorylated groups at the i and i+4 positions with dissociation constants (K(d)) in the micromolar range. Fluorescence titration with an artificially prepared aggregate of the phosphorylated tau protein (p-Tau) revealed that 1 binds strongly to p-Tau (EC(50) = 9 nM). In contrast, the interactions of 1 were weaker toward artificially prepared aggregates of the nonphosphorylated tau protein (n-Tau; EC(50) = 80 nM) and Abeta(1-42) fibrils (EC(50) = 650 nM). Histological imaging of a hippocampus tissue section obtained from an AD patient revealed that 1 fluorescently visualizes deposits of NFTs and clearly discriminates between NFTs and the amyloid plaques assembled from amyloid-beta peptides, confirming our strategy toward the rational design of a molecular probe for the selective fluorescence detection of NFTs in brain tissue sections.

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.

キナーゼプロファイリングのための新しいリン酸アフィニティー電気泳動法

We developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn2+-Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.

Differential Changes in Phosphorylation of Tau at PHF-1 and 12E8 Epitopes During Brain Ischemia and Reperfusion in Gerbils

Cortical neurons are vulnerable to ischemic insult, which may cause cytoskeletal changes and neurodegeneration. Tau is a microtubule-associated protein expressed in neuronal and glial cells. We examined the phosphorylation status of tau protein in the gerbil brain cortex during 5 min ischemia induced by bilateral common carotid artery occlusion followed by reperfusion for 20 min to 7 days. Control brain homogenates contained 63, 65 and 68 kD polypeptides of tau immunoreactive with Alz 50, Tau 14 and Tau 46 antibodies raised against non-phosphorylated tau epitopes. Gerbil tau was also immunoreactive with some (PHF-1 and 12E8) but not all (AT8, AT100, AT180 and AT270) antibodies raised against phosphorylated tau epitopes. PHF-1 recognized a single 68 kD polypeptide and 12E8 bound the 63 kD polypeptide. During 5 min ischemia, PHF-1 immunoreactivity declined to 6%, then recovered to control levels after 20 min of blood recirculation and subsequently increased above control values 3 and 7 days later. In contrast, 12E8 immunoreactivity remained stable during ischemia and reperfusion. Our results suggest that the two phosphorylated epitopes of tau are regulated by different mechanisms and may play different roles in microtubule dynamics. They may also define various pools of neuronal/glial cells vulnerable to ischemia.

The involvement of glycogen synthase kinase-3 and protein phosphatase-2A in lactacystin-induced tau accumulation

Here, we demonstrated that lactacystin inhibited proteasome dose-dependently in HEK293 cells stably expressing tau. Simultaneously, it induces accumulation of both non-phosphorylated and hyperphosphorylated tau and decreases the binding of tau to the taxol-stabilized microtubules. Lactacystin activates glycogen synthase kinsase-3 (GSK-3) and decreases the phosphorylation of GSK-3 at serine-9. LiCl inhibits GSK-3 and thus reverses the lactacystin-induced accumulation of the phosphorylated tau. Lactacystin also inhibits protein phosphase-2A (PP-2A) and it significantly increases the level of inhibitor 1 of PP-2A. These results suggest that inhibition of proteasome by lactacystin induces tau accumulation and activation of GSK-3 and inhibition of PP-2A are involved.

Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease.

To date, there is no reasonable explanation as to why plaques and tangles simultaneously accumulate in Alzheimer's disease (AD). We demonstrate here by Western blotting and ELISA that a stable complex can form between tau and amyloid-beta protein (Abeta). This complex enhances tau phosphorylation by GSK3beta, but the phosphorylation then promotes dissociation of the complex. We have localized the sites of this interaction by using peptide membrane arrays. Abeta binds to multiple tau peptides, especially those in exons 7 and 9. This binding is sharply reduced or abolished by phosphorylation of specific serine and threonine residues. Conversely, tau binds to multiple Abeta peptides in the mid to C-terminal regions of Abeta. This binding is also significantly decreased by GSK3beta phosphorylation of tau. We used surface plasmon resonance to determine the binding affinity of Abeta for tau and found it to be in the low nanomolar range and almost 1,000-fold higher than tau for itself. In soluble extracts from AD and control brain tissue, we detected Abeta bound to tau in ELISAs. We also found by double immunostaining of AD brain tissue that phosphorylated tau and Abeta form separate insoluble complexes within the same neurons and their processes. We hypothesize that in AD, an initial step in the pathogenesis may be the intracellular binding of soluble Abeta to soluble nonphosphorylated tau, thus promoting tau phosphorylation and Abeta nucleation. Blocking the sites where Abeta initially binds to tau might arrest the simultaneous formation of plaques and tangles in AD.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Microtubule-associated protein tau is a substrate of ATP/Mg(2+)-dependent proteasome protease system.

Deposition of hyperphosphorylated microtubule-associated protein tau is a recognized pathological process in Alzheimer's disease (AD) brain, however, the mechanism leading to tau accumulation is still not understood. In the present study, we found that different forms of tau, including phosphorylated tau (PHF-1) and non-phosphorylated tau (Tau-1) as well as total tau (Tau-5) in rat brain cortex extract, were degraded when it was co-incubated with ATP and MgCl(2) at 33 degrees C in vitro, and non-phosphorylated tau at Tau-1 epitope was more accessible to the ATP/Mg(2+)-depended proteolysis. With the increase of ATP and MgCl(2) concentration from 5 mM to 20 mM, increased degradation of tau was observed. ATP/Mg(2+)-induced degradation of tau was blocked by lactacystin, a specific proteasome inhibitor and was enhanced by sodium dodecyl sulphate (SDS), a commonly used in vitro proteasome activator, and polyubiquitinated tau with high molecular weight was detected in the presence of lactacystin. Hyperphosphorylated tau isolated from AD brain (AD p-tau) was also partially degraded when it was incubated with rat brain cortex extract in the present of ATP/Mg(2+), and the degradation of AD p-tau was also enhanced by SDS and was inhibited by lactacystin. This study has demonstrated that tau, both phosphorylated and non-phosphorylated, is a substrate of ATP/Mg(2+)-depended proteasome. To our knowledge, this is the first report providing direct evidence that tau is degraded by 26S proteasome in an ubiquitin- and ATP-dependent manner.

Binding of Tau to Heat Shock Protein 27 Leads to Decreased Concentration of Hyperphosphorylated Tau and Enhanced Cell Survival

Pathological hyperphosphorylated tau is the principal component of paired helical filaments, a pathological hallmark of Alzheimer disease (AD) and a strong candidate for a neurotoxic role in AD and other neurodegenerative disorders. Here we show that heat shock protein 27 (Hsp27) preferentially binds pathological hyperphosphorylated tau and paired helical filaments tau directly but not non-phosphorylated tau. The formation of this complex altered the conformation of pathological hyperphosphorylated tau and reduced its concentration by facilitating its degradation and dephosphorylation. Moreover, Hsp27 rescues pathological hyperphosphorylated tau-mediated cell death. Therefore, Hsp27 is likely to provide a neuroprotective effect in AD and other tauopathies.

Coupled reductions in brain oxidative phosphorylation and synaptic function can be quantified and staged in the course of Alzheimer disease.

In vivo, post-mortem and biopsy data suggest that coupled declines occur in brain synaptic activity and brain energy consumption during the evolution of Alzheimer disease. In the first stage of these declines, changes in synaptic structure and function reduce neuronal energy demand and lead to potentially reversible downregulation of oxidative phosphorylation (OXPHOS) within neuronal mitochondria. At this stage, measuring brain glucose metabolism or brain blood flow in patients, using positron emission tomography (PET), shows that the brain can be almost normally activated in response to stimulation. Thus, therapy at this stage should be designed to re-establish synaptic integrity or prevent its further deterioration. As disease progresses, neurofibrillary tangles with abnormally phosphorylated tau protein accumulate within neuronal cytoplasm, to the point that they co-opt the nonphosphorylated tau necessary for axonal transport of mitochondria between the cell nucleus and the synapse. In this second stage, severe energy depletion and other pathological processes associated with irreversibly downregulated OXPHOS lead to cell death, and the brain cannot normally respond to functional stimulation.

Glycogen Synthase Kinase-3β Is Complexed with Tau Protein in Brain Microtubules

In Alzheimer's disease, microtubule-associated protein tau is hyperphosphorylated by an unknown mechanism and is aggregated into paired helical filaments. Hyperphosphorylation causes loss of tau function, microtubule instability, and neurodegeneration. Glycogen synthase kinase-3beta (GSK3beta) has been implicated in the phosphorylation of tau in normal and Alzheimer's disease brain. The molecular mechanism of GSK3beta-tau interaction has not been clarified. In this study, we find that when microtubules are disassembled, microtubule-associated GSK3beta dissociates from microtubules. From a gel filtration column, the dissociated GSK3beta elutes as an approximately 400-kDa complex. When fractions containing the approximately 400-kDa complex are chromatographed through an anti-GSK3beta immunoaffinity column, tau co-elutes with GSK3beta. From fractions containing the approximately 400-kDa complex, both tau and GSK3beta co-immunoprecipitate with each other. GSK3beta binds to nonphosphorylated tau, and the GSK3beta-binding region is located within the N-terminal projection domain of tau. In vitro, GSK3beta associates with microtubules only in the presence of tau. From brain extract, approximately 6-fold more GSK3beta co-immunoprecipitates with tau than GSK3alpha. These data indicate that, in brain, GSK3beta is bound to tau within a approximately 400-kDa microtubule-associated complex, and GSK3beta associates with microtubules via tau.

Divergent influences of protein kinase C isoforms on intracellular levels of phosphorylated and non-phosphorylated tau immunoreactivity

Stable transfectant subclones of SH-SY-5Y cells expressing antisense-oriented mRNA directed against PKCα (“α-transfectant”) and PKCϵ (“ϵ-transfectant”) expressed reduced levels of target isoforms, yet maintenance of normal levels of heterologous PKC isoforms as compared to those transfected with the plasmid vector only or the parental SH-SY-5Y cell line. Downregulation of PKCα and ϵ had differential effects on phospho-dependent and -independent tau immunoreactivity. Total tau immunoreactivity was reduced in both transfectant subclones as compared to that in the parental line. By contrast, only the ϵ-transfectant contained significantly lower levels of PHF-1 immunoreactivity, while the α-transfectant contained similar levels to that of the parental line. Our use of the complementary antibodies AT-8 and Tau-1, which recognize the same site when phosphorylated or nonphosphorylated, respectively, allowed us to monitor more precisely the relationship between tau phosphorylation and proteolytic degradation. Although significantly reduced in both transfectants, approximately twice as much AT-8 immunoreactivity was present in the α-transfectant as compared to the ϵ-transfectant. Tau-1 immunoreactivity was also drastically reduced in both transfectants. These data provide suggest that site-specific phosphorylation of tau renders it resistant to degradation, and, given the pivotal role of PKC in signal transduction pathways, suggest that a complex interplay of signal transduction events may underscore the onset and/or progression of AD.

Phosphorylation of tau by glycogen synthase kinase 3β affects the ability of tau to promote microtubule self-assembly

To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Axonal origin and purity of growth cones isolated from fetal rat brain

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of ‘growth cone particles’ (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Axonal origin and purity of growth cones isolated from fetal rat brain

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of ‘growth cone particles’ (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans.

The paired helical filament (PHF) is the major component of the neurofibrillary deposits that form a defining neuropathological characteristic of Alzheimer's disease. PHFs are composed of microtubule-associated protein tau, in a hyperphosphorylated state. Hyperphosphorylation of tau results in its inability to bind to microtubules and is believed to precede PHF assembly. However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation. Here we show that non-phosphorylated recombinant tau isoforms with three microtubule-binding repeats form paired helical-like filaments under physiological conditions in vitro, when incubated with sulphated glycosaminoglycans such as heparin or heparan sulphate. Furthermore, heparin prevents tau from binding to microtubules and promotes microtubule disassembly. Finally, we show that heparan sulphate and hyperphosphorylated tau coexist in nerve cells of the Alzheimer's disease brain at the earliest known stages of neurofibrillary pathology. These findings, with previous studies which show that heparin stimulates tau phosphorylation by a number of protein kinases, indicate that sulphated glycosaminoglycans may be a key factor in the formation of the neurofibrillary lesions of Alzheimer's disease.