Protein kinase C-zeta phosphorylates insulin-responsive aminopeptidase in vitro at Ser-80 and Ser-91

Insulin-responsive aminopeptidase (IRAP) colocalizes with glucose transporter type 4 (GLUT4) in adipocytes and is recruited to the plasma membrane in response to insulin. Microinjection of peptides corresponding to the IRAP cytoplasmic domain sequences causes GLUT4 recruitment in adipocytes. Inhibitors of protein kinase C-zeta (PKC-zeta) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-zeta may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the (32)P incorporation into IRAP catalyzed by PKC-zeta in insulin-stimulated cells. There was a small but significant (32)P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-zeta pseudosubstrate, suggesting that PKC-zeta may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-zeta also catalyzed the incorporation of (32)P not only into IRAP in GLUT4 vesicles isolated from rat adipocytes but also into the IRAP cytoplasmic domain inserts in glutathione S-transferase-fusion proteins, demonstrating direct IRAP phosphorylation by PKC-zeta. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the (32)P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the (32)P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP(76-138) and the (32)P labeling at Ser-80 accounted for 80-90% of the total IRAP labeling, suggesting that Ser-80 is the major phosphorylation site in intact IRAP. These findings are consistent with the possibility that the IRAP cytoplasmic domain phosphorylation by PKC-zeta plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.     

Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation

Neurofibrillary tangles, which are major pathological hallmarks of Alzheimer's disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau-tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+- and a Mn2+-dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose-dependent manner. We conclude that TTBK1 is a neuron-specific dual kinase involved in tau phosphorylation at AD-related sites and is also associated with tau aggregation.     

Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease

Alzheimer's disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFTs) formed of tau-based paired helical filaments (PHFs) and extracellular beta-amyloid plaques. The degree of Alzheimer dementia correlates with the severity of PHFs and NFTs. As an intraneuronal accumulation of oxidatively damaged proteins has been found in the brains of patients with AD, a dysfunction of the proteasomal system, which degrades damaged proteins, has been assumed to cause protein aggregation and therefore neurodegeneration in AD. In this study, we revealed that such proteasome dysfunction in AD brain results from the inhibitory binding of PHF-tau to proteasomes. We analysed the proteasome activity in brains from patients with AD and age-matched controls, and observed a significant decrease to 56% of the control level in the straight gyrus of patients with AD. This loss of activity was not associated with a decrease in the proteasome protein. PHF-tau co-precipitated during proteasome immunoprecipitation and proteasome subunits could be co-isolated during isolation of PHFs from AD brain. Furthermore, the proteasome activity in human brains strongly correlated with the amount of co-precipitated PHF-tau during immunoprecipitation of proteasome. Incubation of isolated proteasomes with PHF-tau isolated from AD brain, and with PHFs after in vitro assembly from human recombinant tau protein, resulted in a distinct inhibition of proteasome activity by PHF-tau. As this inhibition of proteasome activity was sufficient to induce neuronal degeneration and death, we suggest that PHF-tau is able directly to induce neuronal damage in the AD brain.     

A novel tubulin-dependent protein kinase forming a paired helical filament epitope on tau

From rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.     

Glycogen synthase kinase 3beta phosphorylates tau at both primed and unprimed sites. Differential impact on microtubule binding

Glycogen synthase kinase 3beta (GSK3beta) phosphorylates substrates, including the microtubule-associated protein tau, at both primed and unprimed epitopes. GSK3beta phosphorylation of tau negatively regulates tau-microtubule interactions; however the differential effects of phosphorylation at primed and unprimed epitopes on tau is unknown. To examine the phosphorylation of tau at primed and unprimed epitopes and how this impacts tau function, the R96A mutant of GSK3beta was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3beta and GSK3beta-R96A phosphorylated tau efficiently in situ. However, expression of GSK3beta-R96A resulted in significantly less phosphorylation of tau at primed sites compared with GSK3beta. Conversely, GSK3beta-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3beta. Prephosphorylating tau with cdk5/p25 impaired the ability of GSK3beta-R96A to phosphorylate tau, whereas GSK3beta-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3beta. Moreover, the amount of tau associated with microtubules was reduced by overexpression of GSK3beta but only when tau was phosphorylated at primed sites, as phosphorylation of tau by GSK3beta-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3beta-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.     

beta-Amyloid induces paired helical filament-like tau filaments in tissue culture

Paired helical filaments (PHF) are the principal pathologic components of neurofibrillary tangles in Alzheimer's disease (AD). To reproduce the formation of PHF in tissue culture, we stably expressed human tau with and without pathogenic mutations in human SH-SY5Y cells and exposed them for 5 days to aggregated synthetic beta-amyloid peptide (A beta 42). This caused a decreased solubility of tau along with the generation of PHF-like tau-containing filaments. These were 20 nm wide and had periodicities of 130-140 nm in the presence of P301L mutant tau or 150-160 nm in the presence of wild-type tau. Mutagenesis of the phosphoepitope serine 422 of tau prevented both the A beta 42-mediated decrease in solubility and the generation of PHF-like filaments, suggesting a role of serine 422 or its phosphorylation in tau filament formation. Together, our data underscore a role of A beta 42 in the formation of PHF-like filaments. Our culture system will be useful to map phosphoepitopes of tau involved in PHF formation and to identify and characterize modifiers of the tau pathology. Further adaptation of the system may allow the screening and validation of compounds designed to prevent PHF formation.     

Interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments

The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.     

Structure of tau protein and assembly into paired helical filaments

Over the past few years the systematic investigation of paired helical filament assembly from tau protein in vitro has become feasible. We review our current understanding of the structure and conformations of tau protein and how this affects tau's assembly into the pathological paired helical filaments in Alzheimer's disease.     

Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease

Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.     

Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.     

p21-activated kinase PAK phosphorylates desmin at sites different from those for Rho-associated kinase

p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.     

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.     

AMP-activated protein kinase phosphorylates cardiac troponin I at Ser-150 to increase myofilament calcium sensitivity and blunt PKA-dependent function

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme central to the regulation of metabolic homeostasis. In the heart AMPK is activated during cardiac stress-induced ATP depletion and functions to stimulate metabolic pathways that restore the AMP/ATP balance. Recently it was demonstrated that AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vitro. We sought to determine if the metabolic regulatory kinase AMPK phosphorylates cTnI at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrates that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. Thus, in addition to the defined role of AMPK as a cardiac metabolic energy gauge, these data demonstrate AMPK Ser-150 phosphorylation of cTnI directly links the regulation of cardiac metabolic demand to myofilament contractile energetics. Furthermore, the blunting effect of cTnI Ser-150 phosphorylation cross-talk can uncouple the effects of myofilament PKA-dependent phosphorylation from β-adrenergic signaling as a novel thin filament contractile regulatory signaling mechanism.     

Casein kinase 1 delta phosphorylates tau and disrupts its binding to microtubules

Tau hyperphosphorylation precedes neuritic lesion formation in Alzheimer's disease, suggesting it participates in the tau fibrillization reaction pathway. Candidate tau protein kinases include members of the casein kinase 1 (CK1) family of phosphotransferases, which are highly overexpressed in Alzheimer's disease brain and colocalize with neuritic and granulovacuolar lesions. Here we characterized the contribution of one CK1 isoform, Ckidelta, to the phosphorylation of tau at residues Ser202/Thr205 and Ser396/Ser404 in human embryonic kidney 293 cells using immunodetection and fluorescence microscopy. Treatment of cells with membrane permeable CK1 inhibitor 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) lowered occupancy of Ser396/Ser404 phosphorylation sites by >70% at saturation, suggesting that endogenous CK1 was the major source of basal phosphorylation activity at these sites. Overexpression of Ckidelta increased CK1 enzyme activity and further raised tau phosphorylation at residues Ser202/Thr205 and Ser396/Ser404 in situ. Inhibitor IC261 reversed tau hyperphosphorylation induced by Ckidelta overexpression. Co-immunoprecipitation assays showed direct association of tau and Ckidelta in situ, consistent with tau being a Ckidelta substrate. Ckidelta overexpression also produced a decrease in the fraction of bulk tau bound to detergent-insoluble microtubules. These results suggest that Ckidelta phosphorylates tau at sites that modulate tau/microtubule binding, and that the expression pattern of Ckidelta in Alzheimer's disease is consistent with it playing an important role in tau aggregation.     

Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.     

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.     

The conformations of filamentous and soluble tau associated with Alzheimer paired helical filaments

Paired helical filaments (PHF) occur in Alzheimer's diseased brains and are known to be composed of the microtubule-associated protein, tau. In the present report, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM) were used to characterize PHF suspended in Tris-buffered saline (TBS), sodium acetate buffer, and water. In TBS the CD spectrum of PHF was observed to have a spectral pattern consistent with 31-37% alpha-helix, 15-20% beta-sheet, 20-23% turn, and 26-29% unordered structure. The TBS sample was found to undergo a cooperative thermal transition between 70 and 75 degrees C, consistent with the changes observed in filament morphology, and it suggests that filamentous tau in the PHF (PHF-tau) makes a substantial contribution to the overall CD. Observed changes in the CD spectrum following removal of PHF by centrifugation suggest that PHF-tau possesses a higher fraction of alpha-helical structure than soluble tau. In acetate buffer, where only straight filaments were observed, the CD was consistent with a marked decrease in the fraction of alpha-helix and an increase in the fraction of beta-sheet relative to the sample in TBS. In water, where only rudimentary filaments remain, the CD was consistent with a Type II or II' beta-turn conformation. Only noncooperative thermal transitions were observed for the PHF samples in acetate buffer and water, consistent with the presence of a heterogeneous population of folded structures. Taken cumulatively, the results are consistent with immunological data showing the presence of folded forms of tau and suggest that phosphorylation or nonproteinaceous components are able to induce conformations of tau other than the random coil conformation previously reported for cloned or purified human tau.     

Deamidation and isoaspartate formation in smeared tau in paired helical filaments. Unusual properties of the microtubule-binding domain of tau

An extensive loss of a selected population of neurons in Alzheimer's disease is closely related to the formation of paired helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160), smeared tau was purified, and its peptide map was compared with that of soluble (normal) tau. A CNBr fragment from soluble tau (CN5; residues 251-419 according to the 441-residue isoform) containing the microtubule-binding domain migrated at 15 and 18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from smeared tau exhibited two larger, unusually broad bands at approximately 30 and approximately 45 kDa, presumably representing dimers and trimers of CN5. In the peptide map of smeared tau-derived CN5, distinct peaks eluting at unusual locations were noted. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other than aspartyl or isoaspartyl peptide were found in the digests of smeared tau-derived CN5, it is likely that site-specific deamidation and isoaspartate formation are involved in its dimerization and trimerization and thus in PHF formation in vivo.     

Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease.

Transglutaminase-catalyzed epsilon(gamma-glutamyl)lysine cross-links exist in Alzheimer's disease (AD) paired helical filament (PHF) tau protein but not normal soluble tau. To test the hypothesis that these cross-links could play a role in the formation of neurofibrillary tangles (NFT), we used single- and double-label immunofluorescence confocal microscopy and immunoaffinity purification and immunoblotting to examine epsilon(gamma-glutamyl)lysine cross-links in AD and control brains. The number of neurons that are immunoreactive with an antibody directed at the epsilon-(gamma-glutamyl)lysine bond was significantly higher in AD cortex compared with age-matched controls and schizophrenics. PHF tau-directed antibodies AT8, MC-1 and PHF-1 co-localized with epsilon(gamma-glutamyl)lysine immunolabeling in AD NFT. Immunoaffinity purification and immunoblotting experiments demonstrated that PHF tau contains epsilon(gamma-glutamyl)lysine bonds in parietal and frontal cortex in AD. In control cases with NFT present in the entorhinal cortex and hippocampus, indicative of Braak and Braak stage II, epsilon(gamma-glutamyl)lysine bonds were present in PHF tau in parietal and frontal cortex, despite the lack of microscopically detectable NFT or senile plaques in these cortical regions. The presence of PHF tau with epsilon(gamma-glutamyl)lysine bonds in brain regions devoid of NFT in stage II (but regions, which would be expected to contain NFT in stage III) suggests that these bonds occur early in the formation of NFT.     

The natively unfolded character of tau and its aggregation to Alzheimer-like paired helical filaments

The abnormal aggregation of the microtubule-associated protein Tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer disease (AD). Tau in solution behaves as a natively unfolded or intrinsically disordered protein while its aggregation is based on the partial structural transition from random coil to beta-structure. Our aim is to understand in more detail the unfolded nature of Tau, to investigate the aggregation of Tau under different conditions and the molecular interactions of Tau in filaments. We show that soluble Tau remains natively unfolded even when its net charge is minimized, in contrast to other unfolded proteins. The CD signature of the random-coil character of Tau shows no major change over wide variations in charge (pH), ionic strength, solvent polarity, and denaturation. Thus there is no indication of a hydrophobicity-driven collapse, neither in the microtubule-binding repeat domain constructs nor in full-length Tau. This argues that the lack of hydrophobic residues but not the net charge accounts for unfolded nature of soluble Tau. The aggregation of the Tau repeat domain (that forms the core of PHFs) in the presence of nucleating polyanionic cofactors (heparin) is efficient in a range of buffers and pH values between approximately 5 and 10 but breaks down beyond that range, presumably because the pattern of charged interactions disappears. Similarly, elevated ionic strength attenuates aggregation, and the temperature dependence is bell-shaped with an optimum around 50 degrees C. Reporter dyes ThS and ANS record the aggregation process but sense different states (cross-beta-structure vs hydrophobic pockets) with different kinetics. Preformed PHFs are surprisingly labile and can be disrupted by denaturants at rather low concentration ( approximately 1.0 M GdnHCl), much less than required to denature globular proteins. Partial disaggregation of Tau filaments at extreme pH values monitored by CD and EM indicate the importance of salt bridges in filament formation. In contrast, Tau filaments are remarkably resistant to high temperature and high ionic strength. Overall, the stability of PHFs appears to depend mainly on directed salt bridges with contributions from hydrophobic interactions as well, consistent with a recent structural model of the PHF core derived from solid state NMR (Andronesi, O. C., von Bergen, M., Biernat, J., Seidel, K., Griesinger, C., Mandelkow, E., and Baldus, M. (2008) Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.