Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease.

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.

Prefibrillar Tau oligomers alter the nucleic acid protective function of Tau in hippocampal neurons in vivo.

The accumulation of DNA and RNA oxidative damage is observed in cortical and hippocampal neurons from Alzheimer's disease (AD) brains at early stages of pathology. We recently reported that Tau is a key nuclear player in the protection of neuronal nucleic acid integrity in vivo under physiological conditions and hyperthermia, a strong inducer of oxidative stress. In a mouse model of tauopathy (THY-Tau22), we demonstrate that hyperthermia selectively induces nucleic acid oxidative damage and nucleic acid strand breaks in the nucleus and cytoplasm of hippocampal neurons that display early Tau phosphorylation but no Tau fibrils. Nucleic acid-damaged neurons were exclusively immunoreactive for prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor and a redox cycler, reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage. This study clearly highlights the existence of an early and critical time frame for hyperthermia-induced Tau oligomerization, which most likely occurs through increased oxidative stress, and nucleic acid vulnerability during the progression of Tau pathology. These results suggest that at early stages of AD, Tau oligomerization triggers the loss of the nucleic acid protective function of monomeric Tau. This study highlights the existence of a short therapeutic window in which to prevent the formation of pathological forms of Tau and their harmful consequences on nucleic acid integrity during the progression of Tau pathology.

TOC1: a valuable tool in assessing disease progression in the rTg4510 mouse model of tauopathy.

All tauopathies result in various forms of cognitive decline and neuronal loss. Although in some diseases, tau mutations appear to cause neurodegeneration, the toxic "form" of tau remains elusive. Tau is the major protein found within neurofibrillary tangles (NFTs) and therefore it seemed rational to assume that aggregation of tau monomers into NFTs was causal to the disease process. However, the appearance of oligomers rather than NFTs coincides much better with the voluminous neuronal loss in many of these diseases. In this study, we utilized the bigenic mouse line (rTg4510) which conditionally expresses P301L human tau. A novel tau antibody, termed Tau Oligomer Complex 1 (TOC1) was employed to probe mouse brains and assess disease progression. TOC1 selectively recognizes dimers/oligomers and appears to constitute an early stage marker of tau pathology. Its peak reactivity is coincident with other well-known early stage pathological markers such as MC1 and the early-stage phospho-marker CP13. TOC1's reactivity depends on the conformation of the tau species since it does not react with monomer under native conditions, although it does react with monomers under SDS-denaturation. This indicates a conformational change must occur within the tau aggregate to expose its epitope. Tau oligomers preferentially form under oxidizing conditions and within this mouse model, we observe tau oligomers forming at an increased rate and persisting much longer, most likely due to the aggressive P301L mutation. With the help of other novel antibodies, the use of this antibody will aid in providing a better understanding of tau toxicity within Alzheimer's disease and other tauopathies.

Prefibrillar tau oligomers in mild cognitive impairment and Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of extracellular amyloid-ß peptide and intracellular tau. Here, we review data suggesting that prefibrillar tau oligomers mediate cognitive decline early in the disease. OBJECTIVE: It was our aim to study the presence of tau-positive pretangle neurons and correlate findings with cognitive test scores. METHODS: Pretangle antibodies (TOC1 and pS422) were applied to tissue containing cholinergic basal forebrain neurons from people who died with a premortem clinical diagnosis of no cognitive impairment, mild cognitive impairment and AD. RESULTS: Data lend support to the concept that tau oligomers are the toxic form of tau, that non-fibillar tau relates to cognitive dysfunction and that the earliest pretangle pathology occurs in neuritic processes. CONCLUSIONS: Clinicopathological findings highlight the importance of studying tau modifications in neuronal soma and neuritic processes, which may be the earliest pathological lesions that correlate with cognitive status.

Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases.

Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2-18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.

In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice.

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Hsp70 alters tau function and aggregation in an isoform specific manner.

Tauopathies are characterized by abnormal aggregation of the microtubule associated protein tau. This aggregation is thought to occur when tau undergoes shifts from its native conformation to one that exposes hydrophobic areas on separate monomers, allowing contact and subsequent association into oligomers and filaments. Molecular chaperones normally function by binding to exposed hydrophobic stretches on proteins and assisting in their refolding. Chaperones of the heat shock protein 70 (Hsp70) family have been implicated in the prevention of abnormal tau aggregation in adult neurons. Tau exists as six alternatively spliced isoforms, and all six isoforms appear capable of forming the pathological aggregates seen in Alzheimer's disease. Because tau isoforms differ in primary sequence, we sought to determine whether Hsp70 would differentially affect the aggregation and microtubule assembly characteristics of the various tau isoforms. We found that Hsp70 inhibits tau aggregation directly and not through inducer-mediated effects. We also determined that Hsp70 inhibits the aggregation of each individual tau isoform and was more effective at inhibiting the three repeat isoforms. Finally, all tau isoforms robustly induced microtubule formation while in the presence of Hsp70. The results presented herein indicate that Hsp70 affects tau isoform dysfunction while having very little impact on the normal function of tau to mediate microtubule assembly. This indicates that targeting Hsp70 to tau may provide a therapeutic approach for the treatment of tauopathies that avoids disruption of normal tau function.

Characterization of prefibrillar Tau oligomers in vitro and in Alzheimer disease.

Neurofibrillary tangles, composed of insoluble aggregates of the microtubule-associated protein Tau, are a pathological hallmark of Alzheimer disease (AD) and other tauopathies. However, recent evidence indicates that neuronal dysfunction precedes the formation of these insoluble fibrillar deposits, suggesting that earlier prefibrillar Tau aggregates may be neurotoxic. To determine the composition of these aggregates, we have employed a photochemical cross-linking technique to examine intermolecular interactions of full-length Tau in vitro. Using this method, we demonstrate that dimerization is an early event in the Tau aggregation process and that these dimers self-associate to form larger oligomeric aggregates. Moreover, using these stabilized Tau aggregates as immunogens, we generated a monoclonal antibody that selectively recognizes Tau dimers and higher order oligomeric aggregates but shows little reactivity to Tau filaments in vitro. Immunostaining indicates that these dimers/oligomers are markedly elevated in AD, appearing in early pathological inclusions such as neuropil threads and pretangle neurons as well as colocalizing with other early markers of Tau pathogenesis. Taken as a whole, the work presented herein demonstrates the existence of alternative Tau aggregates that precede formation of fibrillar Tau pathologies and raises the possibility that these hierarchical oligomeric forms of Tau may contribute to neurodegeneration.

Tau oligomers and tau toxicity in neurodegenerative disease.

AD (Alzheimer's disease) is a progressive neurodegenerative disorder characterized by the extracellular accumulation of amyloid ß-peptide and the intracellular accumulation of tau. Although there is much evidence linking tau to neurodegeneration, the precise mechanism of tau-mediated neurotoxicity remains elusive. The presence of tau-positive pre-tangle neurons lacking neurofibrillary tangles has been reported in AD brain tissue. In order to study this non-fibrillar tau, we generated a novel monoclonal antibody, named TOC1 (tau oligomeric complex 1), which selectively labels tau dimers and oligomers, but does not label filaments. Time-course analysis and antibody labelling indicates that oligomers appear as an early event in AD pathogenesis. Using a squid axoplasm assay, we have demonstrated that aggregated tau inhibits anterograde FAT (fast axonal transport), whereas monomeric tau has no effect. This inhibition requires a small stretch of N-terminal amino acids termed the PAD (phosphatase-activation domain). Using a PAD-specific antibody, TNT1 (tau N-terminal 1), we demonstrate that PAD exposure is increased in diseased neurons and this leads to an increase in FAT inhibition. Antibody co-labelling with the early-AD marker AT8 indicates that, similar to TOC1, TNT1 expression represents an early event in AD pathogenesis. Finally, the effects of the molecular chaperone Hsp70 (heat-shock protein 70) were also investigated within the squid axoplasm assay. We illustrate that Hsp70 preferentially binds to tau oligomers over filaments and prevents anterograde FAT inhibition observed with a mixture of both forms of aggregated tau. Together, these findings support the hypothesis that tau oligomers are the toxic form of tau in neurodegenerative disease.

Progression of tau pathology in cholinergic Basal forebrain neurons in mild cognitive impairment and Alzheimer's disease.

Tau is a microtubule-associated protein that forms neurofibrillary tangles (NFTs) in the selective vulnerable long projection neurons of the cholinergic basal forebrain (CBF) in Alzheimer's disease (AD). Although CBF neurodegeneration correlates with cognitive decline during AD progression, little is known about the temporal changes of tau accumulation in this region. We investigated tau posttranslational modifications during NFT evolution within the CBF neurons of the nucleus basalis (NB) using tissue from subjects with no cognitive impairment, mild cognitive impairment, and AD. The pS422 antibody was used as an early tau pathology marker that labels tau phosphorylated at Ser422; the TauC3 antibody was used to detect later stage tau pathology. Stereologic evaluation of NB tissue immunostained for pS422 and TauC3 revealed an increase in neurons expressing these tau epitopes during disease progression. We also investigated the occurrence of pretangle tau events within cholinergic NB neurons by dual staining for the cholinergic cell marker, p75(NTR), which displays a phenotypic down-regulation within CBF perikarya in AD. As pS422+ neurons increased in number, p75(NTR)+ neurons decreased, and these changes correlated with both AD neuropathology and cognitive decline. Also, NFTs developed slower in the CBF compared with previously examined cortical regions. Taken together, these results suggest that changes in cognition are associated with pretangle events within NB cholinergic neurons before frank NFT deposition.

Tyrosine nitration within the proline-rich region of Tau in Alzheimer's disease.

A substantial body of evidence suggests that nitrative injury contributes to neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative disorders. Previously, we showed in vitro that within the tau protein the N-terminal tyrosine residues (Y18 and Y29) are more susceptible to nitrative modifications than other tyrosine sites (Y197 and Y394). Using site-specific antibodies to nitrated tau at Y18 and Y29, we identified tau nitrated in both glial (Y18) and neuronal (Y29) tau pathologies. In this study, we report the characterization of two novel monoclonal antibodies, Tau-nY197 and Tau-nY394, recognizing tau nitrated at Y197 and Y394, respectively. By Western blot analysis, Tau-nY197 labeled soluble tau and insoluble paired helical filament proteins (PHF-tau) nitrated at Y197 from control and AD brain samples. Tau-nY394 failed to label soluble tau isolated from control or severe AD samples, but labeled insoluble PHF-tau to a limited extent. Immunohistochemical analysis using Tau-nY197 revealed the hallmark tau pathology associated with AD; Tau-nY394 did not detect any pathological lesions characteristic of the disorder. These data suggest that a subset of the hallmark pathological inclusions of AD contain tau nitrated at Y197. However, nitration at Y197 was also identified in soluble tau from all control samples, including those at Braak stage 0, suggesting that nitration at this site in the proline-rich region of tau may have normal biological functions in the human brain.

Selective tau tyrosine nitration in non-AD tauopathies.

Previously, we reported the characterization of two novel antibodies that react with tau nitrated at tyrosine 197 (Tau-nY197) and tyrosine 394 (Tau-nY394) in Alzheimer's disease (AD). In this report, we examined whether tau nitration at these sites also occurs in corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and Pick's disease (PiD), three neurodegenerative tauopathies that contain abundant tau deposits within glial and neuronal cell types but lack amyloid deposition. The reactivity of these antibodies was also compared to two previously characterized antibodies Tau-nY18 and Tau-nY29, specific for tau nitrated at tyrosine 18 and tyrosine 29, respectively. In the present experiments, Tau-nY18 did not label the classical pathological lesions of CBD or PSP but did label the neuronal lesions associated with PiD to a limited extent. In contrast, Tau-nY29 revealed some, but not all classes of tau inclusions associated with both CBD and PSP but did label numerous Pick body inclusions in PiD. Tau-nY197 was restricted to the neuropil threads in both CBD and PSP; however, similar to Tau-nY29, extensive Pick body pathology was clearly labeled. Tau-nY394 did not detect any of the lesions associated with these disorders. In contrast, extensive neuronal and glial tau pathology within these diseases was labeled by Tau-Y197, a monoclonal antibody that reacts within the Y-197-containing proline-rich region of the molecule. Based on our Western and IHC experiments, it appears that nitration of tau at tyrosine 29 is a pathological modification that might be associated with neurodegeneration. Collectively, our data suggest that site-specific tau tyrosine nitration events occur in a disease and lesion-specific manner, indicating that nitration appears to be a highly controlled modification in AD and non-AD tauopathies.

Mild cognitive impairment: pathology and mechanisms.

Mild cognitive impairment (MCI) is rapidly becoming one of the most common clinical manifestations affecting the elderly. The pathologic and molecular substrate of people diagnosed with MCI is not well established. Since MCI is a human specific disorder and neither the clinical nor the neuropathological course appears to follow a direct linear path, it is imperative to characterize neuropathology changes in the brains of people who came to autopsy with a well-characterized clinical diagnosis of MCI. Herein, we discuss findings derived from clinical pathologic studies of autopsy cases who died with a clinical diagnosis of MCI. The heterogeneity of clinical MCI imparts significant challenges to any review of this subject. The pathologic substrate of MCI is equally complex and must take into account not only conventional plaque and tangle pathology but also a wide range of cellular, biochemical and molecular deficits, many of which relate to cognitive decline as well as compensatory responses to the progressive disease process. The multifaceted nature of the neuronal disconnection syndrome associated with MCI suggests that there is no single event which precipitates this prodromal stage of AD. In fact, it can be argued that neuronal degeneration initiated at different levels of the central nervous system drives cognitive decline as a final common pathway at this stage of the dementing disease process.

Peroxynitrite-induced nitrative and oxidative modifications alter tau filament formation.

Tau undergoes numerous posttranslational modifications during the progression of Alzheimer's disease (AD). Some of these changes accelerate tau aggregation, while others are inhibitory. AD-associated inflammation is thought to create oxygen and nitrogen radicals such as peroxynitrite (PN). In vitro, PN can nitrate many proteins, including tau. We have previously demonstrated that tau's ability to form filaments is profoundly affected by treatment with PN and have attributed this inhibition to tyrosine nitration. However, PN is highly reactive and unstable leading to oxidative amino acid modifications through its free radical byproducts. To test whether PN can modify other amino acids in tau via oxidative modifications, a mutant form of the tau protein lacking all tyrosines (5XY → F) was constructed. 5XY → F tau readily forms filaments; however, like wild-type tau the extent of polymerization was greatly reduced following PN treatment. Since 5XY → F tau cannot be nitrated, it was clear that nonnitrative modifications are generated by PN treatment and that these modifications change tau filament formation. Mass spectrometry was used to identify these oxidative alterations in wild-type tau and 5XY → F tau. PN-treated wild-type tau and 5XY → F tau consistently displayed lysine formylation throughout tau in a nonsequence-specific distribution. Lysine formylation likely results from reactive free radical exposure caused by PN treatment. Therefore, our results indicate that PN treatment of proteins in vitro cannot be used to study protein nitration as it likely induces numerous other random oxidative modifications clouding the interpretations of any functional consequences of tyrosine nitration.

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport.

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

Axonal transport defects in neurodegenerative diseases.

Adult-onset neurodegenerative diseases (AONDs) comprise a heterogeneous group of neurological disorders characterized by a progressive, age-dependent decline in neuronal function and loss of selected neuronal populations. Alterations in synaptic function and axonal connectivity represent early and critical pathogenic events in AONDs, but molecular mechanisms underlying these defects remain elusive. The large size and complex subcellular architecture of neurons render them uniquely vulnerable to alterations in axonal transport (AT). Accordingly, deficits in AT have been documented in most AONDs, suggesting a common defect acquired through different pathogenic pathways. These observations suggest that many AONDs can be categorized as dysferopathies, diseases in which alterations in AT represent a critical component in pathogenesis. Topics here address various molecular mechanisms underlying alterations in AT in several AONDs. Illumination of such mechanisms provides a framework for the development of novel therapeutic strategies aimed to prevent axonal and synaptic dysfunction in several major AONDs.

Aging analysis reveals slowed tau turnover and enhanced stress response in a mouse model of tauopathy.

We have extensively analyzed the biochemical and histochemical profiles of the tau protein from the rTg4510 transgenic mouse model in which the animals uniquely develop forebrain tau pathologies similar to those found in human tauopathies. Levels of several soluble phosphorylated tau species were highest at 1 month relative to later time points, suggesting that certain tau hyperphosphorylation events were insufficient to drive tangle formation in young mice. Despite a robust, pre-tangle-like accumulation of phospho-tau in 1-month-old mice, this material was cleared by 3 months, indicating that the young mouse brain either fails to facilitate tau insolubility or possesses an enhanced ability to clear tau relative to the adult. We also found that while heat shock protein expression increased with normal aging, this process was accelerated in rTg4510 mice. Moreover, by exploiting an exon 10 (-) specific antibody, we demonstrated that endogenous mouse tau turnover was slowed in response to human tau over-expression, and that this endogenous tau adopted disease-related properties. These data suggest that a younger brain fails to develop lasting tau pathology despite elevated levels of phosphorylated tau, perhaps because of reduced expression of stress-related proteins. Moreover, we show that the active production of small amounts of abnormal tau protein facilitates dysfunction and accumulation of otherwise normal tau, a significant implication for the pathogenesis of patients with Alzheimer's disease.

Tau 6D and 6P isoforms inhibit polymerization of full-length tau in vitro.

Alzheimer's disease and other tauopathies are characterized by the intracellular accumulation of insoluble filaments of the microtubule-associated protein tau. The six canonical tau isoforms in the adult brain consist of an N-terminal "projection" domain followed by a proline-rich region, a microtubule-binding repeat region, and a C-terminal tail. However, alternative splicing in exon 6 produces an additional set of tau isoforms, termed 6D and 6P, which contain only the N-terminus and part of the proline-rich region. We have previously shown that constructs representing N-terminal fragments of tau, which resemble the naturally occurring 6P and 6D isoforms, inhibit polymerization of the full-length protein in an in vitro filament formation assay and traced the inhibitory activity to amino acids 18-42. Here we report that 6P and 6D tau isoforms inhibit polymerization of full-length tau (hTau40) in a similar manner, likely by stabilizing full-length tau in a soluble conformation. The absence of exons 2 and 3 decreased the effectiveness of the 6D isoforms but not the 6P variants or the N-terminal tau fragments from our previous study, indicating that the 18-42 region is not the sole determinant of inhibitory ability. Finally, this paper demonstrates that inhibition is blocked by pseudophosphorylation of tyrosines 18 and 29, providing a potential link between tyrosine phosphorylation and disease progression. Taken together, these results indicate that the 6P/6D isoforms are potential endogenous inhibitors of tau filament formation and suggest a mechanism by which this ability may be disrupted in disease.

The amino terminus of tau inhibits kinesin-dependent axonal transport: implications for filament toxicity.

The neuropathology of Alzheimer's disease (AD) and other tauopathies is characterized by filamentous deposits of the microtubule-associated protein tau, but the relationship between tau polymerization and neurotoxicity is unknown. Here, we examined effects of filamentous tau on fast axonal transport (FAT) using isolated squid axoplasm. Monomeric and filamentous forms of recombinant human tau were perfused in axoplasm, and their effects on kinesin- and dynein-dependent FAT rates were evaluated by video microscopy. Although perfusion of monomeric tau at physiological concentrations showed no effect, tau filaments at the same concentrations selectively inhibited anterograde (kinesin-dependent) FAT, triggering the release of conventional kinesin from axoplasmic vesicles. Pharmacological experiments indicated that the effect of tau filaments on FAT is mediated by protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK-3) activities. Moreover, deletion analysis suggested that these effects depend on a conserved 18-amino-acid sequence at the amino terminus of tau. Interestingly, monomeric tau isoforms lacking the C-terminal half of the molecule (including the microtubule binding region) recapitulated the effects of full-length filamentous tau. Our results suggest that pathological tau aggregation contributes to neurodegeneration by altering a regulatory pathway for FAT.