Microtubule-binding Repeat Region Research Articles

Role of G326 in Determining the Aggregation Propensity of R3 Tau Repeat: Insights from Studies on R1R3 Tau Construct.

The Microtubule-binding repeat region (MTBR) of Tau has been studied extensively due to its pathological implications in neurodegenerative diseases like Alzheimer's disease. The pathological property of MTBR is mainly due to the R3 repeat's high propensity for self-aggregation, highlighting the critical molecular grammar of the repeat. Utilizing the R1R3 construct (WT) and its G326E mutant (EE), we determine the distinct characteristics of various peptide segments that modulate the aggregation propensity of the R3 repeat using NMR spectroscopy. Through time-dependent experiments, we have identified 317KVTSKCGS324 in R3 repeat as the aggregation initiating motif (AIM) due to its role at the initial stages of aggregation. The G326E mutation induces changes in conformation and dynamics at the AIM, thereby effectively abrogating the aggregation propensity of the R1R3 construct. We further corroborate our findings through MD simulations and propose that AIM is a robust site of interest for tauopathy drug design.

DJ-1 Molecular Chaperone Activity Depresses Tau Aggregation Propensity through Interaction with Monomers.

Tau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer's disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson's disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins.

Sequence-Dependent Conformational Properties of PGGG Motif in Tau Repeats: Insights from Molecular Dynamics Simulations of Narrow Pick Filament.

Tauopathies are a class of neurodegenerative diseases correlated with the presence of pathological Tau fibrils as a diagnostic marker. The microtubule-binding repeat region of Tau protein, which includes R1, R2, R3, and R4 repeats, constitutes the core of these fibrils. Each repeat consists of a semiconserved C-terminal hexapeptide flanked by KxGS and PGGG motifs. Previous studies have shown the influence of these peptides on protein aggregation, yet their repeat-specific properties are less explored. Using molecular dynamics, we probed the sequence-specific influence of the C-terminal hexapeptide (264ENLKHQ269) in determining the compact local conformation of the R1 repeat of the narrow Pick filament (NPF) with a homologous E264G mutation. In addition to that, we also studied the influence of 262S phosphorylation on this conformation as the phosphorylation is proposed to alleviate the pathogenesis of Pick's disease. Interestingly, we determined that E264G mutation induces a conformational shift of 270PGGG273 from a turn to a random coil. This conformational dependence is experimentally verified with the R1R3-E264G mutant construct, which displayed accelerated aggregation compared with the R1R3 wild-type construct. A significant delay in aggregation of the R1R3-G326E mutant further demonstrates the importance of 326G in determining the conformation of the R3 repeat. Thus, we conclude that the conformational properties of the PGGG motif in Tau repeats are strongly dependent on the repeat-specific sequence of the C-terminal hexapeptide.

LRP1 is a master regulator of tau uptake and spread

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer's disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation. 1 Goedert, M., Eisenberg, D. S. & Crowther, R. A. Propagation of Tau Aggregates and Neurodegeneration. Annu Rev Neurosci 40, 189-210, doi:10.1146/annurev-neuro-072116-031153 (2017). 2 Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82, 239-259, doi:10.1007/bf00308809 (1991). 3 de Calignon, A. et al. Propagation of tau pathology in a model of early Alzheimer's disease. Neuron 73, 685-697, doi:10.1016/j.neuron.2011.11.033 (2012). 4 Guo, J. L. et al. Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice. J Exp Med 213, 2635-2654, doi:10.1084/jem.20160833 (2016).

The Two Cysteines of Tau Protein Are Functionally Distinct and Contribute Differentially to Its Pathogenicity in Vivo.

Although Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies, the mechanism that initiates the aggregation of this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro Tau can be induced to form fibrillar filaments by oxidation of its two cysteine residues, generating an intermolecular disulfide bond that promotes dimerization and fibrillization. The recently solved structures of Tau filaments revealed that the two cysteine residues are not structurally equivalent since Cys-322 is incorporated into the core of the fibril, whereas Cys-291 projects away from the core to form the fuzzy coat. Here, we examined whether mutation of these cysteines to alanine affects differentially Tau mediated toxicity and dysfunction in the well-established Drosophila Tauopathy model. Experiments were conducted with both sexes, or with either sex. Each cysteine residue contributes differentially to Tau stability, phosphorylation status, aggregation propensity, resistance to stress, learning, and memory. Importantly, our work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction.SIGNIFICANCE STATEMENT Cysteine-291 and Cysteine-322, the only two cysteine residues of Tau present in only 4-Repeat or all isoforms, respectively, have competing functions: as the key residues in the catalytic center, they enable Tau auto-acetylation; and as residues within the microtubule-binding repeat region are important not only for Tau function but also instrumental in the initiation of Tau aggregation. In this study, we present the first in vivo evidence that their substitution leads to differential consequences on Tau's physiological and pathophysiological functions. These differences raise the possibility that cysteine residues play a potential role in determining the functional diversity between isoforms.

LRP1 is a master regulator of tau uptake and spread.

SummaryThe spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation.

HspB1 and Hsc70 chaperones engage distinct tau species and have different inhibitory effects on amyloid formation

The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.

Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. Graphical Abstract ᅟ.

The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity.

We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate.

The Disparate Effects of two Molecular Chaperones on Tau Amyloid Formation

Tau is an intrinsically disordered protein that binds microtubules and plays important roles in axonal transport and microtubule stability in neurons. It has been implicated in a set of neurodegenerative diseases collectively termed tauopathies, characterized by the dissociation of tau from microtubules and its accumulation in pathological, insoluble amyloid-type aggregates. Molecular chaperones are important players in all amyloid diseases, including tauopathies, as they are part of the cellular response to prevent protein misfolding and aggregation. Tau has been shown to interact with numerous chaperones, although much of the previous work is biological in nature and few interactions have been characterized quantitatively at the molecular level. We seek to better define the interactions between tau and two heat shock proteins, Hsc70 and HSPB1. While both proteins act to prevent tau aggregation and likely play protective roles in tauopathies, their mechanisms of action are distinct. Hsc70 is a 71 kDa ATPase that uses the energy of ATP hydrolysis to assist client proteins to fold to their native structures. In contrast, HSPB1 is a 27 kDa small heat shock protein that forms large, heterogeneous oligomers and functions as a holdase, binding improperly-folded proteins and preventing aggregation. Using NMR spectroscopy, electron microscopy, fluorescence correlation spectroscopy, and other biophysical tools, we characterized the modes by which each protein binds tau and the effects of each protein on the kinetics of tau fibril formation. We show that each protein binds the aggregation-prone microtubule binding repeat region of tau, but they recognize distinct sequences within this region. In addition, the two chaperone proteins have inhibitory effects on fibril formation, but HSPB1 acts primarily during the nucleation phase of fibril formation, whereas Hsc70 has a greater effect on fibril elongation.

Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity.

In Alzheimer's disease, the microtubule-associated protein tau dissociates from the neuronal cytoskeleton and aggregates to form cytoplasmic inclusions. Although hyperphosphorylation of tau serine and threonine residues is an established trigger of tau misfunction and aggregation, tau modifications extend to lysine residues as well, raising the possibility that different modification signatures depress or promote aggregation propensity depending on site occupancy. To identify lysine residue modifications associated with normal tau function, soluble tau proteins isolated from four cognitively normal human brains were characterized by MS methods. The major detectable lysine modification was found to be methylation, which appeared in the form of mono- and di-methyl lysine residues distributed among at least 11 sites. Unlike tau phosphorylation sites, the frequency of lysine methylation was highest in the microtubule-binding repeat region that mediates both microtubule binding and homotypic interactions. When purified recombinant human tau was modified in vitro through reductive methylation, its ability to promote tubulin polymerization was retained, whereas its aggregation propensity was greatly attenuated at both nucleation and extension steps. These data establish lysine methylation as part of the normal tau post-translational modification signature in human brain, and suggest that it can function in part to protect against pathological tau aggregation.

Structural Determinants of Tau Aggregation Inhibitor Potency

Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

PSF Suppresses Tau Exon 10 Inclusion by Interacting with a Stem-Loop Structure Downstream of Exon 10

Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA-protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5' splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.

Tubulin-Independent Tau in Alzheimers Disease and Cancer: Implications for Disease Pathogenesis and Treatment

Microtubule-associated protein tau has long been known for its ability to promote microtubule assembly. A less known feature of tau is its existence as a non-microtubule associated protein. Here we review the interactions of tau with other proteins, some of which interact with the microtubule binding repeat region of tau. The tau interactions with Fyn and with Pin1 have attracted the most attention and both interactions have been thought to have a role in Alzheimer's disease. The fact that tau has unknown cellular functions is further evidenced by its involvement in cell cycle activated neurodegeneration. One possible route for additional investigations stems from the presence of tau in non-neuronal cells where its characteristics have been largely unknown, although there has been a correlation between tau levels and the response of some cancer cells to microtubule-targeting chemotherapy drugs. Our studies of prostate cancer cells indicate that these cells can provide a system with phosphorylated adult tau for functional studies. In fact, structural similarities exist between Alzheimer's disease tau and prostate cancer cell tau, raising the possibility that new tau functions uncovered in prostate cancer cells will have relevance to 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.

Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration

Microtubule associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and related tauopathies; in this form it is the major protein subunit of paired helical filaments (PHF)/neurofibrillary tangles. However, the nature of protein kinases and phosphatases and tau sites involved in this lesion has been elusive. We investigated self-assembly and microtubule assembly promoting activities of hyperphosphorylated tau isolated from Alzheimer disease brain cytosol, the AD abnormally hyperphosphorylated tau (AD P-tau) before and after dephosphorylation by phosphoseryl/phosphothreonyl protein phosphatase-2A (PP-2A), and then rephosphorylation by cyclic AMP-dependent protein kinase (PKA), calcium, calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent protein kinase 5 (cdk5) in different kinase combinations. We found that (i) dephosphorylation of AD P-tau by PP-2A inhibits its polymerization into PHF/straight filaments (SF) and restores its binding and ability to promote assembly of tubulin into microtubules; (ii) rephosphorylation of PP-2A-dephosphorylated AD P-tau by sequential phosphorylation by PKA, CaMKII and GSK-3beta or cdk5, and as well as by cdk5 and GSK-3beta, promotes its self-assembly into tangles of PHF similar to those seen in Alzheimer brain, and (iii) phosphorylation of tau sites required for this pathology are Thr231 and Ser262, along with several sites flanking the microtubule binding repeat region. Phosphorylation of recombinant human brain tau(441) yielded similar results as the PP-2A dephosphorylated AD P-tau, except that mostly SF were formed. The conditions for the abnormal hyperphosphorylation of tau that promoted its self-assembly also induced the microtubule assembly inhibitory activity. These findings suggest that activation of PP-2A or inhibition of either both GSK-3beta and cdk5 or one of these two kinases plus PKA or CaMKII might be required to inhibit Alzheimer neurofibrillary degeneration.

Structural Principles of Tau and the Paired Helical Filaments of Alzheimer’s Disease

Tau, a major microtubule-associated protein in brain, forms abnormal fibers in Alzheimer's disease and several other neurodegenerative diseases. Tau is highly soluble and adopts a natively unfolded structure in solution. In the paired helical filaments of Alzheimer's disease, small segments of tau adopt a beta-conformation and interact with other tau molecules. In the filament core, the microtubule-binding repeat region of tau has a cross-beta structure, while the rest of the protein retains its largely unfolded structure and gives rise to the fuzzy coat of the filaments.

U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy.

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.

Inhibition of tau polymerization by its carboxy-terminal caspase cleavage fragment.

Abnormal aggregation of the microtubule-associated protein, tau, occurs in many neurodegenerative diseases, making it important to understand the mechanisms of tau polymerization. Previous work has indicated that the C-terminal region of tau inhibits polymerization in vitro, and a growing body of evidence implicates caspase cleavage of tau at Asp 421 in the C-terminus as an important inducer of tau polymerization in Alzheimer's disease. In the present study, we provide evidence that the C-terminal peptide fragment produced by caspase cleavage inhibits tau polymerization, suggesting that caspase cleavage of tau enhances its polymerization by removing the inhibitory control element. Moreover, we provide evidence that the peptide assumes an alpha-helical configuration and inhibits tau assembly by interacting with residues 321-375 in the microtubule binding repeat region. These findings indicate that formation of the fibrillar pathologies during the course of Alzheimer's disease may be driven or sustained by apoptotic events leading to caspase activation.

Tau polymerization: role of the amino terminus.

The abnormal polymerization of the tau molecule into insoluble filaments is a seminal event in the neurodegenerative process underlying Alzheimer's disease. Previous experimentation has shown that the microtubule-binding repeat region of the molecule is vital for its ability to polymerize in vitro into filaments similar to those found in Alzheimer's disease. However, it is becoming clear that regions outside the microtubule-binding repeat, such as exons 2 and 3 and the carboxy-terminal tail, can greatly influence its polymerization. Since it has been previously postulated that the amino terminus of tau could be involved in generating pathological conformations in the disease state, its role in the polymerization process was investigated. This report demonstrates that the removal of the amino terminus greatly inhibits the polymerization of the tau molecule, reducing both the rate and extent of polymerization. These results support the hypothesis that the ability of tau to form specific conformations involving the amino terminus is an early event in the formation of tau polymers in the disease state. Furthermore, the mutation of arginine 5 to leucine ((R)5(L)), mimicking an amino-terminal tau mutation found in a single case of FTDP-17, enhances the polymerization of the tau molecule. Therefore, the amino terminus of the tau molecule, while largely overlooked in studies of its polymerization, is a significant contributor to the polymerization process.