Tau Filament Formation Research Articles

Tau and neurodegeneration.

First identified in 1975, tau was implicated in Alzheimer's disease 10 years later. Filamentous tangle inclusions were known to be made of hyperphosphorylated tau by 1991, with similar inclusions gaining recognition for being associated with other neurodegenerative diseases. In 1998, mutations in MAPT, the gene that encodes tau, were identified as the cause of a dominantly inherited form of frontotemporal dementia with abundant filamentous tau inclusions. While this result indicated that assembly of tau into aberrant filaments is sufficient to drive neurodegeneration and dementia, most cases of tauopathy are sporadic. More recent work in experimental systems showed that filamentous assemblies of tau may first form in one brain area, and then spread to others in a prion-like fashion. Beginning in 2017, work on human brains using high-resolution techniques has led to a structure-based classification of tauopathies, which has opened the door to a better understanding of the significance of tau filament formation.

Distinct tau folds initiate templated seeding and alter the post-translational modification profile.

Pathological tau accumulates in the brain in tauopathies such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy and corticobasal degeneration, and forms amyloid-like filaments incorporating various post-translational modifications (PTMs). Cryo-electron microscopic (cryo-EM) studies have demonstrated that tau filaments extracted from tauopathy brains are characteristic of the disease and share a common fold(s) in the same disease group. Furthermore, the tau PTM profile changes during tau pathology formation and disease progression, and disease-specific PTMs are detected in and around the filament core. In addition, templated seeding has been suggested to trigger pathological tau amplification and spreading in vitro and in vivo, although the molecular mechanisms are not fully understood. Recently, we reported that the cryo-EM structures of tau protofilaments in SH-SY5Y cells seeded with patient-derived tau filaments show a core structure(s) resembling that of the original seeds. Here, we investigated PTMs of tau filaments accumulated in the seeded cells by liquid chromatography/tandem mass spectrometry and compared them with the PTMs of patient-derived tau filaments. Examination of insoluble tau extracted from SH-SY5Y cells showed that numerous phosphorylation, deamidation and oxidation sites detected in the fuzzy coat in the original seeds were well reproduced in SH-SY5Y cells. Moreover, templated tau filament formation preceded both truncation of the N-/C-terminals of tau and PTMs in and around the filament core, indicating these PTMs may predominantly be introduced after the degradation of the fuzzy coat.

NMR-Driven Identification of Cinnamon Bud and Bark Components With Anti-Aβ Activity.

The anti-Alzheimer disease (AD) activity reported for an aqueous cinnamon bark extract prompted us to investigate and compare the anti-amyloidogenic properties of cinnamon extracts obtained from both bark and bud, the latter being a very little explored matrix. We prepared the extracts with different procedures (alcoholic, hydroalcoholic, or aqueous extractions). An efficient protocol for the rapid analysis of NMR spectra of cinnamon bud and bark extracts was set up, enabling the automatic identification and quantification of metabolites. Moreover, we exploited preparative reverse-phase (RP) chromatography to prepare fractions enriched in polyphenols, further characterized by UPLC-HR-MS. Then, we combined NMR-based molecular recognition studies, atomic force microscopy, and in vitro biochemical and cellular assays to investigate the anti-amyloidogenic activity of our extracts. Both bud and bark extracts showed a potent anti-amyloidogenic activity. Flavanols, particularly procyanidins, and cinnamaldehydes, are the chemical components of cinnamon hindering Aβ peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Together with the previously reported ability to hinder tau aggregation and filament formation, these data indicate cinnamon polyphenols as natural products possessing multitarget anti-AD activity. Since cinnamon is a spice increasingly present in the human diet, our results support its use to prepare nutraceuticals useful in preventing AD through an active contrast to the biochemical processes that underlie the onset of this disease. Moreover, the structures of cinnamon components responsible for cinnamon anti-AD activities represent molecular templates for designing and synthesizing new anti-amyloidogenic drugs.

Tau Ubiquitination in Alzheimer's Disease.

Paired helical filaments (PHFs) from the Alzheimer's disease (AD) brain are highly ubiquitinated and ubiquitination likely plays a vital role in tau filament formation. Whether tau ubiquitination is the causality or consequence of the disease in AD remains elusive. The following questions are worth considering: What does the extent of tau ubiquitination contribute to tau pathology in AD? Does tau ubiquitination influence aggregation or spreading during disease progression? In addition, tau is polyubiquitinated in nerve growth factor-induced PC12 cells and participates in mitogen-activated protein kinase signaling, in addition to its microtubule stabilization function. Therefore, ubiquitination possibly mediates tau signaling under physiological conditions, but tau aggregation in the pathobiology of AD. Here, we review the advancements in tau ubiquitination and the potential therapeutic effects of targeting tau ubiquitination to alleviate tau pathology in AD.

Microfluidic Protein Imaging Platform: Study of Tau Protein Aggregation and Alzheimer's Drug Response.

Tau protein aggregation is identified as one of the key phenomena associated with the onset and progression of Alzheimer’s disease. In the present study, we performed on-chip confocal imaging of tau protein aggregation and tau–drug interactions using a spiral-shaped passive micromixing platform. Numerical simulations and experiments were performed in order to validate the performance of the micromixer design. We performed molecular modeling of adenosine triphosphate (ATP)-induced tau aggregation in order to successfully validate the concept of helical tau filament formation. Tau aggregation and native tau restoration were realized using an immunofluorescence antibody assay. The dose–response behavior of an Alzheimer’s drug, methylthioninium chloride (MTC), was monitored on-chip for defining the optimum concentration of the drug. The proposed device was tested for reliability and repeatability of on-chip tau imaging. The amount of the tau protein sample used in our experiments was significantly less than the usage for conventional techniques, and the whole protein–drug assay was realized in less than two hours. We identified that intensity-based tau imaging could be used to study Alzheimer’s drug response. In addition, it was demonstrated that cell-free, microfluidic tau protein assays could be used as potential on-chip drug evaluation tools for Alzheimer’s disease.

Role of cysteines in accelerating Tau filament formation

Alzheimer's disease is majorly associated with intracellular accumulation of Tau into paired helical filaments and tangles. The self-aggregated dimeric and oligomeric species of Tau formed are more toxic to neuronal cells and acts as seeds for filament formation. The two cysteine residues and the two hexapeptide regions of full-length Tau play a key role in initialization and filament formation during Tau aggregation. The role of cysteine residues in Tau aggregation has been studied by in-vitro aggregation assay that was measured by Thioflavin S fluorescence to observe the kinetics of aggregation. In this study, we have performed in-vitro aggregation assay with recombinant full-length Tau and the cysteine mutants to understand the mechanism of cysteine independent Tau aggregation. Here, we report that cysteine mutant full-length Tau can aggregate to form filaments under in-vitro conditions. To visualize the polymorphisms of Tau and cysteine mutants under different aggregation conditions anionic cofactor, heparin was employed. Wild-type Tau showed rapid aggregation to form oligomers and filaments. On the other hand, the cysteine mutant delayed the initial Tau aggregation. This indicates the importance of cysteine residues in accelerating initial Tau nucleation for its aggregation. The filament morphology of wild-type and cysteine mutant Tau has been characterized using transmission electron microscopy and high-resolution transmission electron microscopy. Communicated by Ramaswamy H. Sarma

Circular Dichroism Spectroscopy Identifies the β-Adrenoceptor Agonist Salbutamol As a Direct Inhibitor of Tau Filament Formation in Vitro.

Potential drug treatments for Alzheimer's disease (AD) may be found by identifying compounds that block the assembly of the microtubule-associated protein tau into neurofibrillar tangles associated with neuron destabilization and cell death. Here, a small library of structurally diverse compounds was screened in vitro for the ability to inhibit tau aggregation, using high-throughput synchrotron radiation circular dichroism as a novel tool to monitor the structural changes in the protein as it assembles into filaments. The catecholamine epinephrine was found to be the most effective tau aggregation inhibitor of all 88 screened compounds. Subsequently, we tested chemically similar phenolamine drugs from the β-adrenergic receptor agonist class, using conventional circular dichroism spectroscopy, thioflavin T fluorescence, and transmission electron microscopy. Two compounds, salbutamol and dobutamine, used widely in the treatment of respiratory and cardiovascular disease, impede the aggregation of tau in vitro. Dobutamine reduces both the rate and yield of tau filament formation over 24 h; however, it has little effect on the structural transition of tau into β-sheet structures over 24 h. Salbutamol also reduces the yield and rate of filament formation and additionally inhibits tau's structural change into β-sheet-rich aggregates. Salbutamol has a good safety profile and a half-life that facilitates permeation through the blood-brain barrier and could represent an expediated approach to developing AD therapeutics. These results provide the motivation for the in vivo evaluation of pre-existing β-adrenergic receptor agonists as a potential therapy for AD through the reduction of tau deposition.

Understanding the Kinetic Roles of 14-3-3ζ during Tau Filament Formation

The microtubule associated protein tau and 14-3-3ζ protein are two proteins highly expressed in the central nervous system. Tau protein promotes microtubule assembly and aberrant accumulation of tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies including Alzheimer's disease. Extensive studies have suggested that polyanionic inducers, such as heparin, interact with tau directly and induce specific conformational changes within tau, accelerating the amyloidogenesis process. 14-3-3ζ proteins form homodimers and bind their targets through specific pSer/pThr motifs. It has been found that tau protein contains two 14-3-3ζ-binding motifs, i.e., pSer214 and pSer324, and tau protein interacts with 14-3-3ζ protein directly. Furthermore, 14-3-3ζ is able to induce tau aggregation, although it's still controversial whether this process depends on tau phosphorylation. Here, we investigated the kinetic roles of 14-3-3ζ during tau filament formation. We used the K18 constructs of tau that consists of the four microtubule repeats and the pSer324 14-3-3ζ-binding motifs. Our results showed that 14-3-3ζ induced tau K18 aggregation at a rate much larger than that from full-length tau. Kinetics analyses showed that the lag time and rate of aggregation were dependent on the stoichiometry of tau to 14-3-3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14-3-3ζ, phosphorylation only showed marginal effect on 14-3-3ζ mediated tau aggregation. Our results suggest that the formation of the 2:1 complex of tau monomer and 14-3-3ζ dimer plays an important role in the 14-3-3ζ mediated tau aggregation process.

Tau filaments from multiple cases of sporadic and inherited Alzheimer\u2019s disease adopt a common fold

The ordered assembly of tau protein into abnormal filaments is a defining characteristic of Alzheimer’s disease (AD) and other neurodegenerative disorders. It is not known if the structures of tau filaments vary within, or between, the brains of individuals with AD. We used a combination of electron cryo-microscopy (cryo-EM) and immuno-gold negative-stain electron microscopy (immuno-EM) to determine the structures of paired helical filaments (PHFs) and straight filaments (SFs) from the frontal cortex of 17 cases of AD (15 sporadic and 2 inherited) and 2 cases of atypical AD (posterior cortical atrophy). The high-resolution structures of PHFs and SFs from the frontal cortex of 3 cases of AD, 2 sporadic and 1 inherited, were determined by cryo-EM. We also used immuno-EM to study the PHFs and SFs from a number of cortical and subcortical brain regions. PHFs outnumbered SFs in all AD cases. By cryo-EM, PHFs and SFs were made of two C-shaped protofilaments with a combined cross-β/β-helix structure, as described previously for one case of AD. The higher resolution structures obtained here showed two additional amino acids at each end of the protofilament. The immuno-EM findings, which indicated the presence of repeats 3 and 4, but not of the N-terminal regions of repeats 1 and 2, of tau in the filament cores of all AD cases, were consistent with the cryo-EM results. These findings show that there is no significant variation in tau filament structures between individuals with AD. This knowledge will be crucial for understanding the mechanisms that underlie tau filament formation and for developing novel diagnostics and therapies.

Frontotemporal dementia with Parkinsonism linked to chromosome-17 mutations enhance tau oligomer formation.

The P301L mutation in tau, a microtubule-associated protein, causes frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17) that is accompanied by formation of filamentous polymers of tau. The mutation reduces the binding capability of microtubules and enhances tau filament formation. However, it is unclear whether the P301L mutation increases the formation of the intermediates of tau filaments that are suggested to be a toxic species of tau. To determine the amount and structure of the intermediates harboring with the P301L mutation, we purified recombinant versions of wild-type, P301L, and 4 other mutants (i.e., P301S, P301T, V337M, and R406W) tau proteins and analyzed the heparin-induced aggregation of those tau constructs. We found that all of the FTDP-17 mutants increased levels of the intermediate tau oligomers. The sizes were determined by atomic force microscopy and laser light scattering. The V337M and R406W oligomers were similar in size to the wild-type, but the P301L, P301T, and P301S mutants formed smaller oligomers. In a P301L transgenic mouse model, we found tau aggregates that were similar in size to the recombinant tau oligomer. These results indicate that FTDP-17 mutations contribute to the pathogenesis via the increased formation of tau oligomers.

Effect of Acetylation on Liquid‐Liquid Phase Separation and Amyloid Fibril Formation of Tau

The function of proteins depends on their phase behavior. When the concentration of a protein exceeds a critical value, it may undergo liquid‐liquid phase separation, leading to demixing of proteins from solution. The resulted liquid droplets are dense and protein‐rich. The microtubule associated protein tau can enrich tubulin by forming liquid droplets, which promotes the formation of microtubule bundles. On the other hand, aberrant aggregation of Tau into neurofibrillary tangles correlates with the progressive destruction of nerve cells. Therefore, studying the aggregating mechanism of tau and drug design targeting tau fibrils are critical for the therapy of Alzheimer's disease. Recent studies have shown that lysine acetylation plays an important role in modulating tau aggregation and microtubule assembly. Considering the potential correlations between liquid‐liquid phase separation and amyloid formation, here we investigated the effect of lysine acetylation on the phase behavior and function of tau in vitro. We used site‐directed mutagenesis, light scattering, thioflavin T florescence assay, and microrheology to study the liquid‐liquid phase separation and aggregation of tau. Consistent with previous observations, acetylation on K274/K321 (mimicked by K274Q/K321Q) significantly impaired tau aggregation whereas acetylation on K280/K281/K369 (mimicked by K280Q/K281Q/K369Q) significantly promoted tau filament formation as compared with the WT protein. Although acetylation on K274/K321 and K280/K281/K369 differentially affected tau aggregation, both acetylation patterns promoted liquid‐liquid phase separation of tau. Moreover, microrheology characterization and protein concentration determination showed that the droplets formed by acetylated tau were denser than the WT protein, suggesting that more intermolecular interactions were form upon lysine acetylation. These results show that lysine residues within the MT‐binding repeats regulate the phase behavior of tau via complex interactions.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pin1 Knockout Mice: A Model for the Study of Tau Pathology in Alzheimer's Disease.

Pin1 knockout in mice causes age-dependent neuropathy characterized by motor and behavioral deficits, tau hyper phosphorylation, tau filament formation, and neuronal degradation. Here, we describe the methods with mouse behavior test, immunostaining, and immunoblotting to detect many aspects of neurodegeneration in Pin1 knockout mice.

FTDP-17 with Pick body-like inclusions associated with a novel tau mutation, p.E372G.

Mutations in microtubule-associated protein tau gene (MAPT) cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Here, we describe a patient with FTDP-17 and a novel missense mutation in exon 13 of MAPT, p.E372G. We compare clinicopathologic features of this patient to two previously unreported patients with another exon 13 mutation, p.G389R. The patient with the p.E372G mutation was a 40-year-old man with behavioral variant frontotemporal dementia (bvFTD), who subsequently developed agrammatic speech and parkinsonism. One of the FTDP-17 patients with p.G389R mutation presented at age 24 with agrammatic variant of primary progressive aphasia, and subsequently behavioral dysfunction. The other presented at age 53 with bvFTD, followed by agrammatic speech and corticobasal syndrome. Neuropathologic features of FTDP-17 due to p.E372G were similar to those of p.G389R, including tau-immunoreactive Pick body-like neuronal inclusions and swollen, tapering thread-like processes in white matter immunoreactive for 3-repeat and 4-repeat tau. Biochemical analysis of insoluble tau showed similar isoform compositions in p.E372G and p.G389R. Functional studies of the p.E372G mutation showed marked increase in tau filament formation and its reduced ability to promote microtubule assembly. Together these findings indicate that p.E372G is a pathogenic MAPT mutation that causes FTDP-17 similar to p.G389R.

In vitro screen for inhibitors of tau filament formation

In vitro screen for inhibitors of tau filament formation

Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3β enzyme, β-amyloid and tau protein levels.

Cadmium is a neurotoxic compound which induces cognitive alterations similar to those produced by Alzheimer's disease (AD). However, the mechanism through which cadmium induces this effect remains unknown. In this regard, we described in a previous work that cadmium blocks cholinergic transmission and induces a more pronounced cell death on cholinergic neurons from basal forebrain which is partially mediated by AChE overexpression. Degeneration of basal forebrain cholinergic neurons, as happens in AD, results in memory deficits attributable to the loss of cholinergic modulation of hippocampal synaptic circuits. Moreover, cadmium has been described to activate GSK-3β, induce Aβ protein production and tau filament formation, which have been related to a selective loss of basal forebrain cholinergic neurons and development of AD. The present study is aimed at researching the mechanisms of cell death induced by cadmium on basal forebrain cholinergic neurons. For this purpose, we evaluated, in SN56 cholinergic mourine septal cell line from basal forebrain region, the cadmium toxic effects on neuronal viability through muscarinic M1 receptor, AChE splice variants, GSK-3β enzyme, Aβ and tau proteins. This study proves that cadmium induces cell death on cholinergic neurons through blockade of M1 receptor, overexpression of AChE-S and GSK-3β, down-regulation of AChE-R and increase in Aβ and total and phosphorylated tau protein levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on cholinergic neurons and suggest that cadmium could mediate these mechanisms by M1R blockade through AChE splices altered expression.

A novel tau mutation, p.K317N, causes globular glial tauopathy

Globular glial tauopathies (GGTs) are 4-repeat tauopathies neuropathologically characterized by tau-positive, globular glial inclusions, including both globular oligodendroglial inclusions and globular astrocytic inclusions. No mutations have been found in 25 of the 30 GGT cases reported in the literature who have been screened for mutations in microtubule associated protein tau (MAPT). In this report, six patients with GGT (four with subtype III and two with subtype I) were screened for MAPT mutations. They included 4 men and 2 women with a mean age at death of 73 years (55-83 years) and mean age at symptomatic onset of 66 years (50-77 years). Disease duration ranged from 5 to 14 years. All were homozygous for the MAPT H1 haplotype. Three patients had a positive family history of dementia, and a novel MAPT mutation (c.951G>C, p.K317N) was identified in one of them, a patient with subtype III. Recombinant tau protein bearing the lysine-to-asparagine substitution at amino acid residue 317 was used to assess functional significance of the variant on microtubule assembly and tau filament formation. Recombinant p.K317N tau had reduced ability to promote tubulin polymerization. Recombinant 3R and 4R tau bearing the p.K317N mutation showed decreased 3R tau and increased 4R tau filament assembly. These results strongly suggest that the p.K317N variant is pathogenic. Sequencing of MAPT should be considered in patients with GGT and a family history of dementia or movement disorder. Since several individuals in our series had a positive family history but no MAPT mutation, genetic factors other than MAPT may play a role in disease pathogenesis.

Design and synthesis of tacrine-phenothiazine hybrids as multitarget drugs for Alzheimer’s disease

Tacrine is well-known drug for Alzheimer’s disease as an acetylcholinesterase inhibitor. Rember is a bright and promising AD drugs targeting tau protein, and it is currently in Phase III clinical trials. Phenothiazine, the key pharmacophore of Rember, can prevent tau filament formation. In this work, several tacrine-phenothiazine hybrids (T1–T26) were designed for inhibiting acetylcholinesterase and tau protein involved in Alzheimer’s disease. After initial screening with the help of computational chemistry software and Molegro Virtual Docker, three molecules (T5, T18, and T22) were selected for further synthesis and biological evaluation. Next, T5, T18, and T22 were synthesized and evaluated for their acetylcholinesterase and tau hyperphosphorylation inhibition. All the tested compounds had better acetylcholinesterase inhibitory activity compared with tacrine. Among them, compound T5 was found to be the most potent compound with IC50 89 nM. Meanwhile, T5 markedly prevented tau hyperphosphorylation induced by okadaic acid in N2α cell. Its P-tau level was decreased with 39.5 % inhibition when tested at 10−5 M, lower than that Rember (55.7 %). Besides acetylcholinesterase and tau hyperphosphorylation inhibition, T5 can also interact with fibrill beta amyloid using surface plasmon resonance, the data of KD were 5.51 × 10−8 M. All the above results indicated that tacrine-phenothiazine hybrids are potential multitarget directed ligands targeting acetylcholinesterase, tau protein, and beta amyloid.

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.

Medicinal properties of 'true' cinnamon (Cinnamomum zeylanicum): a systematic review.

BackgroundIn traditional medicine Cinnamon is considered a remedy for respiratory, digestive and gynaecological ailments. In-vitro and in-vivo studies from different parts of the world have demonstrated numerous beneficial medicinal effects of Cinnamomum zeylanicum (CZ). This paper aims to systematically review the scientific literature and provide a comprehensive summary on the potential medicinal benefits of CZ.MethodsA comprehensive systematic review was conducted in the following databases; PubMed, Web of Science, SciVerse Scopus for studies published before 31st December 2012. The following keywords were used: “Cinnamomum zeylanicum”, “Ceylon cinnamon”, “True cinnamon” and “Sri Lankan cinnamon”. To obtain additional data a manual search was performed using the reference lists of included articles.ResultsThe literature search identified the following number of articles in the respective databases; PubMed=54, Web of Science=76 and SciVerse Scopus=591. Thirteen additional articles were identified by searching reference lists. After removing duplicates the total number of articles included in the present review is 70. The beneficial health effects of CZ identified were; a) anti-microbial and anti-parasitic activity, b) lowering of blood glucose, blood pressure and serum cholesterol, c) anti-oxidant and free-radical scavenging properties, d) inhibition of tau aggregation and filament formation (hallmarks of Alzheimer’s disease), e) inhibitory effects on osteoclastogenesis, f) anti-secretagogue and anti-gastric ulcer effects, g) anti-nociceptive and anti-inflammatory activity, h) wound healing properties and i) hepato-protective effects. The studies reported minimal toxic and adverse effects.ConclusionsThe available in-vitro and in-vivo evidence suggests that CZ has many beneficial health effects. However, since data on humans are sparse, randomized controlled trials in humans will be necessary to determine whether these effects have public health implications.

Novel mutation in MAPT exon 13 (p.N410H) causes corticobasal degeneration

In order to determine the frequency of microtubule-associated protein tau gene (MAPT) mutations and rare variants in CBD, we performed a systematic sequence analysis of MAPT coding and 3′ untranslated region (3′UTR) in a large cohort of autopsy-confirmed CBD patients (N = 109). This identified a novel MAPT mutation in exon 13, p.N410H, in a case that is neuropathologically indistinguishable from sporadic CBD. On immunoblot, the p.N410H mutation carrier had the same insoluble tau profile as seen in CBD. Additionally, tau expression analysis in brain tissue found a significant increase in the 4R/3R tau mRNA ratio (P = 0.04), indicating that p.N410H disrupts tau isoform homeostasis. Biochemically, recombinant tau protein with p.N410H showed a marked increase in tau filament formation compared to wild-type tau (P < 0.001), had a 19.2% decrease in rate of microtubule assembly (P < 0.05), and a 10.3% reduction in the extent of total microtubule polymerization (P < 0.01). Sequence analysis of the complete MAPT 3′UTR in autopsy-confirmed CBD cases further identified two rare variants with nominally significant association with CBD. An ATC nucleotide insertion (“MAPTv8”) was found in 4.6% of CBD patients compared to 1.2% of controls (P = 0.031, OR = 3.71), and rs186977284 in 4.6% CBD patients, but only 0.9% of controls (P = 0.04, OR = 3.58). Rs186977284 was also present in 2.7% of a large cohort of autopsy-confirmed PSP patients (N = 566) and only 0.9% of an additional control series (P = 0.034, OR = 3.08), extending the association to PSP. Our findings show that mutations in MAPT can cause CBD and MAPT non-coding variants may increase the risk of complex 4R tauopathies.