Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains.

Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer's disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau.

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

An acetylation-phosphorylation switch that regulates tau aggregation propensity and function.

The aberrant accumulation of tau protein is a pathological hallmark of a class of neurodegenerative diseases known as tauopathies, including Alzheimer's disease and related dementias. On the basis of previous observations that tau is a direct substrate of histone deacetylase 6 (HDAC6), we sought to map all HDAC6-responsive sites in tau and determine how acetylation in a site-specific manner affects tau's biophysical properties in vitro Our findings indicate that several acetylation sites in tau are responsive to HDAC6 and that acetylation on Lys-321 (within a KCGS motif) is both essential for acetylation-mediated inhibition of tau aggregation in vitro and a molecular tactic for preventing phosphorylation on the downstream Ser-324 residue. To determine the functional consequence of this HDAC6-regulated phosphorylation event, we examined tau's ability to promote microtubule assembly and found that phosphorylation of Ser-324 interferes with the normal microtubule-stabilizing function of tau. Tau phosphorylation of Ser-324 (pSer-324) has not previously been evaluated in the context of tauopathy, and here we observed increased deposition of pSer-324-positive tau both in mouse models of tauopathy and in patients with Alzheimer's disease. These findings uncover a novel acetylation-phosphorylation switch at Lys-321/Ser-324 that coordinately regulates tau polymerization and function. Because the disease relevance of this finding is evident, additional studies are needed to examine the role of pSer-324 in tau pathobiology and to determine whether therapeutically modulating this acetylation-phosphorylation switch affects disease progression in vivo.

Tau deposition drives neuropathological, inflammatory and behavioral abnormalities independently of neuronal loss in a novel mouse model.

Aberrant tau protein accumulation drives neurofibrillary tangle (NFT) formation in several neurodegenerative diseases. Currently, efforts to elucidate pathogenic mechanisms and assess the efficacy of therapeutic targets are limited by constraints of existing models of tauopathy. In order to generate a more versatile mouse model of tauopathy, somatic brain transgenesis was utilized to deliver adeno-associated virus serotype 1 (AAV1) encoding human mutant P301L-tau compared with GFP control. At 6 months of age, we observed widespread human tau expression with concomitant accumulation of hyperphosphorylated and abnormally folded proteinase K resistant tau. However, no overt neuronal loss was observed, though significant abnormalities were noted in the postsynaptic scaffolding protein PSD95. Neurofibrillary pathology was also detected with Gallyas silver stain and Thioflavin-S, and electron microscopy revealed the deposition of closely packed filaments. In addition to classic markers of tauopathy, significant neuroinflammation and extensive gliosis were detected in AAV1-Tau(P301L) mice. This model also recapitulates the behavioral phenotype characteristic of mouse models of tauopathy, including abnormalities in exploration, anxiety, and learning and memory. These findings indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved and are independent of cell death in this novel AAV-based model of tauopathy, which offers exceptional versatility and speed in comparison with existing transgenic models. Therefore, we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets.

Acetylation of the KXGS motifs in tau is a critical determinant in modulation of tau aggregation and clearance.

The accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of tauopathies, including Alzheimer's disease (AD) and chronic traumatic encephalopathy, but effective therapies directly targeting the tau protein are currently lacking. Herein, we describe a novel mechanism in which the acetylation of tau on KXGS motifs inhibits phosphorylation on this same motif, and also prevents tau aggregation. Using a site-specific antibody to detect acetylation of KXGS motifs, we demonstrate that these sites are hypoacetylated in patients with AD, as well as a mouse model of tauopathy, suggesting that loss of acetylation on KXGS motifs renders tau vulnerable to pathogenic insults. Furthermore, we identify histone deacetylase 6 (HDAC6) as the enzyme responsible for the deacetylation of these residues, and provide proof of concept that acute treatment with a selective and blood-brain barrier-permeable HDAC6 inhibitor enhances acetylation and decreases phosphorylation on tau's KXGS motifs in vivo. As such, we have uncovered a novel therapeutic pathway that can be manipulated to block the formation of pathogenic tau species in disease.

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.

TDP-1/TDP-43 regulates stress signaling and age-dependent proteotoxicity in Caenorhabditis elegans.

TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS-associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16-dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.

Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation.

The abnormal accumulation of the microtubule-binding protein tau is associated with a number of neurodegenerative conditions, and correlates with cognitive decline in Alzheimer's disease. The ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) and the molecular chaperone Hsp90 are implicated in protein triage decisions involving tau, and have consequently been targeted for therapeutic approaches aimed at decreasing tau burden. Here, we present evidence that CHIP binds, ubiquitinates and regulates expression of histone deacetylase 6 (HDAC6). As the deacetylase for Hsp90, HDAC6 modulates Hsp90 function and determines the favorability of refolding versus degradation of Hsp90 client proteins. Moreover, we demonstrate that HDAC6 levels positively correlate with tau burden, while a decrease in HDAC6 activity or expression promotes tau clearance. Consistent with previous research on Hsp90 clients in cancer, we provide evidence that a loss of HDAC6 activity augments the efficacy of an Hsp90 inhibitor and drives client degradation, in this case tau. Therefore, our current findings not only identify HDAC6 as a critical factor for the regulation of tau levels, but also indicate that a multi-faceted treatment approach could more effectively arrest tau accumulation in disease.

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.

TMEM106B core deposition associates with TDP-43 pathology and is increased in risk SNP carriers for frontotemporal dementia.

Genetic variation at the transmembrane protein 106B gene (TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

Robust cytoplasmic accumulation of phosphorylated TDP-43 in transgenic models of tauopathy.

Frontotemporal lobar degeneration (FTLD) has been subdivided based on the main pathology found in the brains of affected individuals. When the primary pathology is aggregated, hyperphosphorylated tau, the pathological diagnosis is FTLD-tau. When the primary pathology is cytoplasmic and/or nuclear aggregates of phosphorylated TAR-DNA-binding protein (TDP-43), the pathological diagnosis is FTLD-TDP. Notably, TDP-43 pathology can also occur in conjunction with a number of neurodegenerative disorders; however, unknown environmental and genetic factors may regulate this TDP-43 pathology. Using transgenic mouse models of several diseases of the central nervous system, we explored whether a primary proteinopathy might secondarily drive TDP-43 proteinopathy. We found abnormal, cytoplasmic accumulation of phosphorylated TDP-43 specifically in two tau transgenic models, but TDP-43 pathology was absent in mouse models of Aß deposition, α-synucleinopathy or Huntington's disease. Though tau pathology showed considerable overlap with cytoplasmic, phosphorylated TDP-43, tau pathology generally preceded TDP-43 pathology. Biochemical analysis confirmed the presence of TDP-43 abnormalities in the tau mice, which showed increased levels of high molecular weight, soluble TDP-43 and insoluble full-length and ~35 kD TDP-43. These data demonstrate that the neurodegenerative cascade associated with a primary tauopathy in tau transgenic mice can also promote TDP-43 abnormalities. These findings provide the first in vivo models to understand how TDP-43 pathology may arise as a secondary consequence of a primary proteinopathy.

The role of eight polymorphisms in three candidate genes in determining the susceptibility, phenotype, and response to anti-TNF therapy in patients with rheumatoid arthritis.

OBJECTIVES: Several single nucleotide polymorphisms (SNPs) have been associated with rheumatoid arthritis (RA) such as peptidylarginine deiminase-4 (PADI4), osteopontin (OPN), and perforin (PRF1) genes. Thus, we aimed at analysing the influence of eight SNPs in these candidate genes on RA susceptibility and their association with laboratory and clinical features in terms of response to anti-TNF therapy. METHODS: We performed a case-control study on 377 Caucasian RA patients and 391 healthy, ethnicity-matched, population-based controls. All subjects were genotyped for PADI4_89/94, PADI4_92, PADI4_104, PADI4_100 in PADI4; -156G/GG and +1239A/C in OPN and A91V and N252S in PRF1 genes. The patients were stratified for shared epitope (SE) HLA-DRB1. rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) were analysed. The patients started anti-TNF treatment and they were evaluated at baseline and after 12 weeks. Disease activity was evaluated with DAS28 and response to treatment with EULAR criteria. RESULTS: A statistically significant association between RA and OPN -156G/GG was found (p=0.023). SE was firmly confirmed to be associated with RA (OR=3.68; p<10-10). No other statistically significant association with clinical and laboratory features were observed. CONCLUSIONS: For the first time, in an Italian cohort, we report the association between -156G/GG in OPN gene and RA susceptibility. Short-term response to anti-TNF therapy was not influenced by the genetic variants studied.

Plasma PolyQ-ATXN3 Levels Associate With Cerebellar Degeneration and Behavioral Abnormalities in a New AAV-Based SCA3 Mouse Model.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited cerebellar ataxia caused by the expansion of a polyglutamine (polyQ) repeat in the gene encoding ATXN3. The polyQ expansion induces protein inclusion formation in the neurons of patients and results in neuronal degeneration in the cerebellum and other brain regions. We used adeno-associated virus (AAV) technology to develop a new mouse model of SCA3 that recapitulates several features of the human disease, including locomotor defects, cerebellar-specific neuronal loss, polyQ-expanded ATXN3 inclusions, and TDP-43 pathology. We also found that neurofilament light is elevated in the cerebrospinal fluid (CSF) of the SCA3 animals, and the expanded polyQ-ATXN3 protein can be detected in the plasma. Interestingly, the levels of polyQ-ATXN3 in plasma correlated with measures of cerebellar degeneration and locomotor deficits in 6-month-old SCA3 mice, supporting the hypothesis that this factor could act as a biomarker for SCA3.

Mutations of FUS gene in sporadic amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). OBJECTIVE: To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). METHODS: Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. RESULTS: Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. CONCLUSIONS: The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.

HDAC6 Interacts With Poly (GA) and Modulates its Accumulation in c9FTD/ALS.

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 (C9orf72), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS.

The AD tau core spontaneously self-assembles and recruits full-length tau to filaments.

Tau accumulation is a major pathological hallmark of Alzheimer's disease (AD) and other tauopathies, but the mechanism(s) of tau aggregation remains unclear. Taking advantage of the identification of tau filament cores by cryoelectron microscopy, we demonstrate that the AD tau core possesses the intrinsic ability to spontaneously aggregate in the absence of an inducer, with antibodies generated against AD tau core filaments detecting AD tau pathology. The AD tau core also drives aggregation of full-length wild-type tau, increases seeding potential, and templates abnormal forms of tau present in brain homogenates and antemortem cerebrospinal fluid (CSF) from patients with AD in an ultrasensitive real-time quaking-induced conversion (QuIC) assay. Finally, we show that the filament cores in corticobasal degeneration (CBD) and Pick's disease (PiD) similarly assemble into filaments under physiological conditions. These results document an approach to modeling tau aggregation and have significant implications for in vivo investigation of tau transmission and biomarker development.

Urine levels of the polyglutamine ataxin-3 protein are elevated in patients with spinocerebellar ataxia type 3.

INTRODUCTION: Accumulation of polyglutamine (polyQ) ataxin-3 (ATXN3) contributes to the pathobiology of spinocerebellar ataxia type 3 (SCA3). Recently, we showed that polyQ ATXN3 is elevated in the plasma and cerebrospinal fluid (CSF) of SCA3 patients, and has the potential to serve as a biological marker for this disease [1]. Based on these findings, we investigated whether polyQ ATXN3 can also be detected in urine samples from SCA3 patients. METHODS: We analyzed urine samples from 30 SCA3 subjects (including one pre-symptomatic subject), 35 subjects with other forms of ataxia, and 37 healthy controls. To quantify polyQ ATXN3 protein levels, we used our previously developed immunoassay. RESULTS: PolyQ ATXN3 can be detected in the urine of SCA3 patients, but not in urine samples from healthy controls or other forms of ataxia. There was a significant statistical association between polyQ ATXN3 levels in urine samples and those in plasma. Further, the levels of polyQ ATXN3 urine associated with an earlier age of SCA3 disease onset. CONCLUSION: As clinical trials for SCA3 advance, urine polyQ ATXN3 protein has potential to be a useful, non-invasive and inexpensive biomarker for SCA3.

Clusterin ameliorates tau pathology in vivo by inhibiting fibril formation.

The molecular chaperone Clusterin (CLU) impacts the amyloid pathway in Alzheimer's disease (AD) but its role in tau pathology is unknown. We observed CLU co-localization with tau aggregates in AD and primary tauopathies and CLU levels were upregulated in response to tau accumulation. To further elucidate the effect of CLU on tau pathology, we utilized a gene delivery approach in CLU knock-out (CLU KO) mice to drive expression of tau bearing the P301L mutation. We found that loss of CLU was associated with exacerbated tau pathology and anxiety-like behaviors in our mouse model of tauopathy. Additionally, we found that CLU dramatically inhibited tau fibrilization using an in vitro assay. Together, these results demonstrate that CLU plays a major role in both amyloid and tau pathologies in AD.