Mutant Tau Research Articles

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro

BackgroundAlzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death.MethodsWe aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively.ResultsWhile ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK.ConclusionsISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.

Plasticity in visual cortex is disrupted in a mouse model of tauopathy

Alzheimer’s disease and other dementias are thought to underlie a progressive impairment of neural plasticity. Previous work in mouse models of Alzheimer’s disease shows pronounced changes in artificially-induced plasticity in hippocampus, perirhinal and prefrontal cortex. However, it is not known how degeneration disrupts intrinsic forms of brain plasticity. Here we characterised the impact of tauopathy on a simple form of intrinsic plasticity in the visual system, which allowed us to track plasticity at both long (days) and short (minutes) timescales. We studied rTg4510 transgenic mice at early stages of tauopathy (5 months) and a more advanced stage (8 months). We recorded local field potentials in the primary visual cortex while animals were repeatedly exposed to a stimulus over 9 days. We found that both short- and long-term visual plasticity were already disrupted at early stages of tauopathy, and further reduced in older animals, such that it was abolished in mice expressing mutant tau. Additionally, visually evoked behaviours were disrupted in both younger and older mice expressing mutant tau. Our results show that visual cortical plasticity and visually evoked behaviours are disrupted in the rTg4510 model of tauopathy. This simple measure of plasticity may help understand how tauopathy disrupts neural circuits, and offers a translatable platform for detection and tracking of the disease.

Loss-of-function and gain-of-function studies refute the hypothesis that tau protein is causally involved in the pathogenesis of Huntington's disease.

Tubulin-associated unit (Tau) is a microtubule-associated protein, whose abnormal phosphorylation and deposition in the brain characterizes a range of neurodegenerative diseases called tauopathies. Recent clinical (post-mortem) and pre-clinical evidence suggests that Huntington's disease (HD), an autosomal dominant neurodegenerative disorder, could be considered as a tauopathy. Studies have found the presence of hyperphosphorylated tau, altered tau isoform ratio and aggregated tau in HD brains. However, little is known about the implication of tau in the development of HD pathophysiology, which includes motor, cognitive and affective symptoms. To shine a light on the involvement of tau in HD, our present study aimed at (i) knocking out tau expression and (ii) expressing a transgene encoding mutant human tau in the R6/1 mouse model of HD. We hypothesized that expression of the mutant human tau transgene in HD mice would worsen the HD phenotype, while knocking out endogenous mouse tau in HD mice would improve some behavioral deficits displayed by HD mice. Our data suggest that neither the expression of a tau transgene nor the ablation of tau expression impacted the progression of the HD motor, cognitive and affective phenotypes. Supporting these behavioral findings, we also found that modulating tau expression had no effect on brain weights in HD mice. We also report that expression of the tau transgene increased the weight of WT and HD male mice, whereas tau ablation increased the weight of HD females only. Together, our results indicate that tau might not be as important in regulating the onset and progression of HD symptomatology as previously proposed.

Biochemical and biophysical features of disease-associated tau mutants V363A and V363I

The comprehension of pathogenetic mechanisms in tauopathy-associated neurodegenerative diseases can be improved by the knowledge of the biochemical and biophysical features of mutated tau proteins. Here, we used the full-length, wild-type tau, the V363A and V363I mutated species, associated with pathology, and the P301L mutated tau as a benchmark. Using several techniques, including small-angle X-ray scattering, atomic force microscopy, thioflavin T binding, and electrophoretic separation, we compared their course from intrinsically disordered monomers in solution to early-stage recruitment in complexes and then aggregates of increasing size over long periods up to the asymptotic aggregative behavior of full-length tau proteins. We showed that diversity in the kinetics of recruitment and aggregate structure occurs from the beginning and spreads all over their pathway to very large objects. The different extents of conformational changes and types of molecular assemblies among the proteins were also reflected in their in vitro toxicity; this variation could correlate with physiopathology in humans, considering that the P301L mutation is more aggressive than V363A, especially V363I. This study identified the presence of aggregation intermediates and corroborated the oligomeric hypothesis of tauopathies.

Structure-Based Design and Biological Evaluation of Novel Caspase-2 Inhibitors Based on the Peptide AcVDVAD-CHO and the Caspase-2-Mediated Tau Cleavage Sequence YKPVD314.

Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.

Biochemical and biophysical features of disease-associated tau mutants V363A and V363I

Biochemical and biophysical features of disease-associated tau mutants V363A and V363I

Generation of an induced pluripotent stem cell line (ICNDXHi001-A) from a patient with frontotemporal dementia carrying a heterozygous mutation c.796C > G (p.L266V) in MAPT

Frontotemporal dementia (FTD) caused by microtubule-associated protein tau (MAPT) mutations is not rare and is almost fully penetrant. However, no disease-modifying treatment for FTD is currently available. Here, we demonstrated the establishment and characterization of a novel human induced pluripotent stem cell (iPSC) line ICNDXHi001-A from a patient with FTD carrying genetic variant MAPT c.796C > G (p.L266V). The generated cell line showed trilineage differentiation potential, expression of pluripotency markers, a normal karyotype, and retention of MAPT mutation. The study provides a useful model to further elucidate the underlying mechanisms of FTD and to facilitate novel therapy development.

Combinatorial model of amyloid β and tau reveals synergy between amyloid deposits and tangle formation.

To illuminate the pathological synergy between Aβ and tau leading to emergence of neurofibrillary tangles (NFT) in Alzheimer's disease (AD), here, we have performed a comparative neuropathological study utilising three distinctive variants of human tau (WT tau, P301L mutant tau and S320F mutant tau). Previously, in non-transgenic mice, we showed that WT tau or P301L tau does not form NFT while S320F tau can spontaneously aggregate into NFT, allowing us to test the selective vulnerability of these different tau conformations to the presence of Aβ plaques. We injected recombinant AAV-tau constructs into neonatal APP transgenic TgCRND8 mice or into 3-month-old TgCRND8 mice; both cohorts were aged 3months post injection. This allowed us to test how different tau variants synergise with soluble forms of Aβ (pre-deposit cohort) or with frank Aβ deposits (post-deposit cohort). Expression of WT tau did not produce NFT or altered Aβ in either cohort. In the pre-deposit cohort, S320F tau induced Aβ plaque deposition, neuroinflammation and synaptic abnormalities, suggesting that early tau tangles affect the amyloid cascade. In the post-deposit cohort, contemporaneous expression of S320F tau did not exacerbate amyloid pathology, showing a dichotomy in Aβ-tau synergy based on the nature of Aβ. P301L tau produced NFT-type inclusions in the post-deposit cohort, but not in the pre-deposit cohort, indicating pathological synergy with pre-existing Aβ deposits. Our data show that different tau mutations representing specific folding variants of tau synergise with Aβ to different extents, depending on the presence of cerebral deposits.

The pathogenic mutation in tau defines the route of tau accumulation at presynapses.

Tau's primary localization is in the axons of neurons. At axons, Tau binds microtubuli and stabilizes them. Many of the pathogenic mutations interfere with binding of Tau to the microtubuli resulting in the relocation of Tau to different neuronal compartments, including the presynapse. Our lab has shown that Tau interact with synaptic vesicles via the synaptic vesicle protein, Synaptogyrin-3 and excessive Tau levels at presynapses results in the sequestration of synaptic vesicles compromising neurotransmission in Tau mutant fly and primary mouse neurons (Zhou, Nat. Commun. 2017; McInnes, Neuron, 2018). In addition, lowering Synaptogyrin-3 expression in TauP301L mutant mice reduces Tau-synaptic vesicle interactions and rescues Tau-mediated memory defects and synapse loss in these mice (Largo-Barrientos,Neuron, 2021). Although, the interaction of Tau with synaptic vesicles is reduced with lowering Synaptogyrin-3 expression, Tau continues to accumulate at presynapses. To reduce Tau levels and keep the presynapse healthy over time, we increased endosomal microautophagy by the expression of Hsc70-4 (Uytterhoeven, Neuron, 2015) in Tau mutant fly neurons . Despite, the overall reduced activity of degradation pathways in pathogenic conditions, we were able to reduce presynaptic TauP301L levels and restore reduced synaptic vesicle mobility and neurotransmission by increasing endosomal microautophagy. However, increasing endosomal microautophagy is not effective in lowering pathogenic TauV337M . Using a fluorescent timer attached to Tau, we show that older Tau is present in TauV337M compared to TauP301L mutant presynapses indicating that the turnover of TauV337M is hindered. On the contrary, with a fluorescence recovery after photobleaching assay we show that TauP301L is more mobile in axons compared to TauV337M indicating that TauP301L detaches more easily from the microtubuli. Together, these data show that both the detachment of Tau from microtubuli and defective turnover of Tau at presynapses contribute to the accumulation of Tau at presynapses and depending on the pathogenic mutation one of the two pathways plays a more prominent role in the accumulation of Tau at presynapses. Hence, these observations have further implications in the development of new therapies acting on Tau.

Pathogenic tauR406W‐induced neurodegeneration and memory deficits rescued by HDAC6‐null mutation in Drosophila melanogaster

Genetic screening was performed by Drosophila melanogaster to observe how different variants of HDAC6 regulate tauR406W mediated neurodegeneration. The versatile Gal4-UAS gene expression system was used to express variants of HDAC6 and tauR406W with the help of pan-eye specific GMR-Gal4, and pan-neuronal elav-Gal4 in the flies. The extent of neurodegeneration was quantified by automated analysis of degenerated tissues, the number of aggregates, immunofluorescence, and also with the help of various behavioral and memory assays. Live imaging was performed on the wing with GCamP6f to study the role of HDAC6 on axonal transport. On expressing the HDAC6 mutants (H237A, H664A) the levels of total tau were increased followed by a decrease in acetylated α-tubulin levels. We have also noticed a reduction in mRNA levels of Atg1, Atg8, and Atg12. On analyzing the various regions of the eye, there was a significant surface degeneration as well as an increase in the number of aggregates. The wings of flies expressing a GCamp6f and eb1-eGFP displayed a defective axonal transport as well as damaged neuronal morphology. Upon expressing HDAC6[KO], there was amelioration in memory deficits, neurodegeneration and the flies regained a better retinal surface morphology. HDAC6 can interact with tau mutations outside the microtubule-binding region (MTBR) and can cause memory deficits, behavioral anomalies. Inhibition of HDAC6 via gene therapy can be an effective therapy to treat Alzheimer's disease and other associated dementias.

Pathogenic tauR406W induced neurodegeneration and memory deficits rescued by HDAC6 null mutation in Drosophila melanogaster

AbstractBackgroundGenetic screening was performed by Drosophila melanogaster to observe how different variants of HDAC6 regulate tauR406W mediated neurodegeneration.MethodThe versatile Gal4‐UAS gene expression system was used to express variants of HDAC6 and tauR406W with the help of pan‐eye specific GMR‐Gal4, and pan‐neuronal elav‐Gal4 in the flies. The extent of neurodegeneration was quantified by automated analysis of degenerated tissues, the number of aggregates, immunofluorescence, and also with the help of various behavioral and memory assays. Live imaging was performed on the wing with GCamP6f to study the role of HDAC6 on axonal transport.ResultOn expressing the HDAC6 mutants (H237A, H664A) the levels of total tau were increased followed by a decrease in acetylated α‐tubulin levels. We have also noticed a reduction in mRNA levels of Atg1, Atg8, and Atg12. On analyzing the various regions of the eye, there was a significant surface degeneration as well as an increase in the number of aggregates. The wings of flies expressing a GCamp6f and eb1‐eGFP displayed a defective axonal transport as well as damaged neuronal morphology. Upon expressing HDAC6[KO], there was amelioration in memory deficits, neurodegeneration and the flies regained a better retinal surface morphology.ConclusionHDAC6 can interact with tau mutations outside the microtubule‐binding region (MTBR) and can cause memory deficits, behavioral anomalies. Inhibition of HDAC6 via gene therapy can be an effective therapy to treat Alzheimer’s disease and other associated dementias.

A Canadian cohort with the IVS10+14 MAPT mutation

AbstractBackgroundThe IVS10+14 microtubule‐associated protein tau gene (MAPT) mutation causes dysregulated protein splicing of MAPT, resulting in abnormal 4‐repeat isoform tau accumulation and associated with neurodegeneration. Three families expressing the IVS10+14 MAPT mutation have been reported who express the IVS10+14 MAPT mutation displaying a high degree of interfamilial heterogeneity in their clinical presentation. Herein, we describe the clinical and neuropathological presentation of a new Canadian cohort with this mutation.MethodClinical and neuropathological records, including neuropsychological and cognitive assessments, and stained neuropathological sections, were obtained for three family members with the IVS10+14 MAPT mutation. The records were reviewed and compared to previously reported families with this mutation.ResultThe Canadian cohort displayed many of the symptoms previously described in cases with the IVS10+14 MAPT mutation, including prominent motor, cognitive, behavioural, and behavioural symptoms. Additionally, consistent with the interfamilial heterogeneity described in literature, our cohort exhibited profound sensory and sleep abnormalities. Neuropathological information was available for two cases, in which one had widespread cortical and subcortical tau deposition and atrophy, while neuropathological changes in the other case was milder with the exception substantia nigral atrophy.ConclusionOur cohort demonstrate that while the IVS10+14 MAPT mutation often results in motor, cognitive and behavioural clinical features, sensory and sleep abnormalities are also manifestations of the mutation. Clinicopathological correlations were able to explain symptom presence and severity for two of the three examined cases. This underlines the need to explore alternate mechanisms and metrics of brain dysfunction in tauopathy in addition to tau burden and neuronal loss. Reference: Maxwell SP, Cash MK, Rockwood K, Fisk JD, Darvesh S. 2021. Clinical and Neuropathological Variability in the Rare IVS10+14 Tau Mutation. Neurobiology of Aging. In Press. DOI: 10.1016/j.neurobiolaging.2021.01.004.

Defective proteostasis in patient-derived iPSC astrocytes and neurons carrying a MAPT IVS10+16 mutation.

Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the toxic accumulation of protein. In a subset of frontotemporal dementia (FTD) cases, mutations in the microtubule-associated protein tau (MAPT) that alter the relative levels of tau isoforms are sufficient to cause tau inclusions in neurons and astroglia and neurodegeneration without the presence of mutated protein (e.g. MAPTIVS10+16). However, the pathogenic events triggered by the expression of the alternatively spliced tau remain poorly understood. To determine whether altered tau splicing induced from MAPT IVS10+16 mutations is sufficient to alter proteostasis in neurons and glia, we used human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes from patients carrying the MAPT IVS10+16 mutation and CRISPR/Cas9, isogenic corrected controls. We found that neurons from MAPT IVS10+16 carriers exhibited significantly higher levels of tau containing 4 microtubule binding repeats (4R tau), deficits in lysosomal trafficking, and acidity relative to isogenic-control neurons. Conversely, astrocytes from MAPT IVS10+16 carriers exhibited morphologically an increase in acidic lysosomes compared to isogenic-control astrocytes. Astrocytes from MAPT IVS10+16 carriers were also larger in size, consistent with cellular hypertrophy observed in brains from FTD-tau patients. Our findings suggest that altered tau splicing induced by the MAPT IVS10+16 mutation is sufficient to cause impaired lysosomal function and altered proteostasis in a cell-type specific manner.

Impact of MAPT mutations on transcriptomic signatures of FTLD brains and patient-derived pluripotent cell models.

Mutations in the microtubule-associated protein tau (MAPT) cause heterogeneous forms of frontotemporal lobar dementia with tau inclusions (FTLD-tau). Yet, the pathogenic events linked to disease remain poorly understood. This study was aimed at identifying genes and pathways that drive to FTLD-tau. To identify the earliest genes and pathways that are dysregulated in FTLD-tau, we detected differentially expressed genes in RNA-seq data generated from induced pluripotent stem cell (iPSC)-derived cortical neurons carrying MAPT R406W, MAPT P301L, and MAPT IVS10+16 and isogenic, controls and 26 brain tissue samples (3 R406W carriers, 7 IVS10+16, 3 P301L, and 13 unrelated controls). We then identified pathological pathways and drug targets that were enriched among the differentially expressed genes. We identified 61 genes that were differentially expressed in iPSC-derived cortical neurons from MAPT R406W carriers compared with isogenic, control neurons and replicated a subset of these genes in brain tissue from MAPT R406W carriers. We identified 15 genes that were differentially expressed in iPSC-derived cortical neurons from MAPT IVS10+16 carriers compared with isogenic, control neurons and replicated a subset of these genes in brain tissue from MAPT IVS10+16 carriers. We identified 37 genes that were differentially expressed in iPSC-derived cortical neurons from MAPT P301L carriers compared with isogenic, control neurons and replicated a subset of these genes in brain tissue from MAPT P301L carriers. Interestingly, there is little overlap among genes differentially expressed for MAPT R406W, MAPT P301L, and MAPT IVS10+16, which suggests that these mutations may lead to tau aggregation and neurodegeneration by different mechanisms. The results from this study demonstrate that iPSC-derived neurons capture molecular processes that occur in human brains and can be used to model disease.

Mapping tau burden and neuronal loss in MAPT-associated frontotemporal lobar degeneration.

Genetic mutations in the microtubule-associated protein tau (MAPT) cause frontotemporal lobar degeneration (FTLD-MAPT), associated with severe frontotemporal atrophy antemortem, and highly heterogeneous tau immunoreactivity postmortem, with differential involvement of 3-repeats (3R) or 4-repeats (4R) tau isoforms. To date, there are limited data objectively measuring tau burden in FTLD-MAPT, or directly comparing specific mutation groups. These novel data can help elucidate patterns of regional spread and associated neuronal loss, and shed light on pathologic heterogeneity in tauopathies. We digitally quantified burden of AT8-positive tau pathology using validated digital methods in a cohort of 35 FTLD-MAPT autopsy cases with 9 different mutations (4R mutations: P301L=16, IVS10+16=4, N279K=1; 3R mutations: G272V=5; L266V=2; 3R+4R mutations: R406W=3; L315R=2; G389R=1; S320F=1). We examined the relationship between quantitative measurements of tau burden and ordinal scores of neuronal loss (0-3) in a maximum of nine available grey matter (GM) regions. Regional analysis of tau burden was performed in a subset of six regions with most available data using linear mixed-effects modeling, adjusting for disease duration and neuronal loss. Overall, digitally quantified GM tau burden correlated positively with neuronal loss (beta=1.7, p<0.001), however a subset of GM samples (39/179, 22%) showed relatively low tau pathology in combination with severe neuronal loss. This was particularly prominent in specific mutations (χ2 =33.0, p<0.001) irrespective of region, i.e. in 7/11 (64%) GM samples of L315R, 11/24 (46%) of G272V, and 2/6 (33%) of S320F, and showed some regional variability (χ2 =34.9, p<0.001) across the cohort, occurring most frequently in the frontal (6/8, 75%) and temporal (9/20, 45%) pole. Regional analysis of tau burden in all FTLD-MAPT found that maximally affected regions were the parahippocampal gyrus (PHG), anterior cingulate cortex (ACC) and temporal pole (TP) (F=53.5, p<0.001). Sub-analyses found that 4R mutations were most affected in PHG and TP (F=29.7, p<0.001), while 3R mutations in ACC (F=13.0, p<0.001), and 3R+4R mutations in ACC and inferior parietal lobule (F=53.6, p<0.001). Heterogeneity in severity and distribution of pathology in FTLD-MAPT may in part be related to underlying mutation and tau isoform conformation, which may help improve the understanding of disease pathogenesis and progression.

A phase 1b, randomized, double‐blind, placebo‐controlled, parallel cohort safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy study of intravenously infused BIIB092 in patients with four different primary tauopathy syndromes

AbstractBackgroundAggregation of insoluble tau is a key autopsy finding in several neurodegenerative conditions including corticobasal syndrome (CBS), non‐fluent variant primary progressive aphasia (nfvPPA), frontotemporal lobar degeneration due to microtubule‐associated protein tau (MAPT) mutation, and traumatic encephalopathy syndrome (TES). Monoclonal‐antibody (mAb) therapies may promote clearance of pathogenic tau species, but differing protein isoforms and conformations may limit the affinity of mAbs for tau epitopes in distinct tauopathy syndromes. A “basket trial” allows for efficient investigation of drug target engagement in distinct diseases sharing common causative molecular alterations. We assessed the safety, tolerability, pharmacokinetic profile, and pharmacodynamic effects of BIIB092 (gosuranemab), a mAb targeting an N‐terminal tau epitope, in a basket trial enrolling participants with 4 different tauopathy syndromes: CBS, nfvPPA, symptomatic MAPT mutations, and TES. We also explored BIIB092’s effects on CSF, MRI and clinical measures of disease severity.MethodN=25 participants were randomized approximately 3:1 to monthly intravenous infusions of 2000 mg BIIB092 (8 CBS, 4 nfvPPA, 4 MAPT, 2 TES) or placebo (CBS, 2 nfvPPA, 1 MAPT, 1 TES) for up to 6 months of double‐blind therapy. Subsequently 14 participants received monthly open label infusions for up to 6 months. Plasma, CSF, exploratory clinical measures, and volumetric imaging were collected at baseline, week 12, and week 24 of the double‐blind portion of the trial.ResultThe study was terminated by Biogen in December 2019 (following negative results in a phase 2 in progressive supranuclear palsy), prior to completion of planned enrollment or treatment in recruited participants. Adverse events were more frequent in participants randomized to BIIB092 (83%) compared to placebo (28%), though no adverse events were attributed to study drug. In all patients randomized to BIIB092, we observed 90% reduction in detectable CSF N‐terminal tau epitopes at weeks 12 (mean ‐96.9%, SD 2.0) and 24 (mean ‐97.0%, SD 1.9) of the blinded treatment period. Analyses of other secondary and exploratory endpoints are underway.ConclusionSix months of BIIB092 treatment was well tolerated and appears to bind to the N‐terminal epitopes in individuals with four different tauopathy syndromes.

Astrocytic BMAL1 regulates protein aggregation in mouse models of alpha‐synuclein and tau pathology

AbstractBackgroundThe circadian clock regulates inflammatory responses in the peripheral immune system, though its function in neuroinflammation is poorly understood. Deletion of the master circadian clock transcription factor BMAL1 abrogates cellular circadian clock function and can be used to probe cell‐type specific functions of the clock. Our lab has previously shown that Bmal1 deletion induces oxidative stress, astrocyte activation, and increased β‐amyloid plaque deposition in mice. We hypothesized that deletion of Bmal1 would increase pathology in other protein aggregation models.MethodsWe generated global inducible Bmal1 knock out mice expressing human P301S mutant tau. Pathology, aggregation state, and gliosis were quantified at nine months of age. We induced synuclein aggregation in global Bmal1 knock out mice using an alpha‐synuclein preformed fibril injection (PFF) model. Three months post‐PFF injection, mice were analyzed for synuclein pathology and gliosis. To determine a cell‐type specific effect, we generated astrocyte‐ and microglia‐specific Bmal1 knock out mice and used these for PFF injections. Pathology and gliosis were analyzed from these mice three months post‐PFF injection. We then generated astrocyte‐specific Bmal1 knock out mice expressing human P301S mutant tau to test the effect of Bmal1 deletion in astrocytes on tau aggregation.ResultsGlobal Bmal1 deletion in the P301S model resulted in a significant decrease in aggregated tau and microglia activation. Global Bmal1 knock out mice injected with PFFs had significantly decreased synuclein pathology and a decrease in microgliosis. Bmal1 deletion specifically in astrocytes was sufficient to reduce both tau and synuclein pathology. Using bioinformatics methods, we discovered an astrocytic candidate gene, Bag3, which may play a role in reducing pathology in our models. We used an in vitro PFF uptake assay and found that Bmal1 knockdown resulted in increased PFF uptake, which was reduced to baseline after simultaneous knockdown of Bag3.Conclusion Bmal1 deletion in astrocytes is sufficient to reduce tau and synuclein pathologies and attenuate microglia activation. This effect may be driven by the upregulation of the macroautophagy chaperone protein, Bag3. Our data suggests that targeting astrocytic protein degradation machinery may be a therapeutic strategy for reducing intra‐neuronal protein aggregates, such as tau and synuclein.

Modeling tauopathy using human-like pathogenic tau seeds.

Compared with traditional transgenic models of tauopathy, there is no confounding effect from the insertion of the exogenous gene and overexpression of the human mutant tau in the tau spreading models. By strategically choosing the location and time of the inoculation, we can spatiotemporally determine the starting point of tau pathology in the model mimicking the development of tauopathy. Moreover, they recapitulate the strain-specific features of tau pathology. Regardless of these advantages, human-like tau spreading models are very much confined due to the limitation on quantity and quality of tau seeds. In vitro seeding assay has shown its great potential of amplifying the pathogenic tau seeds to expand their quantity and increase their quality. In the study, we used biochemically enriched, human-derived, tau aggregates as seeds to template recombinant tau (rT40) in vitro. Using biochemistry and electron microscopy, we examined the biophysical properties of the amplified materials and the human-derived tau seeds. We also interrogated the pathogenicity of the amplified human-like tau strains with orthogonal spreading models of tauopathy. As a result, we observed elongated tau filaments with recombinant T40 attached to the seeds. Compared with the seeds control, the amplified tau strains showed increased pathogenic tau species after seeding. The amplified tau strains recapitulate the pathogenicity and biophysical property of the original seeds. Our data suggest that the in vitro seeded recombinant tau can be recruited onto the seeds and converted into the confirmation of pathological tau seeds in a strain-dependent manner. Along with the conversion, the pathogenicity of different tau strains can be conferred and conserved into the recombinant tau. The study provides an authentic way of amplifying tau strains in vitro as well as a tool to expand our ability to understand tau fibrillization and transmission.

CSF tau phosphorylation occupancy at t217 increases in MAPT R406W mutation carriers without amyloid pathology

AbstractBackgroundTau hyperphosphorylation at Threonine 217 (pT217) in cerebrospinal fluid (CSF) has recently been linked to early amyloidosis and could serve as a highly sensitive biomarker for Alzheimer disease (AD). However, it remains unclear whether pT217 could be modified in other tauopathies. Using immunoprecipitation and mass spectrometry, we compared CSF T217 phosphorylation occupancy (pT217/T217) and amyloid beta (Aβ) 42/40 ratio in cognitively normal controls, and individuals with AD, Progressive Supranuclear Palsy (PSP), Corticobasal Syndrome (CBS), and sporadic and familial Frontotemporal Dementia (FTD).MethodCSF Aβ, tau and phosphorylated tau (p‐tau) were measured using immunoprecipitation and quantitative mass spectrometry methods. Amyloid and tau Positron Emission Topography (PET) imaging were measured in a subset of participants and analyzed for correlation. Quadrant analyses were performed to assess amyloid and p‐tau positivity.ResultWe confirmed the strong association between CSF Aβ 42/40 and pT217 measures in most samples. Individuals with AD had high CSF pT217/T217 and low Aβ 42/40. In contrast, controls and the majority of patients with other tauopathies had low CSF pT217/T217 and normal Aβ 42/40. We identified a subgroup of individuals with increased CSF pT217/T217 and normal Aβ 42/40 ratio relative to controls. Most of these individuals were identified as MAPT R406W mutation carriers, a tau mutation leading to 3R+4R tauopathy similar to AD but without amyloid pathology. Diagnostic accuracies of CSF Aβ 42/40 alone, CSF pT217/T217 alone, and combination of both biomarkers were examined. We show that combination of both biomarkers can separate MAPT R406W carriers from control (AUC=0.960) and other tauopathies that are primarily 4R tauopathies (AUC=0.934).ConclusionWe demonstrate the importance of measuring both CSF Aβ 42/40 and pT217/T217 to improve AD diagnosis and differentiate MAPT R406W mutation carriers. This supports that changes in CSF pT217/T217 ratio is not a specific biomarker for AD but reflects common downstream tau pathology in these two 3R+4R tauopathies.

BAG3 Regulation of RAB35 Mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome Pathway and Tau Clearance

BackgroundDeclining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimer’s disease (AD). Emerging studies implicate impairment of the endosome-lysosome pathway as a significant factor in the pathogenesis of these diseases. Previously, we demonstrated that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression. MethodsMass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3-HSP70-TBC1D10B (EPI64B)-RAB35-HRS regulatory networks. Live-cell imaging was used to study the dynamics of the endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and in P301S tau transgenic mice with BAG3 overexpressed. ResultsThe primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the RAB35 GTPase-activating protein TBC1D10B. We demonstrated that a BAG3-HSP70-TBC1D10B complex attenuates the ability of TBC1D10B to inactivate RAB35. Thus, BAG3 interacts with TBC1D10B to support the activation of RAB35 and recruitment of HRS, initiating endosomal sorting complex required for transport–mediated endosomal tau clearance. Furthermore, TBC1D10B shows significantly less colocalization with BAG3 in AD brains than in age-matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the colocalization of phosphorylated tau with the endosomal sorting complex required for transport III protein CHMP2B and reduced the levels of the mutant human tau. ConclusionsWe identified a novel BAG3-TBC1D10B-RAB35 regulatory axis that modulates endosomal sorting complex required for transport–dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD.