4R Tau Research Articles

A 4R tauopathy develops without amyloid deposits in aged cat brains

Human tauopathies are neurodegenerative diseases with accumulation of abnormally phosphorylated and aggregated tau proteins forming neurofibrillary tangles. We investigated the development of tau pathology in aged cat brains as a model of neurofibrillary tangle formation occurring spontaneously during aging. In 4 of 6 cats aged between 18 and 21 years, we found a somatodendritic accumulation of phosphorylated and aggregated tau in neurons and oligodendrocytes. Two of these 4 cats had no amyloid immunoreactivity. These tau inclusions were mainly composed of 4R tau isoforms and straight filaments and colocalized with the active form of the glycogen synthase kinase-3 (GSK3). Cat brains with a tau pathology showed a significant cortical atrophy and neuronal loss. We demonstrate in this study the presence of a tau pathology in aged cat brains that develop independently of amyloid deposits. The colocalization of the active form of the GSK3 with tau inclusions as observed in human tauopathies suggests that this kinase could be responsible for the abnormal tau phosphorylation observed in aged cat brains, representing a mechanism of tau pathology development shared between a naturally occurring tauopathy in aged cats and human tauopathies.

How to demix Alzheimer-type and PSP-type tau lesions out of their mixture -hybrid approach to dissect comorbidity-

Neurofibrillary tangles (NFTs), are shared between progressive supranuclear palsy (PSP) and Alzheimer disease (AD). Histological distinction of PSP and AD is possible based on the distribution of NFTs. However, neuropathologists may encounter diagnostic difficulty with comorbidity of PSP and AD. In this study, we tried to circumvent this difficulty by analyzing five autopsied brains harboring both PSP and AD pathology. Tau-positive lesions were sorted based on their cell type (neuron versus glia), and tau isoforms: three-repeat (3R) versus four-repeat (4R) tau. 16 regions were selected to map these lesions throughout the brain. 4R-tau lesions were present in all areas examined. Among them, 3R-tau lesions were absent in some areas. These 4R selective (4R+/3R-) areas dictate prototypic distribution of PSP, not usually found in AD, such as pontine nucleus, red nucleus, inferior olivary nucleus, dentate nucleus, globus pallidus and putamen, each contained both glial and neuronal lesions. In contrast, additional 3R-tau lesions were found in hippocampal formation to neocortex, where 3R immunoreactivity (IR) was predominant over the 4R counterpart mainly in neurons as found in AD but not in PSP. Although tau lesions in central grey matter, substantia nigra and locus coeruleus are found in both AD and PSP, 4R-selectivity with glial component suggests PSP origin. Even if the presence of 3 R IR in these areas suggests AD pathology, it does not exclude the involvement of PSP-type lesion because distinction of 4R IR into PSP or AD is not yet possible. Further demixing may be possible if biochemical difference of 4R tau between PSP and AD is identified.

Corticobasal syndrome: neuroimaging and neurophysiological advances.

Corticobasal degeneration (CBD) is a neurodegenerative condition characterized by 4R tau protein deposition in several brain regions that clinically manifests itself as a heterogeneous atypical parkinsonism typically expressed in adulthood. The prototypical clinical phenotype of CBD is corticobasal syndrome (CBS). Important insights into the pathophysiological mechanisms underlying motor and higher cortical symptoms in CBS have been gained by using advanced neuroimaging and neurophysiological techniques. Structural and functional neuroimaging studies often show asymmetric cortical and subcortical abnormalities, mainly involving perirolandic and parietal regions and basal ganglia structures. Neurophysiological investigations including electroencephalography and somatosensory evoked potentials provide useful information on the origin of myoclonus and on cortical sensory loss. Transcranial magnetic stimulation demonstrates heterogeneous and asymmetric changes in the excitability and plasticity of primary motor cortex and abnormal hemispheric connectivity. Neuroimaging and neurophysiological abnormalities in multiple brain areas reflect asymmetric neurodegeneration, leading to asymmetric motor and higher cortical symptoms in CBS.

NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies.

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer’s disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders.

Ordered Assembly of Tau Protein and Neurodegeneration.

Tau filaments with distinct morphologies and/or isoform compositions underlie a large number of human neurodegenerative diseases. Their formation is important, because dominantly inherited mutations in MAPT, the tau gene, cause frontotemporal dementia with abundant filamentous tau inclusions. Assembly of tau may begin in a specific region of the brain, from where it spreads to other areas. It remains to be seen if the molecular species underlying tau aggregate-mediated neurodegeneration and propagation are the same or different. In the brains of mice transgenic for human mutant P301S tau, small tau filaments are the predominant seed-competent species. It has been suggested that different conformers of assembled tau may give rise to different human tauopathies, but until recently, it was not possible to study this directly. Electron cryo-microscopy can now be used to determine high-resolution structures of amyloid filaments from human brain. Paired helical and straight tau filaments of Alzheimer's disease are ultrastructural polymorphs. Each filament core is composed of two identical protofilaments extending from G273/304-E380 (in the numbering of the 441 amino acid isoform of human tau), which adopt a combined cross-β/β-helix structure. They comprise the ends of the first or second microtubule-binding repeat (R1 or R2), the whole of R3 and R4, as well as 12 amino acids after R4. By contrast, the core of the narrow filament of Pick's disease consists of a single protofilament extending from K254-F378 of 3R tau, which adopts a cross-β structure. It comprises the last 21 amino acids of R1, all of R3 and R4, as well as 10 amino acids after R4. Wide tau filaments of Pick's disease, which are in the minority, consist of two narrow filaments packed against each other. The tau filament folds of Alzheimer's and Pick's diseases appear to be conserved between different cases of disease. These findings show that filamentous tau adopts one fold in Alzheimer's disease and a different fold in Pick's disease, establishing the existence of distinct conformers.

Seeding selectivity and ultrasensitive detection of tau aggregate conformers of Alzheimer disease

Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) involve the abnormal accumulation in the brain of filaments composed of both three-repeat (3R) and four-repeat (4R) (3R/4R) tau isoforms. To probe the molecular basis for AD’s tau filament propagation and to improve detection of tau aggregates as potential biomarkers, we have exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking-induced conversion or AD RT-QuIC). The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils made of certain recombinant tau fragments. AD RT-QuIC detected seeding activity in AD (n = 16) brains at dilutions as extreme as 107–1010-fold, but was 102–106-fold less responsive when seeded with brain from most cases of other types of tauopathy with comparable loads of predominant 3R or 4R tau aggregates. For example, AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to the weakest of the AD specimens, and higher than 3 of 4 specimens with 3R/4R primary age-related tauopathy. AD seeds shared properties with the tau filaments found in AD brains, as AD seeds were sarkosyl-insoluble, protease resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity exhibited by AD and CTE tau filaments compared to other types of tauopathies in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides rapid ultrasensitive quantitation of 3R/4R tau-seeding activity as a biomarker.

FTDP-17 Mutations Alter the Aggregation and Microtubule Stabilization Propensity of Tau in an Isoform-Specific Fashion.

More than 50 different intronic and exonic autosomal dominant mutations in the tau gene have been linked to the neurodegenerative disorder frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Although the pathological and clinical presentation of this disorder is heterogeneous among patients, the deposition of tau as pathological aggregates is a common feature. Collectively, FTDP-17 mutations have been shown to alter tau's ability to stabilize microtubules, enhance its aggregation, alter mRNA splicing, or induce its hyperphosphorylation, among other effects. Previous in vitro studies from our lab revealed that these mutations differ markedly from each other in the longest 2N4R isoform of tau. However, it is not entirely known whether the effect of a single mutation varies when compared between different isoforms of tau. Differences in the isoelectric points of the N-terminal region of tau isoforms lead to changes in their biochemical properties, raising the possibility that isoforms could also be disproportionately affected by disease-related mechanisms such as mutations. We therefore performed a comparative study of three FTDP-17 mutations present in different regions of tau (R5L, P301L, and R406W) in the three 4R isoforms of tau. We observed significant differences in the effect these mutations exert on the total amount and kinetics of aggregation, aggregate length distributions, and microtubule stabilizing propensity of 4R tau isoforms for all three selected mutants. These results demonstrate that different combinations of FTDP-17 mutations and tau isoforms are functionally distinct and could have important implications for our understanding of disease and animal models of tauopathies.

Cofactors are essential constituents of stable and seeding-active tau fibrils

Amyloid fibrils are cross-β-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-β-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.

Continental and Antarctic Lichens: isolation, identification and molecular modeling of the depside tenuiorin from the Antarctic lichen Umbilicaria antarctica as tau protein inhibitor

Alzheimer´s disease (AD) is the most common form of dementia involving Aβ and tau protein. So far, AD cure remains elusive, but considering that AD progresses throughout tau pathology, which turns tau protein an appropriate target, besides tau is also included in other neurodegenerative disorders named as tauopathies. Here, we have isolated seventeen compounds belonging to six lichens species. Due to scarce of spectroscopic data of the compound 5,7-dihydroxy-6-methylphthalide, we explained their structural elucidation based on NMR data. In this study, we show that only tenuiorin from Umbilicaria antarctica inhibited 50% of tau 4R at 100 µM. Then, we shown that molecular interactions of tenuiorin with the steric zipper model of the hexapeptide 306VQIVYK311 were studied by docking calculations and the results suggested that tenuiorin forms both hydrogen bonds with lysine and glutamine side chains and forms several hydrophobic interactions with valine and lysine from 306VQIVYK311 motif.

Structural disorder in four-repeat Tau fibrils reveals a new mechanism for barriers to cross-seeding of Tau isoforms

The intracellular deposition of fibrils composed of the microtubule-associated protein Tau is a characteristic feature of Alzheimer's disease (AD) and other fatal neurodegenerative disorders collectively known as tauopathies. Short Tau fibrils spread intracerebrally through transfer between interconnected neurons. Once taken up by a recipient cell, Tau fibrils recruit Tau monomers onto their ends. Based on the number of microtubule-binding repeats, there are two distinct groups of Tau isoforms: three-repeat (3R) Tau and four-repeat (4R) Tau. In AD, all Tau isoforms are deposited, whereas in other tauopathies, only 3R or 4R Tau isoforms are deposited. The molecular basis for these isoform-specific depositions is poorly understood, although conformation-based cross-seeding barriers are key. Here, we used sedimentation assays, EPR spectroscopy, and other structural readouts to better understand the cross-seeding barriers of 4R Tau fibrils. We observed that fibrils formed from truncated Tau (K18), but not full-length Tau (htau40), exhibit a barrier that inhibits 3R Tau recruitment. Investigating an array of differently sized fragments, we found that the Tau C terminus modulates the cross-seeding barrier and that the N terminus plays a synergistic role. Two disease-associated Tau variants, P301S and P301L, also established strong cross-seeding barriers. EPR analysis indicated that fibrils seeded with truncated and mutated Tau, but not htau40, are structurally disordered in the second half of repeat four and onward. These findings suggest that the disorder in this region diminishes the ability of 4R Tau fibrils to recruit 3R Tau monomers, revealing a new mechanism for Tau cross-seeding barriers.

Severity dependent distribution of impairments in PSP and CBS: Interactive visualizations

BackgroundProgressive supranuclear palsy (PSP) -Richardson's Syndrome and Corticobasal Syndrome (CBS) are the two classic clinical syndromes associated with underlying four repeat (4R) tau pathology. The PSP Rating Scale is a commonly used assessment in PSP clinical trials; there is an increasing interest in designing combined 4R tauopathy clinical trials involving both CBS and PSP. ObjectivesTo determine contributions of each domain of the PSP Rating Scale to overall severity and characterize the probable sequence of clinical progression of PSP as compared to CBS. MethodsMulticenter clinical trial and natural history study data were analyzed from 545 patients with PSP and 49 with CBS. Proportional odds models were applied to model normalized cross-sectional PSP Rating Scale, estimating the probability that a patient would experience impairment in each domain using the PSP Rating Scale total score as the index of overall disease severity. ResultsThe earliest symptom domain to demonstrate impairment in PSP patients was most likely to be Ocular Motor, followed jointly by Gait/Midline and Daily Activities, then Limb Motor and Mentation, and finally Bulbar. For CBS, Limb Motor manifested first and ocular showed less probability of impairment throughout the disease spectrum. An online tool to visualize predicted disease progression was developed to predict relative disability on each subscale per overall disease severity. ConclusionThe PSP Rating Scale captures disease severity in both PSP and CBS. Modelling how domains change in relation to one other at varying disease severities may facilitate detection of therapeutic effects in future clinical trials.

4',6-Diamidino-2-Phenylindole Distinctly Labels Tau Deposits.

Tau deposits have distinct biochemical characteristics and vary morphologically based on identification with tau antibodies and several chemical dyes. Here, we report 4',6-diamidino-2-phenylindole (DAPI)-positivity of tau deposits. Furthermore, we investigated the cause for this positivity. DAPI was positive in 3R/4R (3-repeat/4-repeat) tau deposits in Alzheimer's disease, myotonic dystrophy, and neurodegeneration with brain iron accumulation, and in 4R tau deposits in corticobasal degeneration, but negative in 4R tau deposits in frontotemporal dementia with parkinsonism-17 and progressive supranuclear palsy. The peak emission wavelength of DAPI after binding to a tau deposit was similar to that after binding to a nucleus. This DAPI-positivity was conspicuous at the optimum concentration of 2 μg/ml. DAPI-positivity was diminished after formic acid treatment, but preserved after nucleic acid elimination and phosphate moiety blocking. Our results suggest that staining with 2 μg/ml DAPI is a common but useful tool to differentially detect tau deposits in various tauopathies.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

Acetylation of Lysine Residues within the MT‐binding Repeats Specifically Modulates the Structure Ensemble of Tau

The microtubule associated protein tau is expressed primarily in the central nervous system and promotes microtubule (MT) assembly. The aberrant accumulation of tau protein and formation of insoluble aggregates are pathological hallmarks of neurodegenerative tauopathies including Alzheimer's disease. Six tau isoforms are generated by mRNA alternative splicing containing either three (3R tau) or four (4R tau) MT‐binding repeats and various number of N‐terminal exons (0N, 1N and 2N). K18 and K19 are the truncated constructs of 4R tau and 3R tau, consisting of only the four and three repeats, respectively. K18 and K19 form amyloid fibrils with cross‐β structure, constituting the core of tau filaments. Recent studies have shown that acetylation of specific lysine residues within the MT‐binding repeats could either promote or inhibit tau aggregation and tau mediated microtubule assembly. Here, we investigated the effect of lysine acetylation on the structure of tau K18 by a combination of biophysical structure characterization and molecular dynamics simulation. Radius of gyration derived from small angle X‐ray scattering (SAXS) showed that the structures of acetylation mimic tau species (K274Q/K321Q and K280Q/K281Q/K369Q) were more extended than the WT protein. Although the influence of lysine acetylation on the intramolecular interactions was mainly local, long range perturbations were also observed from NMR chemical shift analysis. Molecular dynamic simulations in explicit solvent revealed site specific modulation in the structure ensemble of tau repeats, where K280Q/K281Q mutation led to compaction of repeat R2 and K321Q mutation led to extension of repeat R3. Statistics on the lifetime and distance of residue contacts and secondary structure components were performed to investigate the changes in structure and dynamics upon acetylation. These results show that the effect of acetylation on tau structure was site specific, which may promote or reduce aggregation by exposing or shielding the aggregation prone segments.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.

Modulation of Tau Isoforms Imbalance Precludes Tau Pathology and Cognitive Decline in a Mouse Model of Tauopathy

The microtubule-associated protein tau regulates myriad neuronal functions, such as microtubule dynamics, axonal transport and neurite outgrowth. Tauopathies are neurodegenerative disorders characterized by the abnormal metabolism of tau, which accumulates as insoluble neuronal deposits. The adult human brain contains equal amounts of tauisoforms with three (3R) or four (4R) repeats ofmicrotubule-binding domains, derived from the alternative splicing of exon 10 (E10) in the tau transcript. Several tauopathies are associated with imbalances of tau isoforms, due to splicing deficits. Here, we used a trans-splicing strategy to shift the inclusion of E10 in a mouse model of tauopathy that produces abnormal excess of 3R tau. Modulating the 3R/4R ratio in the prefrontal cortex led to a significant reduction of pathological tau accumulation concomitant with improvement of neuronal firing and reduction of cognitive impairments. Our results suggest promising potential for the use of RNA reprogramming in human neurodegenerative diseases.

Tau Kinetics in Neurons and the Human Central Nervous System

We developed stable isotope labeling and mass spectrometry approaches to measure the kinetics of multiple isoforms and fragments of tau in the human central nervous system (CNS) and in human induced pluripotent stem cell (iPSC)-derived neurons. Newly synthesized tau is truncated and released from human neurons in 3days. Although most tau proteins have similar turnover, 4R tau isoforms and phosphorylated forms of tau exhibit faster turnover rates, suggesting unique processing of these forms that may have independent biological activities. The half-life of tau in control human iPSC-derived neurons is 6.74± 0.45days and in human CNS is 23± 6.4days. In cognitively normal and Alzheimer's disease participants, the production rate of tau positively correlates with the amount of amyloid plaques, indicating a biological link between amyloid plaques and tau physiology.

Progressive Supranuclear Palsy: an Update.

Progressive supranuclear palsy (PSP) is a 4R tau neuropathologic entity. While historically defined by the presence of a vertical supranuclear gaze palsy and falls in the first symptomatic year, clinicopathologic studies identify alternate presenting phenotypes. This article reviews the new PSP diagnostic criteria, diagnostic approaches, and treatment strategies. The 2017 International Parkinson and Movement Disorder Society PSP criteria outline 14 core clinical features and 4 clinical clues that combine to diagnose one of eight PSP phenotypes with probable, possible, or suggestive certainty. Evidence supports the use of select imaging approaches in the classic PSP-Richardson syndrome phenotype. Recent trials of putative disease-modifying agents showed no benefit. The new PSP diagnostic criteria incorporating the range of presenting phenotypes have important implications for diagnosis and research. More work is needed to understand how diagnostic evaluations inform phenotype assessment and identify expected progression. Current treatment is symptomatic, but tau-based therapeutics are in active clinical trials.

Detection of Alzheimer Disease (AD)-Specific Tau Pathology in AD and NonAD Tauopathies by Immunohistochemistry With Novel Conformation-Selective Tau Antibodies.

Aggregation of tau into fibrillar structures within the CNS is a pathological hallmark of a clinically heterogeneous set of neurodegenerative diseases termed tauopathies. Unique misfolded conformations of tau, referred to as strains, are hypothesized to underlie the distinct neuroanatomical and cellular distribution of pathological tau aggregates. Here, we report the identification of novel tau monoclonal antibodies (mAbs) that selectively bind to an Alzheimer disease (AD)-specific conformation of pathological tau. Immunohistochemical analysis of tissue from various AD and nonAD tauopathies demonstrate selective binding of mAbs GT-7 and GT-38 to AD tau pathologies and absence of immunoreactivity for tau aggregates that are diagnostic of corticobasal degenerations (CBD), progressive supranuclear palsy (PSP), and Pick's disease (PiD). In cases with co-occurring AD tauopathy, GT-7 and GT-38 distinguish comorbid AD tau from pathological tau in frontotemporal lobar degeneration characterized by tau inclusions (FTLD-Tau), as confirmed by the presence of both 3 versus 4 microtubule-binding repeat isoforms (3R and 4R tau isoforms, respectively), in AD neurofibrillary tangles but not in the tau aggregates of CBD, PSP, or PiD. These findings support the concept of an AD-specific tau strain. The mAbs described here enable the selective detection of AD tau pathology in nonAD tauopathies.

Isoform-independent and -dependent phosphorylation of microtubule-associated protein tau in mouse brain during postnatal development

Tau is a microtubule (MT)-associated protein that regulates MT dynamics in the axons of neurons. Tau binds to MTs via its C-terminal MT-binding repeats. There are two types of tau, those with three (3R) or four (4R) MT-binding repeats; 4R tau has a stronger MT-stabilizing activity than 3R tau. The MT-stabilizing activity of tau is regulated by phosphorylation. Interestingly, both the isoform and phosphorylation change at the time of neuronal circuit formation during postnatal development; highly phosphorylated 3R tau is replaced with 4R tau, which is less phosphorylated. However, it is not known how the transition of the isoforms and phosphorylation are regulated. Here, we addressed this question using developing mouse brains. Detailed analysis of developing brains revealed that the switch from 3R to 4R tau occurred during postnatal day 9 (P9) to P18 under the same time course as the conversion of phosphorylation from high to low. However, hypothyroidism, which is known to delay brain development, delayed the timing of tau dephosphorylation but not the exchange of isoforms, indicating that isoform switching and phosphorylation are not necessarily linked. Furthermore, we confirmed this finding by using mouse brains that expressed a single isoform of human tau. Human tau, either 3R or 4R, reduced phosphorylation levels during development even though the isoform did not change. We also found that 3R tau and 4R tau were phosphorylated differently in vivo even at the same developmental days. These results show for the first time that the phosphorylation and isoform alteration of tau are regulated differently during mouse development.

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.