Mutant Tau Research Articles

Interplay of pathogenic forms of human tau with different autophagic pathways.

SummaryLoss of neuronal proteostasis, a common feature of the aging brain, is accelerated in neurodegenerative disorders, including different types of tauopathies. Aberrant turnover of tau, a microtubule‐stabilizing protein, contributes to its accumulation and subsequent toxicity in tauopathy patients’ brains. A direct toxic effect of pathogenic forms of tau on the proteolytic systems that normally contribute to their turnover has been proposed. In this study, we analyzed the contribution of three different types of autophagy, macroautophagy, chaperone‐mediated autophagy, and endosomal microautophagy to the degradation of tau protein variants and tau mutations associated with this age‐related disease. We have found that the pathogenic P301L mutation inhibits degradation of tau by any of the three autophagic pathways, whereas the risk‐associated tau mutation A152T reroutes tau for degradation through a different autophagy pathway. We also found defective autophagic degradation of tau when using mutations that mimic common posttranslational modifications in tau or known to promote its aggregation. Interestingly, although most mutations markedly reduced degradation of tau through autophagy, the step of this process preferentially affected varies depending on the type of tau mutation. Overall, our studies unveil a complex interplay between the multiple modifications of tau and selective forms of autophagy that may determine its physiological degradation and its faulty clearance in the disease context.

Chemogenetic locus coeruleus activation restores reversal learning in a rat model of Alzheimer's disease.

See Grinberg and Heinsen (doi:10.1093/brain/awx261) for a scientific commentary on this article. Clinical evidence suggests that aberrant tau accumulation in the locus coeruleus and noradrenergic dysfunction may be a critical early step in Alzheimer’s disease progression. Yet, an accurate preclinical model of these phenotypes that includes early pretangle tau accrual in the locus coeruleus, loss of locus coeruleus innervation and deficits locus coeruleus/norepinephrine modulated behaviours, does not exist, hampering the identification of underlying mechanisms and the development of locus coeruleus-based therapies. Here, a transgenic rat (TgF344-AD) expressing disease-causing mutant amyloid precursor protein (APPsw) and presenilin-1 (PS1ΔE9) was characterized for histological and behavioural signs of locus coeruleus dysfunction reminiscent of mild cognitive impairment/early Alzheimer’s disease. In TgF344-AD rats, hyperphosphorylated tau was detected in the locus coeruleus prior to accrual in the medial entorhinal cortex or hippocampus, and tau pathology in the locus coeruleus was negatively correlated with noradrenergic innervation in the medial entorhinal cortex. Likewise, TgF344-AD rats displayed progressive loss of hippocampal norepinephrine levels and locus coeruleus fibres in the medial entorhinal cortex and dentate gyrus, with no frank noradrenergic cell body loss. Cultured mouse locus coeruleus neurons expressing hyperphosphorylation-prone mutant human tau had shorter neurites than control neurons, but similar cell viability, suggesting a causal link between pretangle tau accrual and altered locus coeruleus fibre morphology. TgF344-AD rats had impaired reversal learning in the Morris water maze compared to their wild-type littermates, which was rescued by chemogenetic locus coeruleus activation via designer receptors exclusively activated by designer drugs (DREADDs). Our results indicate that TgF344-AD rats uniquely meet several key criteria for a suitable model of locus coeruleus pathology and dysfunction early in Alzheimer’s disease progression, and suggest that a substantial window of opportunity for locus coeruleus/ norepinephrine-based therapeutics exists.

Pazopanib Reduces Phosphorylated Tau Levels and Alters Astrocytes in a Mouse Model of Tauopathy.

Hyperphosphorylation and aggregation of tau protein is a critical factor in many neurodegenerative diseases. These diseases are increasing in prevalence, and there are currently no cures. Previous work from our group and others has shown that tyrosine kinase inhibitors (TKIs) can stimulate autophagy, decrease pathological proteins, and improve symptoms in models of neurodegeneration. Here we examined the role of pazopanib in mouse models that express either human mutant P301L tau (TauP301L) or triple mutant amyloid precursor protein (3x-AβPP). The TauP301L mouse expresses P301L tau under the control of a prion promoter in both neurons and astrocytes, reminiscent of some human tauopathies. Pazopanib crosses the blood-brain barrier with no detectable peripheral off-side effects, and decreases p-tau in TauP301L mice. Pazopanib reaches a brain concentration sufficient for inhibition of several tyrosine kinases, including vascular endothelial growth factor receptors (VEGFRs). Further, pazopanib does not affect microglia but reduces astrocyte levels toward nontransgenic controls in TauP301L mice. Pazopanib does not alter amyloid beta levels or astrocytes in 3x-AβPP mice but modulates a number of inflammatory markers (IP-10, MIP-1α, MIP-1β, and RANTES). These data suggest that pazopanib may be involved in p-tau clearance and modulation of astrocytic activity in models of tauopathies.

Progressive Motor Deficit is Mediated by the Denervation of Neuromuscular Junctions and Axonal Degeneration in Transgenic Mice Expressing Mutant (P301S) Tau Protein.

Tauopathies include a variety of neurodegenerative diseases associated with the pathological aggregation of hyperphosphorylated tau, resulting in progressive cognitive decline and motor impairment. The underlying mechanism for motor deficits related to tauopathy is not yet fully understood. Here, we use a novel transgenic tau mouse line, Tau 58/4, with enhanced neuron-specific expression of P301S mutant tau to investigate the motor abnormalities in association with the peripheral nervous system. Using stationary beam, gait, and rotarod tests, motor deficits were found in Tau 58/4 mice already 3 months after birth, which deteriorated during aging. Hyperphosphorylated tau was detected in the cell bodies and axons of motor neurons. At the age of 9 and 12 months, significant denervation of the neuromuscular junction in the extensor digitorum longus muscle was observed in Tau 58/4 mice, compared to wild-type mice. Muscle hypotrophy was observed in Tau 58/4 mice at 9 and 12 months. Using electron microscopy, we observed ultrastructural changes in the sciatic nerve of 12-month-old Tau 58/4 mice indicative of the loss of large axonal fibers and hypomyelination (assessed by g-ratio). We conclude that the accumulated hyperphosphorylated tau in the axon terminals may induce dying-back axonal degeneration, myelin abnormalities, neuromuscular junction denervation, and muscular atrophy, which may be the mechanisms responsible for the deterioration of the motor function in Tau 58/4 mice. Tau 58/4 mice represent an interesting neuromuscular degeneration model, and the pathological mechanisms might be responsible for motor signs observed in some human tauopathies.

Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia.

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

Tau and amyloid-related pathologies in the entorhinal cortex have divergent effects in the hippocampal circuit

The entorhinal cortex (EC) is affected early in Alzheimer's disease, an illness defined by a co-occurrence of tau and amyloid-related pathologies. How the co-occurrence of these pathologies in the EC affects the hippocampal circuit remains unknown. Here we address this question by performing electrophysiological analyses of the EC circuit in mice that express mutant human amyloid precursor protein (hAPP) or tau (hTau), or both in the EC. We show that the alterations in the hippocampal circuit are divergent, with hAPP increasing but hTau decreasing neuronal/circuit excitability. Most importantly, mice co-expressing hAPP and hTau show that hTau has a dominant effect, dampening the excitatory effects of hAPP. Additionally, compensatory synaptic downscaling, in response to increased excitability in EC was observed in subicular neurons of hAPP mice. Based on simulations, we propose that EC interneuron pruning can account for both EC hyperexcitability and subicular synaptic downscaling found in mice expressing hAPP.

Remodeling Tau and Prion Proteins Using Nanochaperons.

There is increasing evidence that tau protein behaves in a prion-like manner in tauopathy. The stabilization of tau protein using a small molecular compound can limit tauopathy associated morbidity that advances with ageing. Here, a lab-on-a-chip experiment is reported, where gold citrate nanoparticles (5 nm, AuNPs) can remodel mutant tau protein (P301L) and prion, thus resolving the mutant tau- and prion-mediated impairment of kinesin cargo transport on microtubules. It is found that tau protein is overexpressed in Alzheimer's disease (AD) patient serum samples and the tau conformational change can also be affected in human serum samples of AD when treated with AuNPs ex vivo. Similarly, AuNPs reorganizing the prion protein and inducing conformational changes of prions in AD serum have been observed, while having no effect on alpha-synuclein in Parkinson patient serum. The mapping of AD serum mediated traffic jams, using particle tracking and mean square displacement analysis, and the observed recovery of uninterrupted processive motor functions by AuNP treatment show that kinesin cargo assays might be a useful method for future ex vivo validation of a targeted therapy against tauopathy before administration, a viable option to combat various neurodegenerative disorders arising from the susceptibility of amyloidogenic proteins toward aggregation.

Abolishing Tau cleavage by caspases at Aspartate421 causes memory/synaptic plasticity deficits and pre-pathological Tau alterations

TAU mutations are genetically linked to fronto-temporal dementia (FTD) and hyper-phosphorylated aggregates of Tau form neurofibrillary tangles (NFTs) that constitute a pathological hallmark of Alzheimer disease (AD) and FTD. These observations indicate that Tau has a pivotal role in the pathogenesis of neurodegenerative disorders. Tau is cleaved by caspases at Aspartate421, to form a Tau metabolite known as δTau; δTau is increased in AD, due to the hyper-activation of caspases in AD brains. δTau is considered a critical toxic moiety underlying neurodegeneration, which initiates and facilitates NFT formation. As Tau is a therapeutic target in neurodegeneration, it is important to rigorously determine whether δTau is a toxic Tau species that should be pharmacologically attacked. To directly address these questions, we have generated a knock-in (KI) mouse called TauDN—that expresses a Tau mutant that cannot be cleaved by caspases. TauDN mice present short-term memory deficits and synaptic plasticity defects. Moreover, mice carrying two mutant Tau alleles show increased total insoluble hyper-phosphorylated Tau in the forebrain. These data are in contrast with the concept that δTau is a critical toxic moiety underlying neurodegeneration, and suggest that cleavage of Tau by caspases represents a negative feedback mechanism aimed to eliminate toxic Tau species. Alternatively, it is possible that either a reduction or an increase in δTau leads to synaptic dysfunction, memory impairments and Tau pathology. Both possibilities will have to be considered when targeting caspase cleavage of Tau in AD therapy.

Tau peptides and tau mutant protein aggregation inhibition by cationic polyethyleneimine and polyarginine.

Tau protein plays a major role in Alzheimer's disease. The tau protein loses its functionality by self-aggregation due to the two six-amino acid sequences VQIVYK and VQIINK of the protein. Hence it is imperative to find therapeutics that could inhibit the self-aggregation of this tau peptide fragments. Here, we study the inhibitory potential of a cationic polymer polyethyleneimine (PEI) and a cationic polypeptide arginine (Arg) on the aggregation of VQIVYK, and GKVQIINKLDL peptides, and tau mutant protein (P301L), found frequently in taupathy. Various characterization methods are employed including thioflavin S, transmission electron microscopy, and dynamic light scattering to study the aggregation/inhibition process in vitro. Results show that PEI and Arg significantly inhibit tau peptides and protein aggregation. The study could be applied to understand tau protein aggregation mechanism in the presence of cationic polymers.

REGULATED RELEASE OF HYPER-PHOSPHORYLATED, AGGREGATED AND SEEDING COMPETENT TAU FROM RODENT BRAINS AND HUMAN SYNAPTOSOMES

Tau is a microtubule binding protein expressed in neuronal axons and nerve terminals. Mutations in tau are known to cause different neurological diseases and tau pathology, in the form of aggregates and tangles, is a hallmark of Alzheimer's disease. Tau pathology in AD starts in the entorhinal cortex but “spreads” along synaptically connected areas as the disease progresses. Understanding how tau is trafficked to the nerve terminal, secreted, and taken up by postsynaptic neurons could lead to the identification of novel mechanisms aimed at halting disease progression in AD. Synaptic structures (synaptosomes) were purified from the brains of transgenic mice harbouring pathological human tau (P301S and tg4510) and from human control and AD subjects. Western blots were used to detect synaptic proteins and tau. STED microscopy was used to evalaute tau sub-cellular localisation. The ability of synaptic tau to “seed” pathological tau aggregation was evaluated in transfected HEK293 and rat cortical neurons. Calcium dependent release was induced by KCl or ionomycin, while botulinum toxins and pharmacological agents were used to cleave SNARE proteins and block exocytosis. Pathologically hyper-phosphorylated (AT8+) aggregated (Sarcosyl-insoluble) and seeding-competent tau was found in synaptosomes from both transgenic mice and human AD brains. Pathological tau was enriched in synaptosomes of the most affected brain areas in the mice, while in human brain synaptosomes the levels of pathological tau were correlated with the patient's Braak stage. STED analysis demonstrated that pathological tau was enriched in the presynaptic compartment. Pathological tau was released in a calcium dependent manner by both KCl and ionomycin. Botulinum toxin A and D were found to cleave synaptosomal SNAP25 and synaptobrevin, respectively, and at the same time inhibit calcium-dependent tau release. All together these data build on previous evidence of pathological tau presence in presynaptic terminals and demonstrate its calcium-dependent release. Pathological tau release requires intact SNARE proteins, suggesting a vesicular, exocytotic, process. This could represent a critical step in the spreading of tau in AD brain. Therapeutic interventions aimed at blocking tau release could be beneficial in reducing tau propagation and disease progression in AD.

RELATIONSHIP BETWEEN PHOSPHO-TAU ACCUMULATION AND STRUCTURAL ALTERATIONS OF NEURONAL GOLGI APPARATUS IN ALZHEIMER’S DISEASE PATIENTS AND P301S TAUOPATHY MOUSE MODEL

The Golgi apparatus (GA) is a highly dynamic organelle involved in the processing and sorting of cellular proteins, like amyloid precursor protein, and in the regulation of neuronal calcium levels. The GA undergoes morphological plasticity processes under different physiological and pathological conditions. Furthermore, in Alzheimer's disease (AD) GA was shown to become fragmented in neocortical and hippocampal neuronal subpopulations. As the microtubule-associated protein tau is present on GA membranes, along with tau phosphorylation-related kinases, and the integrity of the microtubule skeleton is necessary for the structural maintenance of the GA, here we analyze whether the accumulation of hyperphosphorylated tau is related to GA structural alterations. We have analyzed the size (volume and surface area) and morphological state of GA in neurons with and without hyperphosphorylated tau aggregates from temporal neocortex and hippocampus of AD patients and in P301S tauopathy mouse model, using double immunofluorescence techniques, confocal microscopy and 3D quantification techniques. We found that in AD patients, the percentage of temporal neocortical and CA1 hippocampal pyramidal neurons with a highly altered GA is much higher in neurons with neurofibrillary tangles (NFT) than in NFT-free neurons. Quantitative analysis of the surface area and volume of GA elements in neurons revealed that, compared with NFT-free neurons, NFT-bearing neurons had a reduction of approximately one half in neocortical neurons and one third in CA1 neurons. In the P301S mouse model, in the absence of Aβ amyloid aggregates, we found that the overexpression of P301S mutant human tau gene had not structural deleterious effects on GA alterations in cortical neurons. However, phospho-tau aggregation in P301mouse cortical neurons was associated with structural alterations of the GA, as in AD patients. These present findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA including a decrease in the surface area and volume of GA elements. The alterations found in GA likely impact the processing and trafficking of proteins, which could contribute to neuronal dysfunction in AD.

VISUALIZATION OF MICROGLIAL RESPONSE TO TAU-INDUCED NEURODEGENERATION IN A MODEL OF TAUOPATHY

Accumulation of intracellular neurofibrillary tangles (NFTs) consisting of microtubule-associated protein tau is a major hallmark of tau-mediated neurodegenerative diseases referred to as tauopathies. Activation of glial cells, both astrocytes and microglia, has been documented as an early event in the pathogenesis of protein misfolding diseases. However, an alteration between NFT formation and microglial activation was not clearly understood. To investigate the temporospatial relationship between pathological tau accumulation and microglial activation, noninvasive in vivo brain imaging studies on a mouse model of tauopathy was conducted. Furthermore, microglial activation status was examined by cell morphology and immunoreactivity of microglial markers. The rTg4510 transgenic mice expressing P301L mutated tau were examined for volumetric MRI and PET imaging from 2 to 13M of age. PET probes, [11C]PBB3 and [11C]AC-5216 were used for visualizing tau pathology and TSPO (the 18-kDa translocator protein, a neuroinflammation marker), respectively. Tau pathology and microglial activation were subsequently analyzed by immunohistochemistry using postmortem mouse brains. Longitudinal imaging studies revealed age-dependent cortical and hippocampal volume reduction, PBB3 and TSPO accumulations. Retentions of tau and TSPO radioligands in these regions were inversely correlated to forebrain volume. Immunohistochemically, both Iba1 and TSPO signals were increased with aging while immunoreactivity of P2Y12, a marker for visualizing microglia in the ramified state, was significantly reduced in cerebral cortex and hippocampus of rTg4510 mice as early as 2 months of age. Our results indicate that mutant tau deposition accompanied with both TSPO accumulation and forebrain atrophy in the rTg4510 mice. Decrease of a ramified morphology in young rTg4510 mice strongly suggests the microglial response to tau-induced neurodegeneration at an early stage of NFT formation. Further examinations are ongoing to validate whether microglial activation in this mouse model is detrimental or beneficial to protecting neurons.

INTERACTIONS OF ABETA AND TAU IN SYNAPSE LOSS

The collapse of neural networks important for memory and cognition, including death of neurons and degeneration of synapses, causes the debilitating dementia associated with Alzheimer's disease (AD). We have demonstrated that synaptic changes are central to the disease process. Using transgenic animal models expressing APPSwe/PS1dE9, Tg21221 wild-type human tau, and Tg4510 P301L mutant tau, and mice lacking endogenous mouse tau expression, we examined cognitive deficits and the accumulation of pathology and synapse degeneration using high-resolution imaging techniques. We further examined human postmortem brain tissue using array tomography, transmission electron microscopy, and standard stereological techniques to assess synapse degeneration. Both amyloid beta and tau proteins independently contribute to synaptic dysfunction and collapse in transgenic models of AD and frontotemporal dementia. Our recent data from a novel model of Alzheimer's disease combining APPSwe/PS1dE9 and rTg21221 expression on a mouse tau null background indicate that tau and amyloid beta are likely on the same pathway to synapse degeneration. Further, postmortem studies of human brain tissue support a role for both of these proteins in synapse collapse. Understanding mechanisms of synaptic dysfunction and loss will be critical for developing effective synapse-directed therapeutics. Acknowledgements: Funding provided by the European Research Council, Alzheimer's Research UK and the Scottish Government, Alzheimer's Society, a University of Edinburgh Wellcome Trust ISSF, NIH, and an anonymous foundation.

The Distance between N and C Termini of Tau and of FTDP-17 Mutants Is Modulated by Microtubule Interactions in Living Cells.

The microtubule (MT)-associated protein Tau is a natively unfolded protein, involved in a number of neurodegenerative disorders, collectively called tauopathies, aggregating in neurofibrillary tangles (NFT). It is an open question how the conversion from a MT bound molecule to an aggregation-prone Tau species occurs and, also, if and how tauopathy-related mutations affect its behavior in the cell. To address these points, we exploited a genetically encoded FRET sensor based on the full length Tau protein, to monitor in real time Tau conformational changes in different conditions in live cells. By studying the FRET signal we found that soluble Tau molecules, detached from MTs, display an unfolded structure. On the contrary, we observed an increased FRET signal generated by Tau monomers bound to MT, indicating that the association with MTs induced a folding of Tau protein, decreasing the distance between its N and C termini. We exploited the FRET sensor to investigate the impact of FTDP-17 mutations and of phosphorylation-site mutations on Tau folding and mobility in live cells. We demonstrated that the FTDP-17 Tau mutations weaken the interaction of Tau with cellular MTs, shifting the equilibrium towards the soluble pool while, conversely, phosphorylation site mutations shift the equilibrium of Tau towards the MT-bound state and a more closed conformation.

Expression of P301L-hTau in mouse MEC induces hippocampus-dependent memory deficit

Intracellular accumulation of abnormally phosphorylated tau in different types of neurons is a pathological characteristic of Alzheimer’s disease (AD). While tau modification and associated neuronal loss and hypometabolism start in the entorhinal cortex (EC) in early AD patients, the mechanism by which mutant P301L hTau leads to dementia is not fully elucidated. Here, we studied the effects of P301L hTau transduction in the medial EC (MEC) of mice on tau phosphorylation and accumulation, and cognitive deficit. We found that the exogenous mutant tau protein was restricted in MEC without spreading to other brain regions at one month after transduction. Interestingly, expression of the mutant tau in MEC induces endogenous tau hyperphosphorylation and accumulation in hippocampus and cortex, and inhibits neuronal activity with attenuated PP-DG synapse plasticity, leading to hippocampus-dependent memory deficit with intact olfactory function. These findings suggest a novel neuropathological mechanism of early AD, which is initiated by tau accumulation in MEC, and demonstrate a tau pathological model of early stage AD.

Tau association with synaptic vesicles causes presynaptic dysfunction

Tau is implicated in more than 20 neurodegenerative diseases, including Alzheimer’s disease. Under pathological conditions, Tau dissociates from axonal microtubules and missorts to pre- and postsynaptic terminals. Patients suffer from early synaptic dysfunction prior to Tau aggregate formation, but the underlying mechanism is unclear. Here we show that pathogenic Tau binds to synaptic vesicles via its N-terminal domain and interferes with presynaptic functions, including synaptic vesicle mobility and release rate, lowering neurotransmission in fly and rat neurons. Pathological Tau mutants lacking the vesicle binding domain still localize to the presynaptic compartment but do not impair synaptic function in fly neurons. Moreover, an exogenously applied membrane-permeable peptide that competes for Tau-vesicle binding suppresses Tau-induced synaptic toxicity in rat neurons. Our work uncovers a presynaptic role of Tau that may be part of the early pathology in various Tauopathies and could be exploited therapeutically.

Enhanced Tau Aggregation in the Presence of Amyloid β

Amyloid plaques and neurofibrillary tangles co-occur in Alzheimer disease, but with different topological and temporal patterns. Whether these two lesions are independent or pathobiologically related is uncertain. For example, amyloid deposition in the neocortex precedes the spread of tau neurofibrillary tangles from the limbic areas to the cortex. We examined the aggregation properties of tau isolated from human cases with early tau pathology (Braak II) with and without plaques. Using a well-established HEK cell biosensor assay, we show that tau from cases with plaques has an enhanced ability to induce tau aggregates compared to tau from cases without plaques. To further explore this effect, we combined mice carrying the APP/PS1 transgene array that develop plaques with rTg4510 mice carrying the P301L mutant human tau transgene that develop extensive tau pathology with age. The resulting APP/PS1-rTg4510 mice had a threefold increase in tau seeding activity over the rTg4510 strain, without change in tau production or extracellular release. Surprisingly, this effect was observed before overt amyloid deposition. The enhancement of tau aggregation was also apparent by an increase in histological measures of tau pathology in young APP/PS1-rTg4510 mice and an increase in high-molecular-weight tau. Overall, these data provide evidence that amyloid β acts to enhance tau pathology byincreasing the formation of tau species capable of seeding new aggregates.

Amyloid precursor protein reduction enhances the formation of neurofibrillary tangles in a mutant tau transgenic mouse model

Alzheimer's disease is characterized by the presence of 2 neuropathological lesions: neurofibrillary tangles, composed of tau proteins which are highly phosphorylated and phosphorylated on uncommon sites, and amyloid plaques, containing the Aß peptides generated from the amyloid precursor protein (APP). Reduction of some APP proteolytic derivatives in Alzheimer's disease such as sAPPα fragment has been reported and sAPPα has been shown to affect tau phosphorylation. To investigate in vivo the effect of absence of APP protein and its fragments on tau phosphorylation and the formation of neurofibrillary tangles, we have generated mice deleted for APP gene and overexpressing a human mutant tau protein and developing neurofibrillary tangles (APPKOTg30 mice). These APPKOTg30 mice showed more severe motor and cognitive deficits, increased tau phosphorylation, increased load of neurofibrillary tangles, and increased p25/35 ratio in the brain, compared with Tg30 mice. These data suggest that APP and/or its proteolytic derivatives interfere with the formation of neurofibrillary tangles in a transgenic mouse model that will be useful for investigating the relationship between APP and tau.

FTDP‐17 mutants S320F and S352L have subtle differential effects on 4R tau isoform aggregation in vitro

Frontotemporal Dementia with Parkinsonism linked to chromosome‐17 (FTDP‐17) is a class of inherited neurodegenerative diseases caused by mutations in the tau gene. Tau protein is a microtubule associated protein that stabilizes microtubules in neurons. During neurodegeneration, tau protein aggregates to form toxic filaments. Tau isoforms have either three or four 18‐amino acid microtubule binding repeats, known as 3‐repeat tau (3R tau) or 4‐repeat tau (4R tau). The 4R and 3R tau isoforms are further classified into 0N, 1N and 2N isoforms based on the number of N‐terminal exons. Forty four different mutations in various positions of the tau gene have been reported. FTDP‐17 tau mutants 2N4R S320F and S352L cause neuropathology through different mechanisms of tau aggregation. The mutant S320F forms short tau oligomers while S352L forms long tau filaments in vitro. Here, we determined whether the 4R tau isoforms (0N, 1N, 2N) of S320F and S352L affect tau aggregation differently in vitro. We used right angle laser light scattering, thioflavin S florescence assay and transmission electron microscopy (TEM) to study arachidonic acid induced tau aggregation. All wild type 4R tau isoforms (0N, 1N, 2N) showed the same effect on polymerization of tau into filaments upon addition of arachidonic acid. All S320F isoforms (0N, 1N, 2N) showed a significant decrease (p < 0.01) and S352L isoforms (0N, 1N, 2N) showed no significant change in laser light scattering when compared to the corresponding wild type 4R isoforms. However, S320F isoform 1N showed a significant decrease (p < 0.01) in percentage change from the respective wild type isoform when compared to 0N, 2N S320F by one way ANOVA analysis of laser light scattering. S320F isoforms 0N, 2N showed an increase (p < 0.05, 0.01) and 2N S352L also showed an increase (p < 0.001) in thioflavin S fluorescence when compared to the corresponding wild type 4R isoform. TEM images of all 4R S320F filaments showed very short tau fibrils which could be tau oligomers while all S352L filaments showed an increase in length of tau polymer. The 2N S352L filaments were longer than filaments from 0N and 1N isoforms. Though the mutant isoforms have the same effect on tau polymerization, the extent of polymerization varies. These results show that the presence of N‐terminal exons have small, but significant, alterations in the pro‐aggregation properties of FTDP‐17 4R tau.Support or Funding InformationNIH grant R01NS083391

Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy.

With increasingly large numbers of mouse models of human disease dedicated to MRI studies, compromises between in vivo and ex vivo MRI must be fully understood in order to inform the choice of imaging methodology. We investigate the application of high resolution in vivo and ex vivo MRI, in combination with tensor-based morphometry (TBM), to uncover morphological differences in the rTg4510 mouse model of tauopathy. The rTg4510 mouse also offers a novel paradigm by which the overexpression of mutant tau can be regulated by the administration of doxycycline, providing us with a platform on which to investigate more subtle alterations in morphology with morphometry. Both in vivo and ex vivo MRI allowed the detection of widespread bilateral patterns of atrophy in the rTg4510 mouse brain relative to wild-type controls. Regions of volume loss aligned with neuronal loss and pathological tau accumulation demonstrated by immunohistochemistry. When we sought to investigate more subtle structural alterations in the rTg4510 mice relative to a subset of doxycycline-treated rTg4510 mice, ex vivo imaging enabled the detection of more regions of morphological brain changes. The disadvantages of ex vivo MRI may however mitigate this increase in sensitivity: we observed a 10% global shrinkage in brain volume of the post-mortem tissues due to formalin fixation, which was most notable in the cerebellum and olfactory bulbs. However, many central brain regions were not adversely affected by the fixation protocol, perhaps due to our “in-skull” preparation. The disparity between our TBM findings from in vivo and ex vivo MRI underlines the importance of appropriate study design, given the trade-off between these two imaging approaches. We support the utility of in vivo MRI for morphological phenotyping of mouse models of disease; however, for subtler phenotypes, ex vivo offers enhanced sensitivity to discrete morphological changes.