Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform

Filamentous tau aggregates are hallmarks of tauopathies, e.g., frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). Since FTDP-17 tau gene mutations alter levels/functions of tau, we overexpressed the smallest human tau isoform in the CNS of transgenic (Tg) mice to model tauopathies. These mice acquired age-dependent CNS pathology similarto FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Inclusions were present in cortical and brainstem neurons but were most abundant in spinal cord neurons, where they were associated with axon degeneration, diminished microtubules (MTs), and reduced axonal transport in ventral roots, as well as spinal cord gliosis and motor weakness. These Tg mice recapitulate key features of tauopathies and provide models for elucidating mechanisms underlying diverse tauopathies, including Alzheimer's disease (AD).     

An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.     

Tau protein and neurodegeneration

Tau protein is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The identification of mutations in Tau as the cause of FTDP-17 established that dysfunction or misregulation of tau protein is sufficient to cause neurodegeneration and dementia. At an experimental level, the new understanding is leading to the development of good transgenic animal models of the tauopathies.     

Mutations causing neurodegenerative tauopathies

Tau is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). For a long time, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows tau dysfunction. This was resolved when mutations in Tau were found to cause FTDP-17. Currently, 32 different mutations have been identified in over 100 families. About half of the known mutations have their primary effect at the protein level. They reduce the ability of tau protein to interact with microtubules and increase its propensity to assemble into abnormal filaments. The other mutations have their primary effect at the RNA level and perturb the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in a relative overproduction of tau protein with four microtubule-binding domains in the brain. Individual Tau mutations give rise to diseases that resemble progressive supranuclear palsy, corticobasal degeneration or Pick's disease. Moreover, the H1 haplotype of Tau has been identified as a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At a practical level, the new work is leading to the production of experimental animal models that reproduce the essential molecular and cellular features of the human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and nerve cell degeneration.     

Dynactin is required for transport initiation from the distal axon

Dynactin is a required cofactor for the minus-end-directed microtubule motor cytoplasmic dynein. Mutations within the highly conserved CAP-Gly domain of dynactin cause neurodegenerative disease. Here, we show that the CAP-Gly domain is necessary to enrich dynactin at the distal end of primary neurons. While the CAP-Gly domain is not required for sustained transport along the axon, we find that the distal accumulation facilitates the efficient initiation of retrograde vesicular transport from the neurite tip. Neurodegenerative disease mutations in the CAP-Gly domain prevent the distal enrichment of dynactin thereby inhibiting the initiation of retrograde transport. Thus, we propose a model in which distal dynactin is a key mediator in promoting the interaction among the microtubule, dynein motor, and cargo for the efficient initiation of transport. Mutations in?the CAP-Gly domain disrupt the formation of the?motor-cargo complex, highlighting the specific defects in axonal transport that may lead to neurodegeneration.     

Tau alternative splicing in familial and sporadic tauopathies

Six tau isoforms differing in their affinity for microtubules are produced by alternative splicing from the MAPT (microtubule-associated protein tau) gene in adult human brain. Several MAPT mutations causing the familial tauopathy, FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17), affect alternative splicing of exon 10, encoding a microtubule-binding motif. Advanced RNA analysis methods have suggested that levels of exon 10-containing MAPT mRNA are elevated in Alzheimer's disease. Furthermore, the MAPT H1 haplotype, associated with Alzheimer's disease, promotes exon 10 inclusion in MAPT mRNA. Thus an accurate regulation of tau alternative splicing is critical for the maintenance of neuronal viability, and its alteration might be a contributing factor to Alzheimer's disease. Tau alternative splicing could represent a target for therapeutic intervention to delay the progression of pathology in familial as well as sporadic tauopathies.     

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Emergence of directional bias in tau deposition from axonal transport dynamics

Defects in axonal transport may partly underpin the differences between the observed pathophysiology of Alzheimer’s disease (AD) and that of other non-amyloidogenic tauopathies. Particularly, pathological tau variants may have molecular properties that dysregulate motor proteins responsible for the anterograde-directed transport of tau in a disease-specific fashion. Here we develop the first computational model of tau-modified axonal transport that produces directional biases in the spread of tau pathology. We simulated the spatiotemporal profiles of soluble and insoluble tau species in a multicompartment, two-neuron system using biologically plausible parameters and time scales. Changes in the balance of tau transport feedback parameters can elicit anterograde and retrograde biases in the distributions of soluble and insoluble tau between compartments in the system. Aggregation and fragmentation parameters can also perturb this balance, suggesting a complex interplay between these distinct molecular processes. Critically, we show that the model faithfully recreates the characteristic network spread biases in both AD-like and non-AD-like mouse tauopathy models. Tau transport feedback may therefore help link microscopic differences in tau conformational states and the resulting variety in clinical presentations.     

Tau alteration and neuronal degeneration in tauopathies: mechanisms and models

Tau becomes characteristically altered both functionally and structurally in several neurodegenerative diseases now collectively called tauopathies. Although increasing evidence supports that alterations of tau may directly cause neuronal degeneration and cell death, the mechanisms, which render tau to become a toxic agent are still unclear. In addition, it is obscure, whether neurodegeneration in tauopathies occurs via a common mechanism or specific differences exist. The aim of this review is to provide an overview about the different experimental models that currently exist, how they are used to determine the role of tau during degeneration and what has been learnt from them concerning the mechanistic role of tau in the disease process. The review begins with a discussion about similarities and differences in tau alteration in paradigmatic tauopathies such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). The second part concentrates on major experimental models that have been used to address the mechanistic role of tau during degeneration. This will include a discussion of cell-free assays, culture models using cell lines or dissociated neurons, and animal models. How these models aid to understand (i) alterations in the function of tau as a microtubule-associated protein (MAP), (ii) direct cytotoxicity of altered tau protein, and (iii) the potential role of tau aggregation in neurodegenerative processes will be the central theme of this part. The review ends with concluding remarks about a general mechanistic model of the role of tau alteration and neuronal degeneration in tauopathies and future perspectives.     

Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

Tau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552–682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies.     

Correction of alternative splicing of tau in frontotemporal dementia and parkinsonism linked to chromosome 17

Mutations in the human tau gene cause frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17). One of the major disease mechanisms in FTDP-17 is the increased inclusion of tau exon 10 during pre-mRNA splicing. Here we show that modified oligonucleotides directed against the tau exon 10 splice junctions suppress inclusion of tau exon 10. The effect is mediated by the formation of a stable pre-mRNA-oligonucleotide hybrid, which blocks access of the splicing machinery to the pre-mRNA. Correction of tau splicing occurs in a tau minigene system and in endogenous tau RNA in neuronal pheochromocytoma cells and is specific to exon 10 of the tau gene. Antisense oligonucleotide-mediated exclusion of exon 10 has a physiological effect by increasing the ratio of protein lacking the microtubule-binding domain encoded by exon 10. As a consequence, the microtubule cytoskeleton becomes destabilized and cell morphology is altered. Our results demonstrate that alternative splicing defects of tau as found in FTDP-17 patients can be corrected by application of antisense oligonucleotides. These findings provide a tool to study specific tau isoforms in vivo and might lead to a novel therapeutic strategy for FTDP-17.     

Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation

Mutations in the tau gene, which is located on chromosome 17, were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To determine if cognitive deficits could be caused by tau mutations, two transgenic mouse lines were generated expressing a four-repeat isoform of human tau or its mutant, containing one of the FTDP-17 mutations (WILD mice and N279K mice). In open field test, N279K mice showed hyperactivity in locomotion and rearing. In prepulse inhibition test, N279K mice but not Wild mice showed significant deficits. Both transgenic mice, especially N279K mice, showed impairment in acquisition of spatial learning in Morris water maze. Although both N279K mice and Wild mice acquired passive avoidance as well as non-transgenic mice, N279K mice but not Wild mice showed severe deficits in acquisition of active avoidance. Histological analysis of the present mutant mice did not show any signs of neurofibrillary tangle formations in the brain, and cognitive dysfunction seemed to precede such neuropathological changes or occur independently from them. The behavioral phenotype of N279K mice mimics features of human FTDP-17 and provides a basic model for elucidating mechanisms underlying cognitive deficits in not only FTDP-17, but also diverse tauopathies.     

T-817MA, a neuroprotective agent, attenuates the motor and cognitive impairments associated with neuronal degeneration in P301L tau transgenic mice

Tau pathology is implicated in mechanisms of neurodegenerative tauopathies, including Alzheimer’s disease (AD) and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). It has been reported that transgenic mice expressing FTDP-17 mutation P301L of human tau (P301L mice) display extensive tau pathology and exhibit behavioral deficits with aging. In this study, we investigated the effects of T-817MA, a neuroprotective agent, on the motor and cognitive impairments associated with neuronal degeneration in P301L mice. T-817MA prevented the progression of motor deficit and the loss of spinal cord motor neurons in P301L mice. Furthermore, T-817MA significantly attenuated the spatial memory impairment and the reduction in synaptic terminal density in the hippocampal dentate gyrus of P301L mice. These results indicate that T-817MA improved the motor and cognitive impairments as a result of inhibiting neuronal degeneration derived from tau pathology in the P301L mice. Therefore, it is expected that T-817MA has a therapeutic potential for tau-related neurodegenerative diseases such as AD.     

Transgenic animal models of tauopathies

Tauopathies are a group of neurodegenerative disorders that include Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and other related diseases with prominent tau pathology. Research advances in the last several decades have characterized and defined tau neuropathologies of both neuron and glia in these diverse disorders and this has stimulated development of animal models of tauopathies. Indeed, animal models ranging from invertebrate species such as C. elegan to Drosophila melanogaster and mammalian transgenic mouse models of tauopathies have been generated and reported. This review summarizes the salient features of many of the known models of tauopathies.     

FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.     

Brain-penetrant microtubule-stabilizing compounds as potential therapeutic agents for tauopathies

Neurons within the brains of those with AD (Alzheimer's disease) and related neurodegenerative disorders, collectively termed 'tauopathies', contain fibrillar inclusions composed of hyperphosphorylated tau protein. Tau is normally enriched in axons, where it binds and stabilizes MTs (microtubules). Tau hyperphosphorylation and aggregation probably result in reduced MT binding that could affect axonal transport and neuronal function. A possible therapeutic strategy to overcome a loss of tau function in tauopathies is administration of MT-stabilizing agents, such as those used in the treatment of cancer. However, these drugs elicit severe side effects, and most existing MT-stabilizing compounds have poor BBB (blood-brain barrier) permeability, which renders them unsuitable for tauopathy treatment. We identified EpoD (epothilone D) as a brain-penetrant MT-stabilizing agent with preferred pharmacokinetic and pharmacodynamic properties. EpoD was evaluated for its ability to compensate for tau loss-of-function in an established Tg (transgenic) mouse model, using both preventative and interventional dosing paradigms. EpoD at doses much lower than previously used in human cancer patients caused improved axonal MT density and decreased axonal dystrophy in the tau Tg mice, leading to an alleviation of cognitive deficits. Moreover, EpoD reduced the extent of tau pathology in aged tau Tg mice. Importantly, no adverse side effects were observed in the EpoD-treated mice. These results suggest that EpoD might be a viable drug candidate for the treatment of AD and related tauopathies.     

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini-gene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing.     

Calpain-mediated tau cleavage: a mechanism leading to neurodegeneration shared by multiple tauopathies

Tau dysfunction has been associated with a host of neurodegenerative diseases called tauopathies. These diseases share, as a common pathological hallmark, the presence of intracellular aggregates of hyperphosphorylated tau in affected brain areas. Aside from tau hyperphosphorylation, little is known about the role of other posttranslational modifications in tauopathies. Recently, we obtained data suggesting that calpain-mediated tau cleavage leading to the generation of a neurotoxic tau fragment might play an important role in Alzheimer's disease. In the current study, we assessed the presence of this tau fragment in several tauopathies. Our results show high levels of the 17-kDa tau fragment and enhanced calpain activity in the temporal cortex of AD patients and in brain samples obtained from patients with other tauopathies. In addition, our data suggest that this fragment could partially inhibit tau aggregation. Conversely, tau aggregation might prevent calpain-mediated cleavage, establishing a feedback circuit that might lead to the accumulation of this toxic tau fragment. Collectively, these data suggest that the mechanism underlying the generation of the 17-kDa neurotoxic tau fragment might be part of a conserved pathologic process shared by multiple tauopathies.     

Functional characterization of FTDP-17 tau gene mutations through their effects on Xenopus oocyte maturation.

tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wild-type four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, DeltaK280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wild-type Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects of tau gene mutations in a quantitative manner.     

Preparation and characterization of neurotoxic tau oligomers

Tau aggregation is a pathological hallmark of Alzheimer's disease, Parkinson's disease, and many other neurodegenerative disorders known as tauopathies. Tau aggregates take on many forms, and their formation is a multistage process with intermediate stages. Recently, tau oligomers have emerged as the pathogenic species in tauopathies and a possible mediator of amyloid-β toxicity in Alzheimer's disease. Here, we use a novel, physiologically relevant method (oligomer cross-seeding) to prepare homogeneous populations of tau oligomers and characterize these oligomers in vitro. We show that both Aβ and α-synuclein oligomers induce tau aggregation and the formation of β-sheet-rich neurotoxic tau oligomers.