Divergent phosphorylation pattern of tau in P301L tau transgenic mice

Aggregates of hyperphosphorylated tau are prominent in brains of patients with Alzheimer's disease or frontotemporal dementia (FTD). They have been reproduced in animal models following the identification of tau mutations in familial cases of FTD. This includes our previously generated transgenic model, pR5, which expresses FTD (P301L) mutant tau in neurons. The mice are characterized by tau aggregation including tangle (NFT) formation, memory impairment and mitochondrial dysfunction. In 8-month-old mice, S422 phosphorylation of tau is linked to NFT formation, however, a detailed analysis of tau solubility, phosphorylation and aggregation has not been done nor have the mice been monitored until a high age. Here, we undertook an analysis by immunohistochemistry, Gallyas impregnation and Western blotting of brains from 3 month- up to 20 month-old mice. NFTs first appeared at 6 months in the amygdala, followed by the CA1 region of the hippocampus. As the mice get older, the solubility of tau is decreased as determined by sequential extractions. Histological analysis revealed increased phosphorylation at the AT180, AT270 and 12E8 epitopes with ageing. The numbers of AT8-positive neurons increased from 3 to 6 months old. However, whereas S422 appeared only late and concomitantly with NFT formation, the only neurons left with AT8-reactivity at 20 months were those that had undergone NFT formation. As hyperphosphorylated tau continued to accumulate, the lack of AT8-reactivity suggests regulatory mechanisms in specifically dephosphorylating the AT8 epitope in the remaining neurons. Thus, differential regulation of phosphorylation is important for NFT formation in neurodegenerative diseases with tau pathology.     

Oligodendroglial tau filament formation in transgenic mice expressing G272V tau

Genetic evidence indicates that several mutations in tau, including G272V, are linked to frontotemporal dementia with parkinsonism. We expressed this mutation in mouse brains by combining a prion protein promoter-driven expression system with an autoregulatory transactivator loop that resulted in high expression of human G272V tau in neurons and in oligodendrocytes. We show that G272V tau can form filaments in murine oligodendrocytes. Electron microscopy established that the filaments were either straight or had a twisted structure; these were 17-20 nm wide and had a periodicity of approximately 75 nm. Filament formation was associated with tau phosphorylation at distinct sites, including the AT8 epitope 202/205 in vivo. Immunogold electron microscopy of sarcosyl-extracted spinal cords from G272V transgenic mice using phosphorylation-dependent antibodies AT8 or AT100 identified several sparsely gold-labelled 6-nm filaments. In the spinal cord, fibrillary inclusions were also identified by thioflavin-S fluorescent microscopy in oligodendrocytes and motor neurons. These results establish that expression of the G272V mutation in mice causes oligodendroglial fibrillary lesions that are similar to those seen in human tauopathies.     

Accelerated kindling epileptogenesis in Tg4510 tau transgenic mice,but not in tau knockout mice

The biologic processes underlying epileptogenesis following a brain insult are not fully understood, but several lines of evidence suggest that hyperphosphorylation of tau may be an important factor in these processes. To provide further insight into the causal relationship between tau and epileptogenesis, this study applied amygdala kindling to rTg4510 mice that, concurrent with other pathologies, overexpress phosphorylated tau, tau knockout mice, or their respective wild‐type controls. Mice were electrically stimulated twice daily, 5 days per week for 3 weeks. Electroencephalography was recorded to measure the primary afterdischarge duration, and the behavioral progression of kindling‐induced seizures was assessed. rTg4510 mice (n = 10) had increased primary afterdischarge durations (p < 0.001), and significantly more rapid progression of kindling (p < 0.001), compared with wild‐type mice (n = 10). Tau knockout mice (n = 7), however, did not differ from their wild‐type counterparts (n = 8) on any of the seizure outcomes. These results suggest that Tg4510 mice are more vulnerable to epileptogenesis, but that the presence of tau itself is not necessary for kindling epileptogenesis to occur.     

3R tau expression modifies behavior in transgenic mice

Tauopathy is a group of disorders characterized by the accumulation of hyperphosphorylated tau protein in the brain, resulting in dementia. Here, tau‐related behavior was evaluated in a mouse model with brain overexpression of the shortest human tau isoform (0N3R). Two groups of animals [tau‐transgenic (tau‐tg) and control littermates] were tested for learning and memory at 1 and 7 months. In the Morris water maze, all mice learned the task at 1 month of age and did not learn at 7 months. In contrast, at 7 months, the tau‐tg animals demonstrated better retention of the passive avoidance response compared with their control littermates, which did not learn. In the open field test, no differences were measured between transgenic and nontransgenic young mice, but significantly higher locomotion was observed in the 7‐month‐old tau‐tg mice compared with controls. Behavior during the elevated plus maze test was the same at 1 month, but at 7 months increased entrance to the different arms was observed in the tau‐tg group. Tau expression and phosphorylation levels were analyzed at 8 months. In the subcortical brain region associated with passive avoidance behavior, the tau‐tg mice demonstrated increased brain tau expression coupled with reduced relative phosphorylation. In contrast, increased tau expression and phosphorylation were measured in the cerebral cortex of the tau‐tg mice. In conclusion, 7‐8‐month‐old tau‐tg mice overexpressing nonmutated 0N3R human tau isoform demonstrated enhanced behavior in the passive avoidance test, paralleled by relative tau hypophosphorylation in the subcortical brain region. © 2010 Wiley‐Liss, Inc.     

Olfactory dysfunction occurs in transgenic mice overexpressing human tau protein

Disorders of olfaction are among the first clinical signs of neurodegenerative diseases such as Alzheimer's disease (AD) and idiopathic Parkinson's disease (PD). In this study, we employed an odor habituation paradigm to evaluate the olfactory function of T alpha 1-3RT transgenic mice that overexpress tau, a key pathogenic protein in AD, and compared such function to that of wild-type controls who do not overexpress this protein. The T alpha 1-3RT mice, but not the controls, exhibited responses indicative of decreased olfactory function. These data lend support to the notion that tau may be involved in the pathogenesis of the olfactory dysfunction of some neurodegenerative diseases. Future studies need to similarly assess other pathogenic markers, as well as their distribution within various sectors of the brain, to determine the specificity of this phenomenon.     

Nogo receptor interacts with brain APP and Abeta to reduce pathologic changes in Alzheimer's transgenic mice

Pathophysiologic hypotheses for Alzheimer's disease (AD) are centered on the role of the amyloid plaque Abeta peptide and the mechanism of its derivation from the amyloid precursor protein (APP). As part of the disease process, an aberrant axonal sprouting response is known to occur near Abeta deposits. A Nogo to Nogo-66 receptor (NgR) pathway contributes to determining the ability of adult CNS axons to extend after traumatic injuries. Here, we consider the potential role of NgR mechanisms in AD. Both Nogo and NgR are mislocalized in AD brain samples. APP physically associates with the NgR. Overexpression of NgR decreases Abeta production in neuroblastoma culture, and targeted disruption of NgR expression increases transgenic mouse brain Abeta levels, plaque deposition, and dystrophic neurites. Infusion of a soluble NgR fragment reduces Abeta levels, amyloid plaque deposits, and dystrophic neurites in a mouse transgenic AD model. Changes in NgR level produce parallel changes in secreted APP and AB, implicating NgR as a blocker of secretase processing of APP. The NgR provides a novel site for modifying the course of AD and highlights the role of axonal dysfunction in the disease.     

Ongoing in vivo studies with cytoskeletal drugs in tau transgenic mice

Most drug discovery efforts for Alzheimer's disease (AD) have focused on prevention or clearance of beta-amyloid (Abeta) fibrils or oligomers, with far less attention to prevention of tau abnormalities that lead to neurofibrillary tangles (NFTs). Much evidence now indicates that Abeta multimers can trigger neurodegenerative changes that involve formation of dystrophic neurites and cytoskeletal collapse, possibly due loss of microtubule (MT) stabilization by the tau protein. We have found that several MT-stabilizing agents such as Taxol significantly enhanced neuronal survival in the presence of Abeta and identified agents that enter the brain, a necessity for in vivo testing in animal models of tau pathology. Studies were designed to test two agents in the tau mutant (JNPL3) mouse that develops severe motor deficits at about seven months of age, accompanied by neuropathological markers of tau pathology. In addition to using motor performance tests through the planned period of drug administration, we designed a simple appetitive memory test that required a reduction in ad lib food intake. Although the neurochemical data are still being analyzed, we were surprised to find that all of the JNPL3 mice, whether receiving the drug or not, developed no signs of motor impairment up to 10 months of age. This is considerably beyond the age at which free-fed mice survived and suggests that the food restriction alone may have delayed the pathological process. A study is ongoing with free-fed mice to determine if the drug interventions do have any beneficial effects in these mutant mice.     

Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice

Mutant R406W human tau was originally identified in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and causes a hereditary tauopathy that clinically resembles Alzheimer's disease (AD). In the current study, we examined the performance of R406W transgenic (Tg) mice in the forced swimming test, a test with high predictivity of antidepressant efficacy in human depression, and found an enhancement of the immobility time. In contrast, the motor function and anxiety-related emotional response of R406W Tg mice were normal. Furthermore, a selective serotonin reuptake inhibitor (SSRI), fluvoxamine (100 mg/kg, p.o.), significantly reduced this enhancement of the immobility time, whereas a noradrenaline reuptake inhibitor, desipramine, had no effect. In an in vivo microdialysis study, R406W Tg mice exhibited a significantly decreased extracellular 5-hydroxyindoleacetic acid (5-HIAA) level in the frontal cortex and also exhibited a tendency toward a decreased extracellular 5-hydroxytryptamine (5-HT) level. Moreover, fluvoxamine, which reduced the enhancement of the immobility time, significantly increased the extracellular 5-HT level in R406W Tg mice. These results suggest that R406W Tg mice exhibit changes in depression-related behavior involving serotonergic neurons and provide an animal model for investigating AD with depression.     

Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some of these mutations lead to an overproduction of tau isoforms with four microtubule-binding repeats. Here we have expressed the longest four-repeat human brain tau isoform in transgenic mice under the control of the murine Thy1 promoter. Transgenic mice aged 3 weeks to 25 months overexpressed human tau protein in nerve cells of brain and spinal cord. Numerous abnormal, tau-immunoreactive nerve cell bodies and dendrites were seen. In addition, large numbers of pathologically enlarged axons containing neurofilament- and tau-immunoreactive spheroids were present, especially in spinal cord. Signs of Wallerian degeneration and neurogenic muscle atrophy were observed. When motor function was tested, transgenic mice showed signs of muscle weakness. Taken together, these findings demonstrate that overexpression of human four-repeat tau leads to a central and peripheral axonopathy that results in nerve cell dysfunction and amyotrophy. Received: 28 September 1999 / Revised, accepted: 18 November 1999     

Tau transgenic mice as models for cerebrospinal fluid tau biomarkers

Levels of tau in cerebrospinal fluid (CSF) are elevated in Alzheimer's disease (AD) patients. It is believed this elevation is related to the tau pathology and neurodegeneration observed in AD, but not all tauopathies have increased CSF tau. There has been little pre-clinical work to investigate mechanisms of increased CSF tau due to the difficulty in collecting CSF samples from mice, the most commonly used pre-clinical models. We developed methods to collect CSF from mice without contamination from tau in brain tissue, which is approximately 50,000 fold more abundant in brain than CSF. Using these methods, we measured CSF tau from 3xTg, Tg4510, and Tau Alone transgenic mice. All three lines of mice showed age-dependent increases in CSF tau. They varied in phenotype from undetectable to severe tau pathology and neurodegeneration, suggesting that degenerating neurons are unlikely to be the only source of pathologic CSF tau. Overall, CSF tau levels mirrored expression levels and changes of tau in the brain, but they did not always correlate exactly. CSF tau was often more sensitive to changes in brain transgene expression and pathology. In addition, we also developed ELISA assays specific to different regions of the tau protein. We used these assays to provide evidence that CSF tau exists as fragments, with little intact C-terminus and partial loss of the N-terminus. Taken together, these assays and mouse models may be used to facilitate a deeper understanding of CSF tau in neurodegenerative disease.     

Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice.

The formation of neurofibrillary tangles in Alzheimer's disease is preceded by a pretangle stage of hyperphosphorylated tau. To characterize pretangle tau in vivo, we correlated, in human tau transgenic mice, levels of kinases known to phosphorylate tau in vitro with the phosphorylation of tau at specific epitopes. Levels of cyclin-dependent kinase-5 were increased in axons of CA1 pyramidal neurons, where tau was phosphorylated specifically at the AD2 epitope Ser396/Ser404. The 12E8 epitope serine262/serine356 and the AT180 epitope threonine231/serine235 were phosphorylated in dendrites, and colocalized with increased levels of glycogen synthase kinase-3. CA1 neurons phosphorylated tau at more epitopes than dentate gyrus neurons, suggesting that tau phosphorylation is cell type-specific, a possible explanation for the spatial distribution of neurofibrillary tangles.     

Park2-null/tau transgenic mice reveal a functional relationship between parkin and tau

Mutations, haplotypes, and polymorphisms of tau and Park-2 genes constitute risk factors for developing tauopathies. In order to analyze the possible relationship between parkin and tau we generated a double-mutant mouse deficient for Park-2 expression and overexpressing a mutant tau protein (hTauVLW). Mice develop normally, although the median survival rate is considerably reduced with respect to wild type (45%). Aggregates of phosphorylated tau in neurons and reactive gliosis are quite abundant in cortex and hippocampus of these mice. Moreover, while in young transgenic mice the hTauVLW immunostained transgene product is observed in both cell bodies and dendrites, the hTauVLW mutant protein is only detected in the neuronal cell bodies when Park-2 gene is additionally deleted. Moreover, DNA fragmentation was detected by the TUNEL method, and cerebral atrophy is also present in these regions. The levels of phosphorylated tau and Hsp70 are increased in the double-mutant mice, while CHIP expression in hippocampus is lower when the Park-2 gene is deleted. Thus, the combination of Park-2 gene deletion with hTauVLW transgene overexpression in mice produces serious neuropathological effects, which reflect the existence of some relationship between both proteins.     

AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimer's type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.     

Characterization of pathology in transgenic mice over-expressing human genomic and cDNA tau transgenes

To examine the normal cellular function of tau and its role in pathogenesis, we have created transgenic mice that overexpress a tau transgene derived from a human PAC that contains the coding sequence, intronic regions, and regulatory regions of the human gene. All six isoforms of human tau are represented in the transgenic mouse brain at the mRNA and protein level and the human tau is distributed in neurites and at synapses, but is absent from cell bodies. A comparison between the genomic tau mice and mice that overexpress a tau cDNA transgene shows that overall, the distribution of tau is similar in the two lines, but human tau is located in the somatodendritic compartment of many neurons in the cDNA mice. Tau-immunoreactive axonal swellings were found in the spinal cords of the cDNA mice, which correlated with a hind-limb abnormality, whereas neuropathology was essentially normal in the genomic mice up to 8 months of age.     

Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice

Neuropathological features in Alzheimer’s Disease (AD) include the presence of hyperphosphorylated forms of the microtubule-associated tau protein (tau) in hippocampal neurones. Numerous studies indicate a neuroprotective effect of calcium-binding proteins (Ca2+ binding proteins) in neurodegenerative diseases (e.g., AD). Secretagogin is a newly described Ca2+ binding protein that is produced by pyramidal neurones of the human hippocampus. Recently, secretagogin expressing hippocampal neurones were demonstrated to resist tau-induced pathology in AD in contrast to the majority of neighbouring neurones. This suggested a neuroprotective effect of secretagogin in hippocampal neurones. Here, we investigated secretagogin expression in wild type (wt) mice as well as in hemizygous and homozygous P301L tau transgenic (tg) mice, which show pronounced and widespread tau pathology in hippocampal neurones. Secretagogin expression was analyzed at the immunohistochemical and biochemical levels in brains of age-matched wt and hemi- and homozygous tau tg mice. In wt mice hippocampal secretagogin-immunoreactive neurones were invariably detected, while immunoreactivity was much lower (P < 0.001) in tau tg mice. Of note, hippocampal secretagogin immunoreactivity was absent in 62.5% of homozygous tau tg mice. In line with this finding, Western blot analysis demonstrated a significant reduction in protein expression levels of secretagogin in homozygous tau tg compared to wt mice. Our results suggest that increased levels of tau negatively influence secretagogin expression in the hippocampus of tau tg mice.     

Accelerated extinction of conditioned taste aversion in P301L tau transgenic mice

Neurofibrillary tangles, insoluble protein deposits composed of filamentous tau aggregates, are neuropathological hallmarks of Alzheimer's disease and familial frontotemporal dementia (FTDP-17). Transgenic mice expressing the FTDP-17 mutation P301L of tau recapitulate key features of the human pathology, that is, tau proteins aggregate and neurofibrillary tangles begin to appear in the amygdala at 6 months of age. To detect early signs of tau aggregate-associated changes, we investigated behavioral alterations and cognitive deficits in such mice using an amygdala-specific test battery for anxiety-related and cognitive behavior. P301L mice had anxiety levels not different from wild-types, but their exploratory behavior was significantly increased. Acquisition of a fear response to tone and context as well as taste aversion was comparable to wild-types. However, extinction of a conditioned taste aversion was significantly accelerated. We conclude that already aggregation of tau proteins not yet accompanied by massive formation of neurofibrillary tangles causes selective behavioral deficits.     

Abeta deposition does not cause the aggregation of endogenous tau in transgenic mice

In Alzheimer disease, the extracellular deposition of beta-amyloid (Abeta) in the brain is accompanied by the intracellular accumulation of aggregated forms of hyperphosphorylated tau. In developing animal models of AD, the authors and others have been able to reproduce extracellular amyloid pathology in the brains of mice by expressing mutant amyloid precursor proteins (APP). The co-expression of APP with mutant presenilin leads to a dramatic acceleration in Abeta deposition, leading to very high amyloid burdens in mice. In the current study, the authors have examined whether the brains of mice with high burdens of amyloid deposition also contain aggregated forms of tau, using a cellulose acetate filter trap assay. Although discrete accumulations of phosphorylated tau immunoreactivity were apparent in neurites proximal to cored deposits of Abeta, little if any of this tau was in a SDS-resistant state of aggregation. By contrast, the brains of AD patients contained large amounts of aggregated tau. Overall, this study demonstrates that, in mice, deposition of Abeta does not cause endogenous tau to aggregate.     

Truncation of tau protein and its pathological significance in Alzheimer's disease

Abnormal posttranslational modifications of tau protein lead it to aggregate into paired helical filaments in Alzheimer's disease (AD). The mechanisms involved in the early pathological processing of tau and the induction of a polymeric state seem to progress through a sequential pattern of changes mainly involving abnormal phosphorylation, conformational changes and truncation. While proteolytic cleavage of tau protein during the progression of AD has not been comprehensively analyzed, tau is a substrate for several intracellular proteases. Furthermore, abnormal regulation of proteolytic events, including those associated with apoptosis, may generate truncated tau subproducts which in turn may be toxic to neurons per se and capable of polymerization at a faster rate. Accumulation of tau fibrils has long been controversial, with much debate concerning the true toxicity of polymerized tau. The development of different transgenic mice overexpressing tau protein, the generation of cell models expressing tau, and the in vitro polymerization paradigms have significantly enhanced our understanding of the biophysics and pathological properties of tau polymers in AD, as well as in other tau pathologies. This review will discuss the pathological role of truncated tau protein in the context of toxicity and neurofibrillary tangle formation and maturation and its significance in clinical dementia.     

Gait analysis detects early changes in transgenic SOD1(G93A) mice

The effective treatment or cure of motoneuron disease will require understanding the disease processes that precede irreversible cell loss. To study these early stages, and to evaluate potential treatments in relevant animal models, requires a sensitive functional assay. To this end, we sought to determine whether the gait pattern of SOD1 transgenic mice changed prior to overt symptoms. Using a simplified video-based approach we compared the treadmill gait of C57BL/6J and B6.SOD1 transgenic mice at 8 and 10 weeks of age. B6.SOD1 mice had significantly longer stride and stance times than controls by 8 weeks. Consistent with disease progression, hindpaw measures of B6.SOD1 mice showed larger changes than front paws. Differences between control and B6.SOD1 mice increased at 10 weeks, but only because repeat testing caused habituation in control mice to a greater extent than in B6.SOD1 mice. Together the results demonstrate that simplified gait analysis is sensitive to early processes of motor system disease in mice.