Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key?

Summary.  Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of β-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5–6 months prior to the usual onset of β-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1) Premature deposition of β-amyloid in eight month-old, β-amyloid precursor protein (βAPP)-transgenic mice (Kane et al., 2000); 2) augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of β-amyloid in vivo by AD brain extracts suggests pathogenic similarities between β-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. Received June 28, 2001 Accepted August 6, 2001 Published online June 26, 2002     

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.     

Genetically augmenting tau levels does not modulate the onset or progression of Abeta pathology in transgenic mice

The two hallmark pathologies of Alzheimer's disease (AD) are amyloid plaques, composed of the small amyloid-beta (Abeta) peptide, and neurofibrillary tangles, comprised aggregates of the microtubule binding protein, tau. The molecular linkage between these two lesions, however, remains unknown. Based on human and mouse studies, it is clear that the development of Abeta pathology can trigger tau pathology, either directly or indirectly. However, it remains to be established if the interaction between Abeta and tau is bidirectional and whether the modulation of tau will influence Abeta pathology. To address this question, we used the 3xTg-AD mouse model, which is characterized by the age-dependent buildup of both plaques and tangles. Here we show that genetically augmenting tau levels and hyperphosphorylation in the 3xTg-AD mice has no effect on the onset and progression of Abeta pathology. These data suggest that the link between Abeta and tau is predominantly if not exclusively unidirectional, which is consistent with the Abeta cascade hypothesis and may explain why tauopathy-only disorders are devoid of any Abeta pathology.     

Neurogenesis and cell cycle-reactivated neuronal death during pathogenic tau aggregation

The aim of the present study was to investigate the relation between neurogenesis, cell cycle reactivation and neuronal death during tau pathology in a novel tau transgenic mouse line THY-Tau22 with two frontotemporal dementia with parkinsonism linked to chromosome-17 mutations in a human tau isoform. This mouse displays all Alzheimer disease features of neurodegeneration and a broad timely resolution of tau pathology with hyperphosphorylation of tau at younger age (up to 6 months) and abnormal tau phosphorylation and tau aggregation in aged mice (by 10 months). Here, we present a follow-up of cell cycle markers with aging in control and transgenic mice from different ages. We show that there is an increased neurogenesis during tau hyperphosphorylation and cell cycle events during abnormal tau phosphorylation and tau aggregation preceding neuronal death and neurodegeneration. However, besides phosphorylation, other mechanisms including tau mutations and changes in tau expression and/or splicing may be also involved in these mechanisms of cell cycle reactivation. Altogether, these data suggest that cell cycle events in THY-Tau22 are resulting from neurogenesis in young animals and cell death in older ones. It suggests that neuronal cell death in such models is much more complex than believed.     

Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimer's disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.     

Abeta deposition and related pathology in an APP x PS1 transgenic mouse model of Alzheimer's disease

A transgenic mouse bearing mutant transgenes linked to familial forms of Alzheimer's disease (AD) for the amyloid precursor protein and presenilin-1 (TASTPM) showed Abeta plaque deposition and age-related histological changes in associated brain pathology. The Abeta present was of multiple forms, including species with a C-terminus at position 40 or 42, as well as an N-terminus at position 1 or truncated in a pyro-3-glutamate form. Endogenous rodent Abeta was also present in the deposits. Laser capture microdissection extracts showed that multimeric forms of Abeta were present in both plaque and tissue surrounding plaques. Associated with the Abeta deposits was evidence of an inflammatory response characterised by the presence of astrocytes. Also present in close association with the deposits was phosphorylated tau and cathepsin D immunolabelling. The incidence of astrocytes and of phosphorylated tau and cathepsin D load showed that both of these potential disease markers increased in parallel to the age of the mice and with Abeta deposition. Immunohistochemical labelling of neurons in the cortex and hippocampus of TASTPM mice suggested that the areas of Abeta deposition were associated with the loss of neurons. TASTPM mice, therefore, exhibit a number of the pathological characteristics of disease progression in AD and may provide a means for assessment of novel therapeutic agents directed towards modifying or halting disease progression.     

Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model

Filamentous tau inclusions are hallmarks of Alzheimer's disease (AD) and related tauopathies, but earlier pathologies may herald disease onset. To investigate this, we studied wild-type and P301S mutant human tau transgenic (Tg) mice. Filamentous tau lesions developed in P301S Tg mice at 6 months of age, and progressively accumulated in association with striking neuron loss as well as hippocampal and entorhinal cortical atrophy by 9-12 months of age. Remarkably, hippocampal synapse loss and impaired synaptic function were detected in 3 month old P301S Tg mice before fibrillary tau tangles emerged. Prominent microglial activation also preceded tangle formation. Importantly, immunosuppression of young P301S Tg mice with FK506 attenuated tau pathology and increased lifespan, thereby linking neuroinflammation to early progression of tauopathies. Thus, hippocampal synaptic pathology and microgliosis may be the earliest manifestations of neurodegenerative tauopathies, and abrogation of tau-induced microglial activation could retard progression of these disorders.     

Reduced axonal transport and increased excitotoxic retinal ganglion cell degeneration in mice transgenic for human mutant P301S tau

The effects of tau hyperphosphorylation and aggregation on axonal transport were investigated in the optic nerve of mice transgenic for human mutant P301S tau. Transport was examined using cholera toxin B tracing. Retrograde transport was reduced in transgenic mice at 3 and 5 months of age, when compared to C57/Bl6 control mice. Anterograde axonal transport was also reduced in 3-month-old transgenic mice. Mild excitotoxic injury of retinal ganglion cells resulted in greater nerve cell loss in retinas from 3- and 5-month old P301S transgenic mice, when compared to controls. In conjunction with the detection of abnormal tau in the optic nerve in human and experimental glaucoma, the present findings suggest that tau hyperphosphorylation and aggregation may constitute targets for neuroprotective therapies in glaucoma as well as tauopathies.     

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

GRK5 influences the phosphorylation of tau via GSK3β and contributes to Alzheimer's disease

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase whose dysfunction results in cognitive impairment and Alzheimer-like pathology, including tau hyperphosphorylation. However, the mechanisms whereby GRK5 influences tau phosphorylation remain incompletely understood. In the current study, we showed that GRK5 influenced the phosphorylation of tau via glycogen synthase kinase 3β (GSK3β). The activity of both tau and GSK3β in the hippocampus was increased in aged GRK5-knockout mice, which is consistent with what occurs in APP/PS1 transgenic mice. Furthermore, GRK5 regulated the activity of GSK3β and phosphorylated tau in vitro. Regardless of changes of GRK5 protein levels, tau hyperphosphorylation remained reduced after GSK3β activity was inhibited, suggesting that GRK5 may specifically influence tau hyperphosphorylation by modulating GSK3β activity. Taken together, our findings suggest that GRK5 deficiency contributes to the pathogenesis of Alzheimer's disease by influencing the hyperphosphorylation of tau through the activation of GSK3β.     

Trans-synaptic spread of tau pathology in vivo

Tauopathy in the brain of patients with Alzheimer's disease starts in the entorhinal cortex (EC) and spreads anatomically in a defined pattern. To test whether pathology initiating in the EC spreads through the brain along synaptically connected circuits, we have generated a transgenic mouse model that differentially expresses pathological human tau in the EC and we have examined the distribution of tau pathology at different timepoints. In relatively young mice (10-11 months old), human tau was present in some cell bodies, but it was mostly observed in axons within the superficial layers of the medial and lateral EC, and at the terminal zones of the perforant pathway. In old mice (>22 months old), intense human tau immunoreactivity was readily detected not only in neurons in the superficial layers of the EC, but also in the subiculum, a substantial number of hippocampal pyramidal neurons especially in CA1, and in dentate gyrus granule cells. Scattered immunoreactive neurons were also seen in the deeper layers of the EC and in perirhinal and secondary somatosensory cortex. Immunoreactivity with the conformation-specific tau antibody MC1 correlated with the accumulation of argyrophilic material seen in old, but not young mice. In old mice, axonal human tau immunoreactivity, especially at the endzones of the perforant pathway, was greatly reduced. Relocalization of tau from axons to somatodendritic compartments and propagation of tauopathy to regions outside of the EC correlated with mature tangle formation in neurons in the EC as revealed by thioflavin-S staining. Our data demonstrate propagation of pathology from the EC and support a trans-synaptic mechanism of spread along anatomically connected networks, between connected and vulnerable neurons. In general, the mouse recapitulates the tauopathy that defines the early stages of AD and provides a model for testing mechanisms and functional outcomes associated with disease progression.     

Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology

Accumulation of amyloid-beta (Abeta) is one of the earliest molecular events in Alzheimer disease (AD), whereas tau pathology is thought to be a later downstream event. It is now well established that Abeta exists as monomers, oligomers, and fibrils. To study the temporal profile of Abeta oligomer formation in vivo and to determine their interaction with tau pathology, we used the 3xTg-AD mice, which develop a progressive accumulation of plaques and tangles and cognitive impairments. We show that SDS-resistant Abeta oligomers accumulate in an age-dependent fashion, and we present evidence to show that oligomerization of Abeta appears to first occur intraneuronally. Finally, we show that a single intrahippocampal injection of a specific oligomeric antibody is sufficient to clear Abeta pathology, and more importantly, tau pathology. Therefore, Abeta oligomers may play a role in the induction of tau pathology, making the interference of Abeta oligomerization a valid therapeutic target.     

Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates cognitive decline in transgenic mice with plaques and tangles

Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-beta (Abeta) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Abeta pathology. We describe improved cognitive function following a reduction in both soluble Abeta and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Abeta alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Abeta immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.     

Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain

Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, differential glutamatergic responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as 3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal "stubs," and Abeta(40) and Abeta(42) peptides in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected, the Abeta(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late neuropathological and clinical aspects of Alzheimer's disease.     

Stabilization of the cyclin-dependent kinase 5 activator,p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.     

Expression of human FE65 in amyloid precursor protein transgenic mice is associated with a reduction in beta-amyloid load

FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non-neuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and beta-amyloid (Abeta) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Abeta deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Abeta accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP-CTFs. The reduced accumulation of Abeta in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Abeta42 level observed at 4 months of age and to the lower APP-CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.     

Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity

In Alzheimer's disease and tauopathies, tau protein aggregates into neurofibrillary tangles that progressively spread to synaptically connected brain regions. A prion‐like mechanism has been suggested: misfolded tau propagating through the brain seeds neurotoxic aggregation of soluble tau in recipient neurons. We use transgenic mice and viral tau expression to test the hypotheses that trans‐synaptic tau propagation, aggregation, and toxicity rely on the presence of endogenous soluble tau. Surprisingly, mice expressing human P301Ltau in the entorhinal cortex showed equivalent tau propagation and accumulation in recipient neurons even in the absence of endogenous tau. We then tested whether the lack of endogenous tau protects against misfolded tau aggregation and toxicity, a second prion model paradigm for tau, using P301Ltau‐overexpressing mice with severe tangle pathology and neurodegeneration. Crossed onto tau‐null background, these mice had similar tangle numbers but were protected against neurotoxicity. Therefore, misfolded tau can propagate across neural systems without requisite templated misfolding, but the absence of endogenous tau markedly blunts toxicity. These results show that tau does not strictly classify as a prion protein.     

Stress-induced hyperphosphorylation of tau in the mouse brain

We previously showed that starvation causes reversible hyperphosphorylation of tau in the mouse brain. To explore possible involvement of stress in tau hyperphosphorylation quantitative analysis of phosphorylated tau in four brain regions of mice subjected to cold water stress (CWS) was made by immunoblot analyses using phosphorylation-dependent antibodies directed to eight sites on tau known to be hyperphosphorylated in the brain of Alzheimer's disease (AD) patients. Ser199, Ser202/Thr205, Thr231/Ser235 were hyperphosphorylated 20 and 40 min after CWS. The response was pronounced in the hippocampus and cerebral hemisphere, but weak in the cerebellum in parallel with the regional vulnerability in AD. Among the regulatory phosphorylation of protein kinases studied, a transient phosphorylation of tau protein kinase I/glycogen synthase kinase 3beta at Ser9 was most conspicuous.     

Lithium treatment of APPSwDI/NOS2-/- mice leads to reduced hyperphosphorylated tau, increased amyloid deposition and altered inflammatory phenotype

Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2-/- mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2-/- mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimer's disease.     

Humanized Tau Mice with Regionalized Amyloid Exhibit Behavioral Deficits but No Pathological Interaction

Alzheimer’s disease (AD) researchers have struggled for decades to draw a causal link between extracellular Aβ aggregation and intraneuronal accumulation of microtubule-associated protein tau. The amyloid cascade hypothesis posits that Aβ deposition promotes tau hyperphosphorylation, tangle formation, cell loss, vascular damage, and dementia. While the genetics of familial AD and the pathological staging of sporadic disease support this sequence of events, attempts to examine the molecular mechanism in transgenic animal models have largely relied on models of other inherited tauopathies as the basis for testing the interaction with Aβ. In an effort to more accurately model the relationship between Aβ and wild-type tau in AD, we intercrossed mice that overproduce human Aβ with a tau substitution model in which all 6 isoforms of the human protein are expressed in animals lacking murine tau. We selected an amyloid model in which pathology was biased towards the entorhinal region so that we could further examine whether the anticipated changes in tau phosphorylation occurred at the site of Aβ deposition or in synaptically connected regions. We found that Aβ and tau had independent effects on locomotion, learning, and memory, but found no behavioral evidence for an interaction between the two transgenes. Moreover, we saw no indication of amyloid-induced changes in the phosphorylation or aggregation of human tau either within the entorhinal area or elsewhere. These findings suggest that robust amyloid pathology within the medial temporal lobe has little effect on the metabolism of wild type human tau in this model.