In vivo spreading of tau pathology

Recent findings have suggested that tau pathology may spread in the brain by a prion-like mechanism. In this issue of Neuron, de Calignon et al. (2012) recreated an early stage of neurofibrillary tangle pathology to show that tau aggregates initially generated in a circumscribed area spread throughout the brain and lead to neurodegeneration.     

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.     

Long-term treadmill exercise attenuates tau pathology in P301S tau transgenic mice

Background

Recent epidemiological evidence suggests that modifying lifestyle by increasing physical activity could be a non-pharmacological approach to improving symptoms and slowing disease progression in Alzheimer’s disease and other tauopathies. Previous studies have shown that exercise reduces tau hyperphosphorylation, however, it is not known whether exercise reduces the accumulation of soluble or insoluble tau aggregates and neurofibrillary tangles, which are both neuropathological hallmarks of neurodegenerative tauopathy. In this study, 7-month old P301S tau transgenic mice were subjected to 12-weeks of forced treadmill exercise and evaluated for effects on motor function and tau pathology at 10 months of age.

Results

Exercise improved general locomotor and exploratory activity and resulted in significant reductions in full-length and hyperphosphorylated tau in the spinal cord and hippocampus as well as a reduction in sarkosyl-insoluble AT8-tau in the spinal cord. Exercise did not attenuate significant neuron loss in the hippocampus or cortex. Key proteins involved in autophagy—microtubule-associated protein 1A/1B light chain 3 and p62/sequestosome 1 —were also measured to assess whether autophagy is implicated in the exercised-induced reduction of aggregated tau protein. There were no significant effects of forced treadmill exercise on autophagy protein levels in P301S mice.

Conclusions

Our results suggest that forced treadmill exercise differently affects the brain and spinal cord of aged P301S tau mice, with greater benefits observed in the spinal cord versus the brain. Our work adds to the growing body of evidence that exercise is beneficial in tauopathy, however these benefits may be more limited at later stages of disease.

     

Site-specific nitration differentially influences tau assembly in vitro

Previously, we reported that the microtubule-associated tau protein, the major constituent of neurofibrillary tangles (NFTs) in Alzheimer's brain, undergoes site-selective nitration by peroxynitrite (ONOO-) and that this event inhibits tau polymerization in vitro [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we extend our analysis of tau nitration to include mutant tau proteins singly nitrated at each residue targeted by ONOO- in vitro (Tyr18, Tyr29, Tyr197, and Tyr394). Using our polymerization paradigm, we demonstrate that site-specific Tyr nitration differentially alters the rate and/or extent of tau assembly and generates robust changes in filament morphology. As determined by quantitative electron microscopy, select nitration of residues Tyr29 and Tyr197 increases the average length of synthetic tau filaments but does not alter the steady-state polymer mass. In contrast, site-specific nitration of residues Tyr18 and Tyr394 decreases the average length and/or number of synthetic filaments, resulting in a significant reduction in filamentous mass and an increase in tau critical concentration. Intriguingly, affinity measurements demonstrate that nitrative modifications do not preclude formation of the Alz-50 epitope, a pathological tau conformation detectable in authentic paired helical filaments (PHFtau). In fact, the Alz-50 antibody binds filaments assembled from nitrated mutant tau with higher avidity than wild-type filaments, even in instances where the overall filamentous mass is reduced. Taken together, our results suggest that site-specific nitration modulates the nucleation and/or elongation capacity of assembly-competent tau and that assumption of the Alz-50 conformation may be necessary, but not sufficient, to induce filament formation.     

Regulation of tau pathology by the microglial fractalkine receptor

Aggregates of the hyperphosphorylated microtubule-associated protein tau (MAPT) are an invariant neuropathological feature of tauopathies. Here, we show that microglial neuroinflammation promotes MAPT phosphorylation and aggregation. First, lipopolysaccharide-induced microglial activation promotes hyperphosphorylation of endogenous mouse MAPT in nontransgenic mice that is further enhanced in mice lacking the microglial-specific fractalkine receptor (CX3CR1) and is dependent upon functional toll-like receptor 4 and interleukin-1 (IL-1) receptors. Second, humanized MAPT transgenic mice lacking CX3CR1 exhibited enhanced MAPT phosphorylation and aggregation as well as behavioral impairments that correlated with increased levels of active p38 MAPK. Third, in?vitro experiments demonstrate that microglial activation elevates the level of active p38 MAPK and enhances MAPT hyperphosphorylation within neurons that can be blocked by administration of an interleukin-1 receptor antagonist and a specific p38 MAPK inhibitor. Taken together, our results suggest that CX3CR1 and IL-1/p38 MAPK may serve as novel therapeutic targets for human tauopathies.     

Knockout of 5‐lipoxygenase prevents dexamethasone‐induced tau pathology in 3xTg mice

Emerging evidence suggests that dysregulation stress hormones, such as glucocorticoids, in aged persons put them at a higher risk to develop Alzheimer's disease (AD). However, the mechanisms underlying such vulnerability remain to be unraveled. Pharmacologic inhibition of 5‐lipoxygenase (5LO), an active player in AD pathogenesis whose protein level increases with aging in the human, has been shown to blunt glucocorticoid‐mediated amyloid β (Ab) formation in vitro. In this article, we investigated the role of this pathway in modulating the development of the corticosteroid‐dependent AD‐like phenotype in the triple transgenic mice (3xTg). Dexamethasone was administered for 1 week to 3xTg or 3xTg genetically deficient for 5LO (3xTg/5LO^?/?) mice, and its effect on memory, amyloid‐β and tau levels, and metabolism assessed. At the end of the treatment, we observed that dexamethasone did not induce changes in behavior. Compared with controls, treated mice did not show significant alterations in brain soluble Aβ levels. While total tau protein levels were unmodified in all groups, we found that dexamethasone significantly increased tau phosphorylation at S396, as recognized by the antibody PHF‐13, which was specifically associated with an increase in the GSK3β activity. Additionally, dexamethasone‐treated mice had a significant increase in the tau insoluble fraction and reduction in the postsynaptic protein PDS‐95. By contrast, these modifications were blunted in the 3xTg/5LO^?/? mice. Our findings highlight the functional role that 5LO plays in stress‐induced AD tau pathology and support the hypothesis that pharmacologic inhibition of this enzyme could be a useful tool for individuals with this risk factor.     

Cofilin promotes tau pathology in Alzheimer’s disease

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Substrate-specific reduction of PP2A activity exaggerates tau pathology

Phosphorylation of the microtubule-associated protein tau is regulated by the balanced interplay of kinases and phosphatases. Disturbance of this balance causes hyperphosphorylation of tau and neurofibrillary tangle formation in Alzheimer’s disease brain. Here, we crossed Dom5 mice that express a substrate-specific dominant negative mutant form, L309A Cα, of protein phosphatase 2A (PP2A) with neurofibrillary-tangle-forming P301L mutant tau transgenic pR5 mice. This exacerbated the tau pathology of pR5 mice significantly. Double-transgenic Dom5/pR5 mice showed 7-fold increased numbers of hippocampal neurons that specifically phosphorylated the pathological S422 epitope of tau. They showed 8-fold increased numbers of tangles compared to pR5 mice, in agreement with our previous finding that tangle formation is correlated with and preceded by phosphorylation of tau at the S422 epitope. This suggests that, in addition to kinases, PP2A and its regulatory subunits may be a therapeutic target for Alzheimer’s disease.     

Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease. The major neurofibrillary tangle component is tau that is truncated at Asp421 (Δtau), hyperphosphorylated and aggregates into insoluble paired helical filaments. Alzheimer's disease brains also exhibit signs of inflammation manifested by activated astrocytes and microglia, which produce cytotoxic agents among them prostaglandins. We show that prostaglandin (PG) J2, an endogenous product of inflammation, induces caspase-mediated cleavage of tau, generating Δtau, an aggregation prone form known to seed tau aggregation prior to neurofibrillary tangle formation. The initial event observed upon PGJ2-treatment of human neuroblastoma SK-N-SH cells was the build-up of ubiquitinated (Ub) proteins indicating an early disruption of the ubiquitin-proteasome pathway. Apoptosis kicked in later, manifested by caspase activation and caspase-mediated cleavage of tau at Asp421 and poly (ADP-ribose) polymerase. Furthermore, cathepsin inhibition stabilized Δtau suggesting its lysosomal clearance. Upon PGJ2-treatment tau accumulated in a large perinuclear aggregate. In rat E18 cortical neuronal cultures PGJ2-treatment also generated Δtau detected in dystrophic neurites. Levels of Δtau were diminished by caspase 3 knockdown using siRNA. PGD2, the precursor of PGJ2, produced some Δtau. PGE2 generated none. Our data suggest a potential sequence of events triggered by the neurotoxic product of inflammation PGJ2 leading to tau pathology. The accumulation of Ub proteins is an early response. If cells fail to overcome the toxic effects induced by PGJ2, including accumulation of Ub proteins, apoptosis kicks in triggering caspase activation and tau cleavage, the clearance of which by cathepsins could be compromised culminating in tau pathology. Our studies are the first to provide a mechanistic link between inflammation and tau pathology.     

The relationship between subcortical tau pathology and Alzheimer's disease

The stepwise progression of tau pathology [NFTs (neurofibrillary tangles) and NTs (neuropil threads)] in AD (Alzheimer's disease) is generally assumed to begin in the transentorhinal region (entorhinal stage) from which it progresses to the hippocampus (limbic stage) and to neocortical regions (neocortical stage). This stepwise progression is reflected in the NFT Braak stages. However, it has been shown recently that tau pathology is frequently seen in subcortical nuclei, in particular the LC (locus coeruleus) in over 90% of individuals under 30 years of age, suggesting that AD-associated tau pathology begins in the LC and not in the transentorhinal region. On the other hand, only minimal amounts of tau pathology are seen in the LC in cases with considerable entorhinal tau pathology, while the severity of tau pathology in the LC significantly increases with increasing NFT Braak stages. These findings suggest that the LC becomes increasingly involved during AD progression rather than representing the site initially affected. Further studies are warranted to answer the question of whether tau pathology in the LC of young individuals is associated with AD or whether it rather reflects non-specific neuronal damage.     

Propagation of tau pathology in a model of early Alzheimer's disease

Neurofibrillary tangles advance from layer II of the entorhinal cortex (EC-II) toward limbic and association cortices as Alzheimer's disease evolves. However, the mechanism involved in this hierarchical pattern of disease progression is unknown. We describe a transgenic mouse model in which overexpression of human tau P301L is restricted to EC-II. Tau pathology progresses from EC transgene-expressing neurons to neurons without detectable transgene expression, first to EC neighboring cells, followed by propagation to neurons downstream in the synaptic circuit such as the dentate gyrus, CA fields of the hippocampus, and cingulate cortex. Human tau protein spreads to these regions and coaggregates with endogenous mouse tau. With age, synaptic degeneration occurs in the entorhinal target zone and EC neurons are lost. These data suggest that a sequence of progressive misfolding of tau proteins, circuit-based transfer to new cell populations, and deafferentation induced degeneration are part of a process of tau-induced neurodegeneration.     

Seed-competent tau monomer initiates pathology in a tauopathy mouse model

Tau aggregation into ordered assemblies causes neurodegenerative tauopathies. We previously reported that tau monomer exists in either inert (M_i) or seed-competent (M_s) conformational ensembles and that M_s encodes strains, that is, unique, self-replicating, biologically active assemblies. It is unknown if disease begins with M_s formation followed by fibril assembly or if M_s derives from fibrils and is therefore an epiphenomenon. Here, we studied a tauopathy mouse model (PS19) that expresses full-length mutant human (1N4R) tau (P301S). Insoluble tau seeding activity appeared at 2 months of age and insoluble tau protein assemblies by immunoblot at 3 months. Tau monomer from mice aged 1 to 6 weeks, purified using size-exclusion chromatography, contained soluble seeding activity at 4 weeks, before insoluble material or larger assemblies were observed, with assemblies ranging from n = 1 to 3 tau units. By 5 to 6 weeks, large soluble assemblies had formed. This indicated that the first detectable pathological forms of tau were in fact M_s. We next examined posttranslational modifications of tau monomer from 1 to 6 weeks. We detected no phosphorylation unique to M_s in PS19 or human Alzheimer’s disease brains. We conclude that tauopathy begins with formation of the M_s monomer, whose activity is phosphorylation independent. M_s then self assembles to form oligomers before it forms insoluble fibrils. The conversion of tau monomer from M_i to M_s thus constitutes the first detectable step in the initiation of tauopathy in this mouse model, with obvious implications for the origins of tauopathy in humans.     

Clearing tau pathology with Abeta immunotherapy--reversible and irreversible stages revealed

The report by Oddo and colleagues in this issue of Neuron demonstrates for the first time that clearance of amyloid also results in the removal of early-stage tau pathology in mice that develop both amyloid plaques and neurofibrillary tangles (NFT), the two hallmark lesions of Alzheimer's disease (AD). This result supports a primary role for Abeta in AD etiology.     

From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases

The most common degenerative diseases of the human brain are characterized by the presence of abnormal filamentous inclusions in affected nerve cells and glial cells. These diseases can be grouped into two classes, based on the identity of the major proteinaceous components of the filamentous assemblies. The filaments are made of either the microtubule-associated protein tau or the protein alpha-synuclein. Importantly, the discovery of mutations in the tau gene in familial forms of frontotemporal dementia and of mutations in the alpha-synuclein gene in familial forms of Parkinson's disease has established that dysfunction of tau protein and alpha-synuclein can cause neurodegeneration.     

Cultured cell and transgenic mouse models for tau pathology linked to beta-amyloid

The two histopathological signatures of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles, prompting speculation that a causal relationship exists between the respective building blocks of these abnormal brain structures: the beta-amyloid peptides (Abeta) and the neuron-enriched microtubule-associated protein called tau. Transgenic mouse models have provided in vivo evidence for such connections, and cultured cell models have allowed tightly controlled, systematic manipulation of conditions that influence links between Abeta and tau. The emerging evidence supports the view that amyloid pathology lies upstream of tau pathology in a pathway whose details remain largely mysterious. In this communication, we review and discuss published work about the Abeta-tau connection. In addition, we present some of our own previously unpublished data on the effects of exogenous Abeta on primary brain cultures that contain both neurons and glial cells. We report here that continuous exposure to 5 microM non-fibrillar Abeta40 or Abeta42 kills primary brain cells by apoptosis within 2-3 weeks, Abeta42 is more toxic and selective for neurons than Abeta40, and Abeta42, but not Abeta40, induces a transient increase in neurons that are positive for the AD-like PHF1 epitope. These findings demonstrate the greater potency of Abeta42 than Abeta40 at inducing tau pathology and programmed cell death, and corroborate and extend reports that tau-containing cells are more sensitive to Abeta peptides than cells that lack or express low levels of tau.     

Synaptic oligomeric tau in Alzheimer’s disease — A potential culprit in the spread of tau pathology through the brain

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Mouse genetic background influences severity of immune responses following trauma-hemorrhage

Studies have shown that following bacterial infection or endotoxin administration, immune functions are regulated differently in mice of different genetic background. Since the susceptibility to sepsis following trauma-hemorrhage is dependant on the severity of injury, it is important to determine whether genetic background of the animal influence immune functions after trauma-hemorrhage. The aim of our studies, therefore, was to assess differences in the immune functions in genetically different strains of age-matched C3H/HeN and C57BL/6 male mice following trauma-hemorrhage. The analysis for immune functions included: proliferation of splenocyte and bone-marrow cells, IL-2 and IFN-gamma release by splenocytes, and TNF-alpha and IL-10 release by splenic, peritoneal, liver (Kupffer cell), and bone-marrow macrophages. The results show significant differences in splenocyte and bone-marrow functions, and in the release of the mediators of immune function by immune competent cells: (a) between the two genetic strains, and (b) in each mouse strain following trauma-hemorrhage. Thus, genetic background appears to significantly influence the severity of immune responses in males following trauma-hemorrhage.