Amy M. Pooler

Physiological release of endogenous tau is stimulated by neuronal activity

Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans‐synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA‐induced release of tau is calcium‐dependent. Blocking pre‐synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA‐mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease.

Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture.

Alzheimers disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Dynamic association of tau with neuronal membranes is regulated by phosphorylation

Tau is an abundant cytosolic protein which regulates cytoskeletal stability by associating with microtubules in a phosphorylation-dependent manner. We have found a significant proportion of tau is located in the membrane fraction of rat cortical neurons and is dephosphorylated, at least at Tau-1 (Ser199/Ser202), AT8 (Ser199/Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Inhibition of tau kinases casein kinase 1 (CK1) or glycogen synthase kinase-3 decreased tau phosphorylation and significantly increased amounts of tau in the membrane fraction. Mutation of serine/threonine residues to glutamate to mimic phosphorylation in the N-terminal, but not C-terminal, region of tau prevented its membrane localization in transfected cells, demonstrating that the phosphorylation state of tau directly impacts its localization. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn also induced tau dephosphorylation but did not affect its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that trafficking of tau between the cytosol and the neuronal membrane is dynamically regulated by tau phosphorylation through a mechanism dependent on fyn expression.

The intersection of amyloid beta and tau in glutamatergic synaptic dysfunction and collapse in Alzheimer's disease

The synaptic connections that form between neurons during development remain plastic and able to adapt throughout the lifespan, enabling learning and memory. However, during aging and in particular in neurodegenerative diseases, synapses become dysfunctional and degenerate, contributing to dementia. In the case of Alzheimers disease (AD), synapse loss is the strongest pathological correlate of cognitive decline, indicating that synaptic degeneration plays a central role in dementia. Over the past decade, strong evidence has emerged that oligomeric forms of amyloid beta, the protein that accumulates in senile plaques in the AD brain, contribute to degeneration of synaptic structure and function. More recent data indicate that pathological forms of tau protein, which accumulate in neurofibrillary tangles in the AD brain, also cause synaptic dysfunction and loss. In this review, we will present the case that soluble forms of both amyloid beta and tau protein act at the synapse to cause neural network dysfunction, and further that these two pathological proteins may act in concert to cause synaptic pathology. These data may have wide-ranging implications for the targeting of soluble pathological proteins in neurodegenerative diseases to prevent or reverse cognitive decline.

Amyloid accelerates tau propagation and toxicity in a model of early Alzheimer's disease

IntroductionIn early stages of Alzheimer’s disease (AD), neurofibrillary tangles (NFT) are largely restricted to the entorhinal cortex and medial temporal lobe. At later stages, when clinical symptoms generally occur, NFT involve widespread limbic and association cortices. At this point in the disease, amyloid plaques are also abundantly distributed in the cortex. This observation from human neuropathological studies led us to pose two alternative hypotheses: that amyloid in the cortex is permissive for the spread of tangles from the medial temporal lobe, or that these are co-occurring but not causally related events simply reflecting progression of AD pathology.ResultsWe now directly test the hypothesis that cortical amyloid acts as an accelerant for spreading of tangles beyond the medial temporal lobe. We crossed rTgTauEC transgenic mice that demonstrate spread of tau from entorhinal cortex to other brain structures at advanced age with APP/PS1 mice, and examined mice with either NFTs, amyloid pathology, or both. We show that concurrent amyloid deposition in the cortex 1) leads to a dramatic increase in the speed of tau propagation and an extraordinary increase in the spread of tau to distal brain regions, and 2) significantly increases tau-induced neuronal loss.ConclusionsThese data strongly support the hypothesis that cortical amyloid accelerates the spread of tangles throughout the cortex and amplifies tangle-associated neural system failure in AD.

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau

Recent reports have demonstrated that interactions between the microtubule‐associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to β‐amyloid (Aβ) in models of Alzheimer’s disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aβ‐induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src‐homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213–216) motif in tau plays an important role in mediating the interaction of tau with Fyn‐SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn‐SH3, is influenced by Fyn‐mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn‐SH2–tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent‐resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn‐dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn‐mediated synaptic dysfunction that occurs in response to elevated Aβ levels in neurodegenerative disease.

Functional implications of the association of tau with the plasma membrane

Tau is an abundant microtubule-associated protein which regulates the stability of the cytoskeleton. Tau binds microtubules directly through microtubule-binding domains in its C-terminus. However, tau is not only located in the cytosol of cells, but also associated with other intracellular domains, including the plasma membrane, suggesting that tau may have additional functions other than stabilizing the neuronal cytoskeleton. Localization of tau at the cell surface appears to be dependent on interactions of the N-terminal projection domain of tau. Furthermore, membrane-associated tau is dephosphorylated at serine/threonine residues, suggesting that the phosphorylation state of tau regulates its intracellular trafficking. Dephosphorylation of tau may increase the association of tau with trafficking proteins which target tau to the plasma membrane. Thus it is possible that the hyperphosphoryation of tau may contribute to the pathogenesis of Alzheimers disease by promoting the formation of neurofibrillary tangles from cytosolic tau, and also by inhibiting additional tau functions through disruption of its targeting to the plasma membrane.

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

In Alzheimers 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.

Studying synapses in human brain with array tomography and electron microscopy

Postmortem studies of synapses in human brain are problematic because of the axial resolution limit of light microscopy and the difficulty in preserving and analyzing ultrastructure with electron microscopy (EM). Array tomography (AT) overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes ∼3 d per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve the structure in human samples allows complementary ultrastructural studies. Incorporation of AT and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts in order to develop clinicopathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental disease and psychiatric illness.

Propagation of tau pathology in Alzheimer's disease: identification of novel therapeutic targets

Accumulation and aggregation of the microtubule-associated protein tau are a pathological hallmark of neurodegenerative disorders such as Alzheimer’s disease (AD). In AD, tau becomes abnormally phosphorylated and forms inclusions throughout the brain, starting in the entorhinal cortex and progressively affecting additional brain regions as the disease progresses. Formation of these inclusions is thought to lead to synapse loss and cell death. Tau is also found in the cerebrospinal fluid (CSF), and elevated levels are a biomarker for AD. Until recently, it was thought that the presence of tau in the CSF was due to the passive release of aggregated tau from dead or dying tangle-bearing neurons. However, accumulating evidence from different AD model systems suggests that tau is actively secreted and transferred between synaptically connected neurons. Transgenic mouse lines with localized expression of aggregating human tau in the entorhinal cortex have demonstrated that, as these animals age, tau becomes mislocalized from axons to cell bodies and dendrites and that human tau-positive aggregates form first in the entorhinal cortex and later in downstream projection targets. Numerous in vitro and in vivo studies have provided insight into the mechanisms by which tau may be released and internalized by neurons and have started to provide insight into how tau pathology may spread in AD. In this review, we discuss the evidence for regulated tau release and its specific uptake by neurons. Furthermore, we identify possible therapeutic targets for preventing the propagation of tau pathology, as inhibition of tau transfer may restrict development of tau tangles in a small subset of neurons affected in early stages of AD and therefore prevent widespread neuron loss and cognitive dysfunction associated with later stages of the disease.