Tara L. Spires-Jones

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 Alzheimers 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.

Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease.

Senile plaques accumulate over the course of decades in the brains of patients with Alzheimer’s disease. A fundamental tenet of the amyloid hypothesis of Alzheimer’s disease is that the deposition of amyloid-β precedes and induces the neuronal abnormalities that underlie dementia. This idea has been challenged, however, by the suggestion that alterations in axonal trafficking and morphological abnormalities precede and lead to senile plaques. The role of microglia in accelerating or retarding these processes has been uncertain. To investigate the temporal relation between plaque formation and the changes in local neuritic architecture, we used longitudinal in vivo multiphoton microscopy to sequentially image young APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice. Here we show that plaques form extraordinarily quickly, over 24 h. Within 1–2 days of a new plaque’s appearance, microglia are activated and recruited to the site. Progressive neuritic changes ensue, leading to increasingly dysmorphic neurites over the next days to weeks. These data establish plaques as a critical mediator of neuritic pathology.

Oligomeric amyloid β associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques

Synapse loss correlates with a cognitive decline in Alzheimers disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid β (Aβ) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric Aβ surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric Aβ. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric Aβ surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 μm from a plaque in an approximately linear fashion (linear regression, r2 = 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric Aβ associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric Aβ. The proportion of excitatory synapses associated with Aβ correlates with decreasing density (correlation, −0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric Aβ, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of “plaque” vs. “oligomeric Aβ” as the synaptotoxic species in the brain of AD patients.

Caspase activation precedes and leads to tangles

Studies of post-mortem tissue have shown that the location of fibrillar tau deposits, called neurofibrillary tangles (NFT), matches closely with regions of massive neuronal death, severe cytological abnormalities, and markers of caspase activation and apoptosis, leading to the idea that tangles cause neurodegeneration in Alzheimer’s disease and tau-related frontotemporal dementia. However, using in vivo multiphoton imaging to observe tangles and activation of executioner caspases in living tau transgenic mice (Tg4510 strain), we find the opposite: caspase activation occurs first, and precedes tangle formation by hours to days. New tangles form within a day. After a new tangle forms, the neuron remains alive and caspase activity seems to be suppressed. Similarly, introduction of wild-type 4-repeat tau (tau-4R) into wild-type animals triggered caspase activation, tau truncation and tau aggregation. Adeno-associated virus-mediated expression of a construct mimicking caspase-cleaved tau into wild-type mice led to the appearance of intracellular aggregates, tangle-related conformational- and phospho-epitopes, and the recruitment of full-length endogenous tau to the aggregates. On the basis of these data, we propose a new model in which caspase activation cleaves tau to initiate tangle formation, then truncated tau recruits normal tau to misfold and form tangles. Because tangle-bearing neurons are long-lived, we suggest that tangles are ‘off pathway’ to acute neuronal death. Soluble tau species, rather than fibrillar tau, may be the critical toxic moiety underlying neurodegeneration.

The Intersection of Amyloid Beta and Tau at Synapses in Alzheimer’s Disease

The collapse of neural networks important for memory and cognition, including death of neurons and degeneration of synapses, causes the debilitating dementia associated with Alzheimers disease (AD). We suggest that synaptic changes are central to the disease process. Amyloid beta and tau form fibrillar lesions that are the classical hallmarks of AD. Recent data indicate that both molecules may have normal roles at the synapse, and that the accumulation of soluble toxic forms of the proteins at the synapse may be on the critical path to neurodegeneration. Further, the march of neurofibrillary tangles through brain circuits appears to take advantage of recently described mechanisms of transsynaptic spread of pathological forms of tau. These two key phenomena, synapse loss and the spread of pathology through the brain via synapses, make it critical to understand the physiological and pathological roles of amyloid beta and tau at the synapse.

Amyloid β Induces the Morphological Neurodegenerative Triad of Spine Loss, Dendritic Simplification, and Neuritic Dystrophies through Calcineurin Activation

Amyloid β (Aβ)-containing plaques are surrounded by dystrophic neurites in the Alzheimers disease (AD) brain, but whether and how plaques induce these neuritic abnormalities remain unknown. We tested the hypothesis that soluble oligomeric assemblies of Aβ, which surround plaques, induce calcium-mediated secondary cascades that lead to dystrophic changes in local neurites. We show that soluble Aβ oligomers lead to activation of the calcium-dependent phosphatase calcineurin (CaN) (PP2B), which in turn activates the transcriptional factor nuclear factor of activated T cells (NFAT). Activation of these signaling pathways, even in the absence of Aβ, is sufficient to produce a virtual phenocopy of Aβ-induced dystrophic neurites, dendritic simplification, and dendritic spine loss in both neurons in culture and in the adult mouse brain. Importantly, the morphological deficits in the vicinity of Aβ deposits in a mouse model of AD are ameliorated by CaN inhibition, supporting the hypothesis that CaN–NFAT are aberrantly activated by Aβ and that CaN–NFAT activation is responsible for disruption of neuronal structure near plaques. In accord with this, we also detect increased levels of an active form of CaN and NFATc4 in the nuclear fraction from the cortex of patients with AD. Thus, Aβ appears to mediate the neurodegeneration of AD, at least in part, by activation of CaN and subsequent NFAT-mediated downstream cascades.

Tau pathophysiology in neurodegeneration: a tangled issue.

Neurodegenerative tauopathies are marked by their common pathologic feature of aggregates formed of hyperphosphorylated tau protein, which are associated with synapse and neuronal loss. Changes in tau conformation result in both loss of normal function and gain of fibrillogenicity that leads to aggregation. Here, we discuss the pathophysiology of tau and emerging evidence of how changes in this protein might ultimately lead to neuronal death. In particular, based on recent evidence, we propose that a non-apoptotic caspase-associated form of death is occurring in tauopathy.

The Synaptic Accumulation of Hyperphosphorylated Tau Oligomers in Alzheimer Disease Is Associated With Dysfunction of the Ubiquitin-Proteasome System

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD.

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood–brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a “Nissl stain” contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimers disease using (traditionally) non–BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ∼18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.

Apolipoprotein E4 effects in Alzheimer’s disease are mediated by synaptotoxic oligomeric amyloid-β

The apolipoprotein E ε4 gene is the most important genetic risk factor for sporadic Alzheimers disease, but the link between this gene and neurodegeneration remains unclear. Using array tomography, we analysed >50000 synapses in brains of 11 patients with Alzheimers disease and five non-demented control subjects and found that synapse loss around senile plaques in Alzheimers disease correlates with the burden of oligomeric amyloid-β in the neuropil and that this synaptotoxic oligomerized peptide is present at a subset of synapses. Further analysis reveals apolipoprotein E ε4 patients with Alzheimers disease have significantly higher oligomeric amyloid-β burden and exacerbated synapse loss around plaques compared with apolipoprotein E ε3 patients. Apolipoprotein E4 protein colocalizes with oligomeric amyloid-β and enhances synaptic localization of oligomeric amyloid-β by >5-fold. Biochemical characterization shows that the amyloid-β enriched at synapses by apolipoprotein E4 includes sodium dodecyl sulphate-stable dimers and trimers. In mouse primary neuronal culture, lipidated apolipoprotein E4 enhances oligomeric amyloid-β association with synapses via a mechanism involving apolipoprotein E receptors. Together, these data suggest that apolipoprotein E4 is a co-factor that enhances the toxicity of oligomeric amyloid-β both by increasing its levels and directing it to synapses, providing a link between apolipoprotein E ε4 genotype and synapse loss, a major correlate of cognitive decline in Alzheimers disease.