The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from presynaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the presynaptic compartment in AD.

Much evidence indicates that soluble amyloid beta (Aβ) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimer's disease (AD) synapses accumulate both Aβ and p-tau. The present experiments use peptide-specific xMAP assays and Western blotting to identify the Aβ peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aβ40 peptide levels did not vary, but both Aβ42 and Aβ oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared to detergent extracts. In Western blots, a ladder of SDS-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kD and larger. Multiple oligomers, including monomer, small oligomers, a 56 kD assembly, and APP were correlated with the Aβ level measured in flow cytomety-purified synaptosomes. These results suggest that multiple APP processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.

Like amyloid beta (Aβ) oligomers, tau aggregates are increasingly recognized as potential key toxic intermediates in Alzheimer’s disease and as therapeutic targets. P-tau co-localizes with Aβ in cortical AD synapses and may contribute to synapse dysfunction and loss. Flow cytometry analysis of synaptosomes from AD compared to aged cognitively normal cortex demonstrates increased immunolabeling for three p-tau antibodies (AT8, PHF-1 and pS422), indicating phosphorylation at multiple tau epitopes. Sequential extraction experiments show increased soluble p-tau in AD synapses, but a sizable pool of p-tau requires detergent solubilization, suggesting endosomal/lysosomal localization. P-tau is co-localized with Aβ in individual synaptosomes in dual labeling experiments, and flow cytometry sorting of Aβ-positive synaptosomes from an AD case reveals a marked enrichment of p-tau aggregates. The p-tau enrichment, a 76-fold increase over the initial homogenate, is consistent with sequestration of p-tau in internal synaptic compartments. Western analysis of a series of AD and normal cases shows SDS-stable tau oligomers in the dimer/trimer size range in AD samples. These results indicate that widespread synaptic p-tau pathology accompanies Aβ accumulations in surviving synaptic terminals, particularly in late-stage AD.

The signal transduction molecule, Stat1, is critical for the expression of type I and II interferon (IFN)-responsive genes in most cells; however, we previously showed that primary hippocampal mouse neurons express low basal Stat1, with delayed and attenuated expression of IFN-responsive genes. Moreover, IFNγ-dependent resolution of a neurotropic viral challenge in permissive mice is Stat1-independent. Here, we show that exogenous INFγ has no deleterious impact on neuronal viability, and staurosporine-induced apoptosis in neurons is significantly blunted by the addition of INFγ, suggesting that INFγ confers a pro-survival signal in neurons. To identify the pathways induced by INFγ in neurons, the activation of alternative signal transducers associated with INFγ signaling was assessed. Rapid and pronounced activation of extracellular signal regulated kinase (Erk1/2) was observed in neurons, compared to a modest response in fibroblasts. Moreover, the absence of Stat1 in primary fibroblasts led to enhanced Erk activation following IFNγ addition, implying that the cell-specific availability of signal transducers can diversify the cellular response following IFN engagement.

Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for APP and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.