Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease.

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 pre-synaptic 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 pre-synaptic compartment in AD. Results demonstrate the abundance of tau, mainly C-terminal truncated tau, in synaptic terminals in aged control and in Alzheimer's disease (AD) samples. Tau fragments and dimers/oligomers are prominent in AD synapses. Following depolarization, tau release is potentiated in AD nerve terminals compared to aged controls. We hypothesize (i) endosomal release of the different tau peptides from AD synapses, and (ii) together with phosphorylation, fragmentation of synaptic tau exacerbates tau aggregation, synaptic dysfunction, and the spread of tau pathology in AD. Aß = amyloid-beta.

Levels of soluble apolipoprotein E/amyloid-ß (Aß) complex are reduced and oligomeric Aß increased with APOE4 and Alzheimer disease in a transgenic mouse model and human samples.

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-ß (Aß) levels. Evidence suggests physical interactions between apoE and Aß are partially responsible for these functional effects. However, the apoE/Aß complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aß in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aß and an increase in soluble Aß, specifically oligomeric Aß (oAß), are associated with APOE4 and AD. Previously, soluble Aß42 and oAß levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aß levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aß levels isoform-specifically modulate soluble oAß clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aß levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aß levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aß42 levels decreased in AD patients compared with controls, oAß levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aß modulates oAß levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

Extensive p-tau pathology and SDS-stable p-tau oligomers in Alzheimer's cortical synapses.

Like amyloid beta (Aß) oligomers, tau aggregates are increasingly recognized as potential key toxic intermediates in Alzheimer's disease (AD) and as therapeutic targets. P-tau co-localizes with Aß in cortical AD synapses and may contribute to synapse dysfunction and loss. Flow cytometry analysis of synaptosomes from AD compared with 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 Aß in individual synaptosomes in dual labeling experiments, and flow cytometry sorting of Aß-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 Aß accumulations in surviving synaptic terminals, particularly in late-stage AD.

Apolipoprotein E level and cholesterol are associated with reduced synaptic amyloid beta in Alzheimer's disease and apoE TR mouse cortex.

The apolipoprotein E4 allele (APOE4) contributes to Alzheimer's disease (AD) risk and APOE2 is protective, but the relevant cellular mechanisms are unknown. We have used flow cytometry analysis to measure apolipoprotein E (apoE) and amyloid beta peptide (Aß) levels in large populations of synaptic terminals from AD and aged cognitively normal controls, and demonstrate that modest but significant increases in soluble apoE levels accompany elevated Aß in AD cortical synapses and in an APP/PS1 rat model of AD. Dual labeling experiments document co-localization of apoE and Aß in individual synapses with concentration of Aß in a small population of apoE-positive synapses in both AD and controls. Consistent with a clearance role, the apoE level was higher in Aß-positive synapses in control cases. In aged targeted replacement mice expressing human apoE, apoE2/4 synaptic terminals demonstrated the highest level of apoE and the lowest level of Aß compared to apoE3/3 and apoE4/4 lines. In apoE2/4 terminals, the pattern of immunolabeling for apoE and Aß closely resembled the pattern in human control cases, and elevated apoE was accompanied by elevated free cholesterol in apoE2/4 synaptic terminals. These results are consistent with a role for APOE in Aß clearance in AD synapses, and suggest that optimal lipidation of apoE2 compared to E3 and E4 makes an important contribution to Aß clearance and synaptic function.