Isolation of synaptic terminals from Alzheimer's disease cortex

Abstract

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.

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

“…; Sokolow et al . ). High levels of tau labeling (~ 75% positive terminals) are illustrated in representative aged normal control (Fig.…”

“…; Sokolow et al . ,b,c). Synaptosome particles contain synaptic structures in situ , including mitochondria and exocytotic elements; the preparation has been used for decades to study neurotransmitter release and synaptic function (Dunkley et al .…”

“…; Sokolow et al . ). In the present study, we assessed total levels of tau protein and its cleaved fragments in surviving nerve terminals (i.e., with intact membranes) from cryopreserved human AD and control samples (Gylys et al .…”

“…We have previously demonstrated that p-tau co-localizes with Ab in AD synapses and that p-tau species are mainly oligomeric (Fein et al 2008;Henkins et al 2012;Sokolow et al 2012b). In the present study, we assessed total levels of tau protein and its cleaved fragments in surviving nerve terminals (i.e., with intact membranes) from cryopreserved human AD and control samples (Gylys et al 2004b;Sokolow et al 2012c).…”

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

“…42 In addition, preaggregated Ab does not show up on flow dot plots, 43 and synaptosomes physically sorted on size show a uniform population of spherical particles without visible artifacts. 44 Representative dot plots from the flow cytometric analysis are shown in Figure 1; Ab immunofluorescence is plotted against forward scatter, which is proportional to particle size. Two variables are reported for the fluorescence of each sample: the size of the positive fraction (% positive) and the brightness of fluorescence (relative fluorescence unit).…”