Isolation of synaptic terminals from Alzheimer's disease cortex.
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 amyloid precursor protein (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.
UCLA School of Nursing, Los Angeles, California 90095, USA. ssokolow@sonnet.ucla.edu
Sophie
S
Sokolow
Kristen M
KM
Henkins
Iris A
IA
Williams
Harry V
HV
Vinters
Ingrid
I
Schmid
Gregory M
GM
Cole
Karen H
KH
Gylys
D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology*; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000096:biosynthesis; D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry / Q000473:pathology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008297:Male; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D017729:Presynaptic Terminals / Q000473:pathology*; D013574:Synaptosomes / Q000473:pathology* / Q000502:physiology; D016875:tau Proteins / Q000032:analysis* / Q000737:chemistry
D016229:Amyloid beta-Peptides; D016875:tau Proteins