Detection, Visualization and Quantification of Protein Complexes in Human Alzheimer’s Disease Brains using Proximity Ligation Assay

Abstract

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer’s disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that hyperphosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

IQGAP2 regulates blood-brain barrier immune dynamics

“…Human brain tissue was obtained at autopsy and prepared as previously described ( 85 ). De-identified brain tissue was obtained from the Vanderbilt Brain and Biospecimen Bank at Vanderbilt University Medical Center.…”

“…Human tissue: Immunohistochemical labeling in hippocampal tissue slices was performed as previously described (85,86) with minor modifications. Tissue slices were incubated with primary antibodies for 48 hours at 4 o C followed by secondary antibodies for 2 hours at room temperature.…”