Studying Unconventional Secretion of Misfolded Proteins in Cultured Cells and Primary Neurons

Abstract

Protein misfolding poses a significant threat to the fitness of eukaryotic cells, particularly for neurons facing environmental stress. To effectively triage and remove defective and unwanted proteins, cells have evolved diverse protein quality control (PQC) mechanisms relying on proteasome- and endolysosome-mediated degradation systems. Defects in PQC functions are linked to various human diseases including many aging-associated neurodegenerative diseases. Misfolding-associated protein secretion (MAPS) is a recently reported PQC mechanism that eliminates misfolded cytosolic proteins by an unconventional secretory pathway using an endo-vesiclular network. This process implicates DNAJC5, a chaperone that escorts misfolded cargos to intracellular vesicles to facilitate their secretion. Cargos of DNAJC5 include Parkinson's and Alzheimer's disease-associated proteins known to undergo cell-to-cell transmission during disease progression. Thus, elucidating how these proteins are secreted may reveal novel therapeutic targets for these diseases. Here we describe a collection of methods used to detect either the basal or induced secretion of misfolded proteins from cell lines and cultured primary neurons.

Keywords: Alzheimer’s disease; DNAJC5/CLN4/CSPα; Misfolding-associated protein secretion (MAPS); Parkinson’s disease; Primary murine hippocampal neurons; Protein quality control (PQC); Tau; Unconventional protein secretion (UPS); α-Synuclein; β-Galactosidase/LacZ assay.