The proper folding of secretory proteins in the endoplasmic reticulum (ER) is vital to a variety of cellular and organismal functions. However, when ER protein folding capacity is compromised or ER luminal conditions become unfavorable, aggregated and/or misfolded proteins accumulate in the ER resulting in "ER stress". To restore ER protein homeostasis ("proteostasis") and prevent the trafficking of non-native protein conformations to secretory environments, cells have evolved a highly conserved tripartite signal transduction pathway known as the Unfolded Protein Response (UPR). Activation of each arm of the UPR (IRE1, ATF6, and PERK) results in the production of distinct transcription factors which transcriptionally
remodel ER proteostasis pathways to combat ER stress. UPR activation has been shown to contribute to the pathogenesis of cancer, metabolic and neurological disorders. Recently, in a co-culture/ dual-species model of human aging neurons (grown on a feeder layer of mouse glia), UPR activation resulted in impaired action potential evoked synchronous neurotransmitter release, a hallmark of neurodegenerative disease. To comprehensively characterize temporal changes in UPR target protein expression throughout neuronal aging, we have implemented a DIA LC-MS/MS proteomics approach. A pipeline in R using DIA-NN result files as an input was then developed and validated to obtain species-specific protein quantification from the coculture model system. In comparison to western blot analysis, this method provides speciesspecific information to understand how the proteomes of neurons and glia change with age and how protein expression in glia changes in the presence and absence of neurons. To assess the status of UPR, previously described and novel proteins were then selected to monitor aggregate activation of these defined target protein sets, resulting in a high throughput assay to assess branch-specific pathway activation. Lastly, to explore how an increase in intracellular calcium affects neurons with age, an NMDAR antagonist was applied to modulate calcium concentration in neurons. Overall, a systematic proteomics evaluation of the three branches of the UPR throughout neuronal aging will inform studies to pharmacologically manipulate the UPR to restore synaptic transmission in the aging brain.