Differential inclusion formation of an aggregation-prone protein reveals differences in the proteostasis capacity of...

Maintaining proteome integrity is essential for cellular function and survival. Disruptions in proteostasis lead to the aggregation of proteins into inclusions, a process that underlies many neurodegenerative diseases. To quantitatively assess the proteostasis capacity of neuronal cells, we employed an aggregation-prone double mutant form of firefly luciferase (denoted Fluc^DM) as a reporter protein. We compared two commonly used neuronal cell lines, mouse neuroblastoma cells (Neuro-2a) and a motor neuron-like hybrid line (NSC-34), to evaluate their ability to prevent the aggregation of proteins into intracellular inclusions. We observed a significantly greater propensity of Fluc^DM to form inclusions in NSC-34 cells compared to Neuro-2a cells. This suggests a reduced capacity of NSC-34 cells for managing aggregation-prone proteins. Proteomic profiling of Fluc^DM inclusions purified from both cell types revealed cell-type-specific engagement of the proteostasis machinery with aggregation-prone proteins. Comparing the proteomic profiles of key arms of the proteostasis network between these two cell lines revealed that the endoplasmic reticulum (ER) unfolded protein response is differentially expressed. This study establishes a quantitative platform for assessing cellular proteostasis capacity and underscores the importance of cell-type context in proteome maintenance. These insights have implications for understanding the selective vulnerability of neurons in protein misfolding disorders.