Methodology to Investigate Protein-Protein Interactions of Jun During Neuronal Differentiation of PC12 Cells

Abstract

In humans and other mammals, injury to the central nervous system (CNS) can cause a permanent loss of neuronal function, leading to cognitive defects, limb paralysis, and other neurological disabilities. In contrast, studies have shown that some non-mammalian vertebrates like zebrafish, have the remarkable ability to functionally regenerate axons after CNS injury by reactivating and sustaining the expression of regeneration-associated genes (RAGs). Some RAGs encode transcriptional regulators that dimerize to control downstream gene expression necessary for functional axonal recovery. Our lab has previously identified Jun as an important transcriptional regulator of regeneration after optic nerve injury in zebrafish. After axon injury in the peripheral nervous system, Jun is necessary for functional axonal regeneration and has been shown to heterodimerize to regulate gene expression. However, it is not known if these same binding partners interact with Jun during CNS regeneration. To investigate this, we have created a fusion protein of Jun tethered to one of the most recent generations of proximity labeling ligases, APEX2. Unlike traditional methods to investigate protein-protein interactions (PPIs), proximity labeling can also detect transient interactions, such as post-translational modifications performed by kinases, as well as stable binding seen in transcription complex formation. Using the nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells as a model, the PPIs of Jun were identified during the early stage of axon extension using proximity labeling, followed by mass spectrometry. Elucidating the PPIs of Jun will aid in developing new therapeutic approaches in human CNS regeneration.