Traumatic brain injury (TBI) is an incredibly heterogenous pathology, yet many of the current mechanisms under exploration involve a common phenotype, traumatic axonal injury (TAI), and a commonly overlooked sensor, the Endoplasmic Reticulum (ER). TBI research shows that there are immediate and prolonged disturbances in calcium signaling, oxidative stress, metabolic deficits, lipid synthesis, and protein folding – all of which rely on, or effected by, the ER. Neuronal ER is unique in that it is the largest organelle with one continuous membrane. It adapts to the changing demands of its environment be that for protein synthesis, calcium storage, or inter-organelle communication. While the ER is equipped with a stress fighting response, the Unfolded Protein Response (UPR), this pathway can either adapt to stress or initiate apoptosis. Accordingly, studies attempting to successfully balance ER stress are met with mixed results. Variability has been attributed to the involvement of other stress pathways, interventional windows, variation in injury type/severity, etc. Taking a new perspective, the hypothesis of this thesis proposes that the inconsistency of ER stress interventions is less about inter-cellular responses and more about intra-cellular differences. Thus, I hypothesized that ER stress, and its shared mechanisms, would vary between cell soma and axon.
Using a closed-head weight drop mouse model of TBI that induces axonal injury to the optic nerve, termed traumatic optic neuropathy, this body of work reveals the importance of the ER as it relates to axon injury responses. To start, we characterized the cell loss, degeneration, and functional deficits associated with our model followed by confirmation of both acute and chronic ER stress (Chapter 2). We then utilized three interventions to determine the response of the cell soma compared to the axon (i.e., rough ER versus smooth ER respectively). First, we used an indirect change to the oxidative environment of the eye/brain (i.e., brief oxygen exposure; Chapter 3). From these data, the PERK pathway of the UPR was most sensitive. So, we compared two previously tested drugs that affect PERK’s downstream phosphorylation target eIF2a (Chapter 4). Chapters 3 and 4 revealed the importance of ER sensitivity to redox imbalance, which was confirmed in chapter 5, as well as distinct responses to changes in ER stress based not only on soma versus axon but also when examining proximal axon versus the distal degenerating axon. Finally, because chapters 3 and 4 lacked visualization, antibody specificity, and clarity on the location of ER stress, chapter 5 utilized fluorescence microscopy to further pinpoint where, and in which cells, ER stress occurred. Altogether, this work confirmed that somatic ER responds differently from axonal ER explaining some of the variability in other TBI literature. Most interestingly, though, this work revealed a larger importance for a particular ER stress molecule, ATF4, and its potentially non-genomic role in glial cells around the location of axonal injury that warrants future studies to clarify the multicellular importance of ER stress and atypical expression of ATF4.