Poster Z19 - Involvement of the endoplasmic reticulum stress response in stretch-grown dorsal root ganglion axons

Introduction: Peripheral nerve injuries (PNI) are a common cause of long-term disability in the United States with severe outcomes leading to chronic pain and a loss of sensory and motor function. This creates an impaired quality of life and a large social and economic burden with no current treatments able to restore full functional abilities. Past research for treatment of PNI focus on the first stage of growth, growth-cone mediated growth, however, due to its peak rate at 1 mm/day in humans it is not fast enough to treat larger PNI. The second phase of growth, termed “stretch-growth,” can sustain a faster growth rate i.e., 4 mm/day, making it a possible treatment option for larger PNI. In recent in vitro and in vivo studies, the endoplasmic reticulum’s (ER) stress response, termed the unfolded protein response (UPR), has been shown to play a beneficial role in axon regeneration, however, the role it plays in stretch-growth remains unknown. One of the UPRs signaling pathways is activated by the transmembrane protein, Protein Kinase RNA (PKR)-like ER kinase (PERK), and its downstream signaling effects, such as the phosphorylation of eukaryotic initiation factor 2 alpha (P-eIF2α), have been seen to promote axonal regeneration. This pathway also leads to the upregulation of activating transcription factor 3 (ATF3) which is seen to be upregulated in all PNI and modulates the transcriptional response to injury by creating an intrinsic growth state.

Materials and

Methods: Primary DRG neurons will be isolated from rat embryos taken from an E16 Sprague-Dawley pregnant female rat and seeded onto a custom-built elongator. They will be incubated at 37°𝐶 and 5% CO2 in static conditions for 5 days by growth cone to establish connections on aclar substrates. On DIV 5, axons will begin pretension for 24 hours, and at DIV 6 will be ramped from 1 mm/day to 4 mm/day, as previously established to support stretch-growth, in an incubator at 37°𝐶 and 5% CO2. DRG neurons will be seeded in Neurobasal Plus medium, 2% B-27, 0.5 mM L-glutamine 1% gentamicin, 20 ng/mL Nerve Growth Factor, 20 𝜇𝑀 FdU, and 20 𝜇𝑀 Uridine with media changes every 2-3 days. At DIV 12 the cell somas and axons will be extracted in RIPPA buffer with a protease inhibitor and prepared for a western blot. A western blot will be performed for P-PERK, P-eIF2α, and ATF3. The bands will be standardized and quantified for expression. Statistical analysis will be conducted using one-way ANOVA with Tukey’s post-hoc analysis (p < 0.05).

Results, Conclusions, and Discussions:

Results: To determine if the UPR is responding to stretch, previously published RNA microarray data following the same experimental protocol was reexamined to determine activation of the PERK pathway. These data show that eIF2α and ATF3 are significantly upregulated after stretch with a fold change average of 1.6 (p <.001) and 3.2 (p <.0001), respectively. Preliminary data using a western blot and immunohistochemistry was used to confirm the protein expression of eIF2α and ATF3. A western blot of eIF2α confirmed protein expression at DIV 12 (Fig. 1). Immunohistochemistry investigating the protein expression of eIF2α (Fig. 2) and ATF3 (Fig. 3) showed a brighter fluorescence at DIV 12 compared to non-stretched controls. Following the completion of the western blots looking at the protein expression of stretch-growth compared to controls, the blots will be standardized using a loading control of β-actin to normalize the protein expression and determine any variance during loading. Densiometric quantification will be done using a Bio-Rad GS800 calibrated densitometer and will be analyzed using Bio-Rad Quantity One 1D software.

Conclusions: RNA microarray data shows that eIF2α and ATF3 are significantly upregulated at DIV 12, suggesting that the PERK pathway of the ER stress response may be active during stretch-growth. Preliminary data using immunohistochemistry and western blots to examine protein expression illustrate an increase eIF2α and ATF3 compared to controls, coinciding with PNI literature. Following the completion of the western-blots, the statistical difference in the PERK pathways protein expression will be quantified after stretch-growth. This work will support our hypothesis that the unfolded protein response of the PERK pathway is activated during stretch-growth. Future work includes inhibiting the PERK pathway to investigate the ability of the axons to stretch and quantifying changes in protein expression.

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