Alterations in mRNA and Protein Following Spinal Cord Injury in Humans

Abstract

PURPOSE: There is a rapid loss of muscle mass following spinal cord injury (SCI). There are no known treatment options available to attenuate losses in muscle mass, given the lack of understanding of how SCI affects signaling pathways within the musculoskeletal system in humans. This work expanded on a preliminary microarray study that explored global changes in gene expression to identify pathways affected by SCI. The purpose of this work was two fold: 1) To examine alterations in mRNA for components of the ubiquitin proteasome pathway (UPP), metallothioneins (MTs) and an inflammatory response gene (secretory leukocyte protease inhibitor: SLPI) 2d and 5d post-SCI and 2) To examine alterations in protein levels for components of the AKT and UPP pathway, MTs, and SLPI. METHODS: Biopsies were taken from the vastus lateralis muscle of 5 patients 2d and 5d post-SCI. Global changes in gene expression were analyzed using Affymetrix gene chips. mRNA levels of candidate genes selected from this analysis were confirmed via quantitative Real Time Polymerase Chain Reaction (qRT-PCR). When alterations in mRNA were confirmed via qRT-PCR, Western blotting (WB) was used to assess protein products of candidate genes and components of the AKT pathway. Immunohistochemistry (IHC) was then used to confirm alterations in protein products observed via WB. RESULTS: 50 genes met inclusion criteria for gene chip analysis (fold-change >2.0; p <0.001; chronically up or down regulated at 2d and 5d). UPP components (UBE2E, PSMD11, Atrogin-1) were upregulated 2.0 to 11.3-fold. MTs (1F, 1H, 1A) were upregulated 3.9 to 20.7-fold, and SLPI was upregulated 44-fold. qRT-PCR confirmed gene chip results. qRT-PCR was used to explore the expression of MuRF-1, a component of the UPP shown to be dramatically upregulated in animals post-SCI. MuRF-1 expression was increased 4.9–6.5-fold. WB revealed that total AKT was unchanged but AKTser473 was decreased at 2d post-SCI (34%, p<0.05) but not 5d. MT and PSMD11 protein increased, 16 and 18% respectively, by 5d post-SCI. IHC analysis confirmed WB results. CONCLUSIONS: This work identifies specific signaling pathways that are affected in the first days post-SCI, likely contributing to losses in muscle mass in humans. The observed temporal pattern of change in mRNA and protein suggest that following SCI, there is an initial stress (decreased AKTser473, increased MT) and inflammatory response (SLPI), which is followed by increased activity of the UPP. These alterations are important biomarkers for future work investigating ways to manipulate these pathways post-SCI to attenuate losses in muscle mass. Acknowledgements: Christopher Reeve Paralysis Foundation.