Alterations in mRNA expression and protein products following spinal cord injury in humans

Abstract

We examined the effects of spinal cord injury (SCI) on alterations in gene expression and respective protein products in human skeletal muscle 2 days and 5 days post-SCI. Biopsies were taken from skeletal muscle of 9 men and 1 woman (n = 10) (43.9 +/- 6.7 years) 2 days and 5 days post-SCI and from 5 healthy young men who served as controls (20.4 +/- 0.5 years). Global changes in gene expression were analysed using Affymetrix GeneChips on a subsample of subjects (n = 3). Candidate genes were then pursued via qRT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes. Immunohistochemistry (IHC) was used to localize proteins. Groups of transcripts showing the greatest percentage of altered expression, the most robust fold-changes, and indicative of involvement of an entire pathway using the GeneChip included genes involved in the ubiquitin proteasome pathway (UPP), metallothionein function, and protease inhibition. qRT-PCR analysis confirmed increases in gene expression for UPP components (UBE3C, Atrogin-1, MURF1, and PSMD11), the metallothioneins (MT1A, MT1F, MT1H), and the protease inhibitor, SLPI (P < 0.05) at 2 days and 5 days post-SCI. Protein levels of the proteasome subunit (PSMD11) and the metallothioneins were increased 5 days post-SCI. Protein levels of UBE3C, Atrogin-1, MURF1 and SLPI were unchanged (P > 0.05). IHC showed increased staining for PSMD11 and the metallothioneins 5 days post-SCI, along the peripheral region of the cells. IHC also showed altered staining for Atrogin-1 at 5 days post-SCI along the membrane region. Thus, there was a profound increase in gene expression of UPP components, the metallothioneins, and the protease inhibitor, SLPI, within 5 days of SCI. Increased protein levels for PSMD11 and the metallothioneins 5 days post-SCI, specifically along the cell periphery, indicate that proteins in this region may be early targets for degradation post-SCI.

Figure 1

Global changes in gene expression at two and five days post-SCI: microarray analysis Candidate genes grouped according to biological function based on GO criteria (https://www.affymetrix.com/analysis/netaffx/go_analysis_netaffx4.affx). Genes were considered candidate genes if they met the following criteria: (chronically up- or downregulated at two and five days post-SCI, P < 0.01, fold-change > 1.5).

Figure 3

Western blotting for E3 ligases A–C, Western blotting results for the E3 ligases (A: Atrogin-1; B: MURF1; C: UBE3C). Protein levels for Atrogin-1, MURF1, and UBE3C were not different at two (D2 Leg) or five (D5 Leg) days post-SCI compared to healthy controls (P > 0.05). Representative blots are below each graph and in each case, the control value has been assigned the value 100%. Values represent means ±s.e.m.

Figure 2

Western blotting for PSMD11 Protein levels of PSMD11 increased ∼20% five days post-SCI compared to healthy controls (P < 0.001) and compared to two days post-SCI (P < 0.05). A representative blot (4 controls, 4 SCI patients day 2 (D2 Leg), and 3 SCI patients Day 5 (D5 Leg)) is below the graph and the control value has been assigned the value 100%. Values represent means ± standard error (s.e.m.). *P < 0.05 versus control and two days post-SCI. Arrow indicates approximate molecular weight (kDa) of the band of interest, according to protein standard.

Figure 4

Western blotting for metallothionein I and II Metallothionein protein levels were not changed two days post-SCI (D2 Leg), but by five days post-SCI (D5 Leg), protein levels increased ∼17% compared to healthy controls (P < 0.05) and ∼18% five days post-SCI compared to two days post-SCI (P < 0.05). A representative blot is below the graph and the control value has been assigned the value 100%. Values represent means ±s.e.m.*P < 0.05 versus control and two days post-SCI.

Figure 5

Western blotting for SLPI Protein levels for SLPI were not different at two (D2 Leg) or five (D5 Leg) days post-SCI compared to healthy controls (P > 0.05). A representative blot is below the graph and the control value has been assigned the value 100%. Values represent means ±s.e.m.

Figure 6

Western blotting for GAPDH GAPDH was used as a loading control each time a protein was analysed using Western blotting. To do this, membranes were stripped and reprobed for antibodies specific for GAPDH. Representative blot illustrates that equal amounts of protein were loaded per lane, and that protein levels for GAPDH were not affected by SCI. The control value has been assigned the value 100%. Values represent means ±s.e.m.

Figure 7

Immunohistochemistry for PSMD11 A (control), B (two days post-SCI) and C (five days post-SCI) are representative images of muscle biopsies stained for nuclei (blue), dystrophin (green) and PSMD11 (red). The last panel in each row (A–C) is the merged image for nuclei, dystrophin and PSMD11. White arrows indicate the most intense region of staining for PSMD11. Scale bar equals 50 μm.

Figure 8

Immunohistochemistry for Atrogin-1 A (control), B (two days post-SCI) and C (five days post-SCI) are representative images of muscle biopsies stained for nuclei (blue), dystrophin (green) and Atrogin-1 (red). The last panel in each row (A–C) is the merged image for nuclei, dystrophin and Atrogin-1. White arrows indicate the most intense region of staining for Atrogin-1. Scale bar equals 50 μm.

Figure 9

Immunohistochemistry for metallothionein I and II Merged representative images of muscle biopsies (control, two days post-SCI, and five days post-SCI, respectively) stained for nuclei (blue) and metallothionein (green). White arrows indicate the most intense region of staining for metallothionein. Scale bar equals 50 μm.

Figure 10

Immunohistochemistry for SLPI A (control), B (two days post-SCI) and C (five days post-SCI) are representative images of muscle biopsies stained for nuclei (blue), dystrophin (green) and SLPI (red). The last panel in each row (A–C) is the merged image for nuclei, dystrophin and SLPI. White arrows indicate the most intense region of staining for SLPI at five days post-SCI. Scale bar equals 50 μm.