Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration

Abstract

Aging is a main risk factor for intervertebral disc (IVD) degeneration, the main cause of low back pain. FOXO transcription factors are important regulators of tissue homeostasis and longevity. Here, we determined the expression pattern of FOXO in healthy and degenerated human IVD and the associations with IVD degeneration during mouse aging. FOXO expression was assessed by immunohistochemistry in normal and degenerated human IVD samples and in cervical and lumbar IVD from 6-, 12-, 24-, and 36-month-old C57BL/6J mice. Mouse spines were graded for key histological features of disc degeneration in all the time points and expression of two key FOXO downstream targets, sestrin 3 (SESN3) and superoxide dismutase (SOD2), was assessed by immunohistochemistry. Histological analysis revealed that FOXO proteins are expressed in all compartments of human and mouse IVD. Expression of FOXO1 and FOXO3, but not FOXO4, was significantly deceased in human degenerated discs. In mice, degenerative changes in the lumbar spine were seen at 24 and 36 months of age whereas cervical IVD showed increased histopathological scores at 36 months. FOXO expression was significantly reduced in lumbar IVD at 12-, 24-, and 36-month-old mice and in cervical IVD at 36-month-old mice when compared with the 6-month-old group. The reduction of FOXO expression in lumbar IVD was concomitant with a decrease in the expression of SESN3 and SOD2. These findings suggest that reduced FOXO expression occurs in lumbar IVD during aging and precedes the major histopathological changes associated with lumbar IVD degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2682-2691, 2017.

Keywords: FOXO; aging; intervertebral disc.

Figure 1

FOXO expression in human intervertebral discs (IVD). A) Representative images of FOXO1, −3, and −4 immunohistochemical staining in the nucleus pulposus and annulus fibrosus of lumbar IVD from normal donors and donors with disc degeneration (n=3 per group). Magnification bar = 100 μm. B) Quantification of FOXO1, −3, and −4 immunopositive cells in IVD from normal donors and donors with disc degeneration (n=3 per group). Data is presented as percentage of positive cells. All values are mean ± 95% CI.

Figure 2

Age-related changes in mouse cervical intervertebral discs (IVD). A) Representative images of cervical IVD from 6, 12, 24, and 36-month-old mice (n=6 per group) stained with safranin-O. Magnification bar = 100 μm. B) Histopathological scores in the nucleus pulposus/annulus fibrosus (NP/AF) and endplates (EP) of cervical IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). C) Measurement of total disc height in cervical IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). All values are mean ± 95CI. D) Quantification of cellularity in the NP and AF of cervical IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). E) Quantification of NP area occupied by NP cells in cervical IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). All values are mean ± 95% CI.

Figure 3

Age-related changes in mouse lumbar intervertebral discs (IVD). A) Representative images of lumbar IVD from 6, 12, 24, and 36-month-old mice (n=8 per group) stained with safranin-O. Magnification bar = 100 μm. B) Histopathological scores in the nucleus pulposus/annulus fibrosus (NP/AF) and endplates (EP) of lumbar IVD from 6, 12, 24, and 36-month-old mice (n=8 per group). C) Measurement of total disc height in lumbar IVD from 6, 12, 24, and 36-month-old mice (n=8 per group). D) Quantification of cellularity in the NP and AF of lumbar IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). E) Quantification of NP area occupied by NP cells in lumbar IVD from 6, 12, 24, and 36-month-old mice (n=6 per group). All values are mean ± 95% CI.

Figure 4

Changes in FOXO expression in mouse cervical intervertebral discs (IVD) during aging. A) Representative images of FOXO1 and FOXO3 immunohistochemical staining in cervical IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). Magnification bar = 50 μm. B–C) Quantification of FOXO1 and FOXO3 immunopositive cells in the nucleus pulposus/annulus fibrosus (NP/AF) (B) and endplates (EP) (C) of cervical IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). All values are mean ± 95% CI.

Figure 5

Changes in FOXO expression in mouse lumbar intervertebral discs (IVD) during aging. A) Representative images of FOXO1 and FOXO3 immunohistochemical staining in lumbar IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). Magnification bar = 50 μm. B–C) Quantification of FOXO1 and FOXO3 immunopositive cells in the nucleus pulposus/annulus fibrosus (NP/AF) (B) and endplates (EP) (C) of lumbar IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). All values are mean ± 95% CI.

Figure 6

Changes in SESN3 and SOD2 expression in mouse intervertebral discs (IVD) during aging. A) Representative images of SESN3 and SOD2 immunohistochemical staining in lumbar IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). Magnification bar = 50 μm. B) Quantification of SESN3 and SOD2 immunopositive cells in the nucleus pulposus of lumbar IVD from 6, 12, 24, and 36-month-old mice (n=5 per group). All values are mean ± 95% CI.