Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs

Abstract

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

Figure 1. Genotoxic stress-induced loss of intervertebral disc proteoglycan matrix

A, Safranin O and fast green staining of disc sections of IR-treated Ercc1^−/Δ mice and their Wt littermates. Red, proteoglycan staining. Arrows indicate the NP region of the disc. The black bars represent 100 μm B, Quantitation of NP GAG. GAG levels from nucleus puloposus tissues of IR-treated and untreated mice were measured using 1,9-dimethylmethylene assay against GAG standards (chondroitin-6-sulfate) and normalized to total DNA content. * = p < 0.05 from six independent assays. C, Coronal sections of mouse discs were immunostained for aggrecan (brown) and counterstained with aqueous hematoxylin and blue Scott’s tap water. Decreased immunodetection of nucleus pulposus aggrecan was observed in Wt and Ercc1^−/Δ mice treated with either IR or MEC. The bar represents 100 μm.

Figure 2. Chronic exposure to IR and MEC decreased new matrix protein synthesis in intervertebral discs

Proteoglycan synthesis as measured by ³⁵S-sulfate incorporation in intervertebral discs of unexposed mice (C) and mice exposed to mechlorethamine (MEC) or ionizing radiation (IR) to induce nuclear DNA damage. * = p < 0.05 from six independent assays (3 mice each analyzed in duplicate).

Figure 3. Effects of IR and MEC exposure on cellular senesecence in mouse intervetebral discs

A, immunohistochemical detection of p16^INK4a, a senescence marker, to distinguish senescent (brown, arrow) from non-senescent (blue) cells. B, Quantitation of the percent p16^INK4a immunopositive cells in the nucleus pulposus of unexposed mice (C) and mice exposed to mechlorethamine (MEC) or ionizing radiation (IR). Top graph, Wt mice. Bottom graph, Ercc1^−/Δ mice. * = p < 0.05 from nine random fields.

Figure 4. Enhanced apoptosis in intervertebral discs of IR- and MEC-exposed mice

A, TUNEL assay to identify apoptotic cells (green) in disc tissue. Insets, nuclear DAPI stain (blue) to reveal tissue cellularity. B, Quantitation of the percent TUNEL positive cells in the annulus fibrosus of unexposed mice (C) and mice exposed to mechlorethamine (MEC) or ionizing radiation (IR). Top graph, Wt mice. Bottom graph, Ercc1^−/Δ mice. * = p < 0.05 from nine random fields.

Fig. 5. Immunohistochemical detection of aggrecanase and aggrecan degradation products

Sagittal disc sections were analyzed using an anti-ADAMTS4 antibody (top) and antibodies recognizing the C-terminal neo-epitope generated by aggrecanase cleavage in the interglobular domain. ADAMTS4 protein expression (reddish brown, arrow) was dramatically up-regulated throughout the nucleus puloposus of both Wt and Ercc1^−/Δ mice following treatment of IR or MEC. MEC, but not IR exposure, also induced aggrecanase-mediated proteolysis of aggrecan (reddish brown, arrows), as evident by increased immunodetection of the aggrecan NVTEGE neo-epitope.

Figure 6. Local ionizing radiation exposure induced loss of intervertebral disc proteoglycan matrix

Safranin O and fast green staining of sections of discs isolated from the lumbar and tail region of IR-exposed Wt mice. Red, proteoglycan staining. The black bars represent 100 μm.