ADAMTS4 and ADAMTS5 knockout mice are protected from versican but not aggrecan or brevican proteolysis during spinal cord injury

Abstract

The chondroitin sulfate proteoglycans (CSPGs) aggrecan, versican, and brevican are large aggregating extracellular matrix molecules that inhibit axonal growth of the mature central nervous system (CNS). ADAMTS proteoglycanases, including ADAMTS4 and ADAMTS5, degrade CSPGs, representing potential targets for ameliorating axonal growth-inhibition by CSPG accumulation after CNS injury. We investigated the proteolysis of CSPGs in mice homozygous for Adamts4 or Adamts5 null alleles after spinal cord injury (SCI). ADAMTS-derived 50-60 kDa aggrecan and 50 kDa brevican fragments were observed in Adamts4-/-, Adamts5-/-, and wt mice but not in the sham-operated group. By contrast Adamts4-/- and Adamts5-/- mice were both protected from versican proteolysis with an ADAMTS-generated 70 kDa versican fragment predominately observed in WT mice. ADAMTS1, ADAMTS9, and ADAMTS15 were detected by Western blot in Adamts4-/- mice' spinal cords after SCI. Immunohistochemistry showed astrocyte accumulation at the injury site. These data indicate that aggrecan and brevican proteolysis is compensated in Adamts4-/- or Adamts5-/- mice by ADAMTS proteoglycanase family members but a threshold of versican proteolysis is sensitive to the loss of a single ADAMTS proteoglycanase during SCI. We show robust ADAMTS activity after SCI and exemplify the requirement for collective proteolysis for effective CSPG clearance during SCI.

Figure 1

Aggrecan cleavage in Adamts4−/− and Adamts5−/− mice following SCI. Aggrecan fragments (~50 and 60 kDa) detected with the anti-NITEGE antibody are seen in wild-type, Adamts4−/−, and Adamts5−/− mice 1 day after SCI (arrows, top panel). Lower molecular weight species (~25 kDa) are also seen in most groups (asterisks, top panel). No aggrecanase activity was detected in sham-operated mice. β-Actin was used as a loading control (bottom panel).

Figure 2

Versican cleavage in Adamts4−/− and Adamts5−/− mice following SCI. Versican fragments (~70 kDa) detected with the anti-DPEAAE antibody are seen in wild-type but not Adamts4−/− and Adamts5−/− mice 7 days after SCI (top panel, arrow). No versicanase activity was detected in sham-operated mice. β-Actin was used as a loading control (bottom panel).

Figure 3

Brevican cleavage in Adamts4−/− and Adamts5−/− mice following SCI. Full-length brevican (~145 kDa) and brevican fragments (~50 kDa) detected with the anti-brevican antibody are seen in wild-type, Adamts4−/−, and Adamts5−/− mice 1 and 7 days after SCI (arrows). All groups showed diminished brevican cleavage at 7 days after injury. Minimal brevicanase activity was detected in sham-operated mice.

Figure 4

ADAMTS proteoglycanases are expressed during SCI. (a) ADAMTS1 is expressed in spinal cord tissue of Adamts4−/− mice 1 and 7 days after injury, m = mature, asterisk = autocatalytic fragment. (b) ADAMTS9 is expressed in spinal cord tissue of Adamts4−/− mice in the sham-operated group 1 and 7 days after injury, z = zymogen (inactive), asterisks = uncharacterized fragments. (c) ADAMTS15 is expressed in spinal cord tissue of Adamts4−/− mice in the sham-operated group 1 and 7 days after injury, z = zymogen (inactive), asterisk = uncharacterized fragments. In all cases β-actin was used as a loading control (bottom panels). (d) Spinal cord injury triggered astrocyte accumulation (reactive astrocytes) at the injury site compared with sham-operated mice. Red = astrocyte, blue = nucleus. Scale bar = 50 μm.