Neuroprotective Effects of Chaperonin Containing TCP1 Subunit 2 (CCT2) on Motor Neurons Following Oxidative or Ischemic Stress

Chaperonin containing TCP1 (CCT) is an essential protein that controls proteostasis following spinal cord damage. In particular, CCT2 plays an important role in neuronal death in various neurological disorders; however, few studies have investigated the effects of CCT2 on ischemic damage in the spinal cord. In the present study, we synthesized a cell-permeable Tat-CCT2 fusion protein and observed its effects on H₂O₂-induced oxidative damage in NSC34 motoneuron-like cells and in the spinal cord after ischemic injury. Tat-CCT2, but not its control protein CCTs, was delivered into NSC34 cells in a concentration- and incubation time-dependent manner, and a clear cytosolic location of the delivered protein was observed. In addition, the delivered protein gradually degraded, and nearly control levels were observed 24 h after Tat-CCT2 treatment. Tat-CCT2 treatment significantly ameliorated 200 µM H₂O₂-induced neuronal damage in NSC34 cells at 8.0 µM protein treatment. Additionally, Tat-CCT2 significantly ameliorated H₂O₂-induced reactive oxygen species formation and DNA fragmentation. In the rabbit spinal cord, Tat-CCT2 was efficiently delivered into the spinal cord 4 h after 0.125 mg/kg protein treatment. In addition, treatment with Tat-CCT2 significantly improved the neurological scores based on the Tarlov criteria 24 and 72 h after ischemia/reperfusion. Moreover, the number of surviving neurons in the ventral horn of the spinal cord was significantly increased in the Tat-CCT2-treated group 3 and 7 days after ischemia compared to vehicle-treated group. Treatment with Tat-CCT2 alleviated the ischemia-induced oxidative stress and ferroptosis-related factor (malondialdehyde, 8-iso-prostaglandin F2α, and high mobility group box 1) and pro-inflammatory cytokine (interleukin-1β, interleukin-6, and tumor necrosis factor-α) releases in the ventral horn of the spinal cord 8 and 24 h after ischemia/reperfusion. In addition, Tat-CCT2 treatment significantly ameliorated ischemia-induced microglial activation in the ventral horn of spinal cord 24 h after reperfusion. These results suggest that Tat-CCT2 mitigates ischemia-induced neuronal damage in the spinal cord.