Mechanism of Action for NNZ-2566 Anti-inflammatory Effects Following PBBI Involves Upregulation of Immunomodulator ATF3

The tripeptide glycine–proline–glutamate analogue NNZ-2566 (Neuren Pharmaceuticals) demonstrates neuroprotective efficacy in models of traumatic brain injury. In penetrating ballistic-like brain injury (PBBI), it significantly decreases injury-induced upregulation of inflammatory cytokines including TNF-α, IFN-γ, and IL-6. However, the mechanism by which NNZ-2566 acts has yet to be determined. The activating transcription factor-3 (ATF3) is known to repress expression of these inflammatory cytokines and was increased at the mRNA and protein level 24-h post-PBBI. This study investigated whether 12 h of NNZ-2566 treatment following PBBI alters atf3 expression. PBBI alone significantly increased atf3 mRNA levels by 13-fold at 12 h and these levels were increased by an additional fourfold with NNZ-2566 treatment. To confirm that changes in mRNA translated to changes in protein expression, ATF3 expression levels were determined in vivo in microglia/macrophages, T cells, natural killer cells (NKCs), astrocytes, and neurons. PBBI alone significantly increased ATF3 in microglia/macrophages (820 %), NKCs (58 %), and astrocytes (51 %), but decreased levels in T cells (48 %). NNZ-2566 treatment further increased ATF3 protein expression in microglia/macrophages (102 %), NKCs (308 %), and astrocytes (13 %), while reversing ATF3 decreases in T cells. Finally, PBBI increased ATF3 levels by 55 % in neurons and NNZ-2566 treatment further increased these levels an additional 33 %. Since increased ATF3 may be an innate protective mechanism to limit inflammation following injury, these results demonstrating that the anti-inflammatory and neuroprotective drug NNZ-2566 increase both mRNA and protein levels of ATF3 in multiple cell types provide a cellular mechanism for NNZ-2566 modulation of neuroinflammation following PBBI.