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

Abstract

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.

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Acknowledgments

We would like to thank Matthew Bombard and Weihong Yang for their excellent assistance with surgical procedures. These studies were supported in part by a cooperative research and development agreement with Neuren Pharmaceuticals Ltd. (W81XWH-05-0074). This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or reflecting true views of the Department of the Army or the Department of Defense.

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No competing financial interests exist.

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Correspondence to Casandra M. Cartagena.

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Supplimental Figure 1. Hematoxylin and eosin staining of sham (upper panel) and PBBI (lower panel) coronal sections 24-h post-injury. (Bar = 2 mm). (TIFF 27772 kb)

Supplimental Figure 2. Confocal Microscopy of ATF3 and OX42 protein expression in Microglia 24-h post-injury. (Bar = 50 μm). (TIFF 4491 kb)

Supplimental Figure 3. Confocal Microscopy of ATF3 and CD3 protein expression in T Cells 24-h post-injury. (Bar = 50 μm). (TIFF 5362 kb)

Supplimental Figure 4. Confocal Microscopy of ATF3 and NKC protein expression in Natural Killer Cells 24-h post-injury. (Bar = 50 μm). (TIFF 3768 kb)

Supplimental Figure 5. Confocal Microscopy of ATF3 and GFAP protein expression in Astrocytes 24-h post-injury. (Bar = 50 μm). (TIFF 4294 kb)

Supplimental Figure 6. Confocal Microscopy of ATF3 and NeuN protein expression in Neurons 24-h post-injury. (Bar = 50 μm). (TIFF 1827 kb)

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Cartagena, C.M., Phillips, K.L., Williams, G.L. et al. Mechanism of Action for NNZ-2566 Anti-inflammatory Effects Following PBBI Involves Upregulation of Immunomodulator ATF3. Neuromol Med 15, 504–514 (2013). https://doi.org/10.1007/s12017-013-8236-z

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Keywords

  • NNZ-2566
  • Penetrating ballistic-like brain injury
  • Traumatic brain injury