NeuroMolecular Medicine

, Volume 15, Issue 3, pp 504–514 | Cite as

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

  • Casandra M. CartagenaEmail author
  • Katie L. Phillips
  • Garry L. Williams
  • Melissa Konopko
  • Frank C. Tortella
  • Jitendra R. Dave
  • Kara E. Schmid
Original Paper


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.


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



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.

Conflict of interest

No competing financial interests exist.

Supplementary material

12017_2013_8236_MOESM1_ESM.tif (27.1 mb)
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)
12017_2013_8236_MOESM2_ESM.tif (4.4 mb)
Supplimental Figure 2. Confocal Microscopy of ATF3 and OX42 protein expression in Microglia 24-h post-injury. (Bar = 50 μm). (TIFF 4491 kb)
12017_2013_8236_MOESM3_ESM.tif (5.2 mb)
Supplimental Figure 3. Confocal Microscopy of ATF3 and CD3 protein expression in T Cells 24-h post-injury. (Bar = 50 μm). (TIFF 5362 kb)
12017_2013_8236_MOESM4_ESM.tif (3.7 mb)
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)
12017_2013_8236_MOESM5_ESM.tif (4.2 mb)
Supplimental Figure 5. Confocal Microscopy of ATF3 and GFAP protein expression in Astrocytes 24-h post-injury. (Bar = 50 μm). (TIFF 4294 kb)
12017_2013_8236_MOESM6_ESM.tif (1.8 mb)
Supplimental Figure 6. Confocal Microscopy of ATF3 and NeuN protein expression in Neurons 24-h post-injury. (Bar = 50 μm). (TIFF 1827 kb)


  1. Amiry-Moghaddam, M., Otsuka, T., Hurn, P. D., Traystman, R. J., Haug, F. M., Froehner, S. C., et al. (2003). An alpha-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain. Proceedings of the National Academy of Sciences of the United States of America, 100(4), 2106–2111.PubMedCrossRefGoogle Scholar
  2. Bickerdike, M. J., Thomas, G. B., Batchelor, D. C., Sirimanne, E. S., Leong, W., Lin, H., et al. (2009). NNZ-2566: A Gly-Pro-Glu analogue with neuroprotective efficacy in a rat model of acute focal stroke. Journal of the Neurological Sciences, 278(1–2), 85–90.PubMedCrossRefGoogle Scholar
  3. Bienvenu, T. C., Busti, D., Magill, P. J., Ferraguti, F., & Capogna, M. (2012). Cell-type-specific recruitment of amygdala interneurons to hippocampal theta rhythm and noxious stimuli in vivo. [Research Support, Non-U.S. Gov’t]. Neuron, 74(6), 1059–1074.PubMedCrossRefGoogle Scholar
  4. Byram, S. C., Serpe, C. J., Pruett, S. B., Sanders, V. M., & Jones, K. J. (2003). Natural killer cells do not mediate facial motoneuron survival after facial nerve transection. Brain, Behavior, and Immunity, 17(6), 417–425.PubMedCrossRefGoogle Scholar
  5. Cartagena, C. M., Ahmed, F., Burns, M. P., Pajoohesh-Ganji, A., Pak, D. T., Faden, A. I., et al. (2008). Cortical injury increases cholesterol 24S hydroxylase (Cyp46) levels in the rat brain. Journal of Neurotrauma, 25(9), 1087–1098.PubMedCrossRefGoogle Scholar
  6. Chen, B. P., Liang, G., Whelan, J., & Hai, T. (1994). ATF3 and ATF3 delta Zip. Transcriptional repression versus activation by alternatively spliced isoforms. Journal of Biological Chemistry, 269(22), 15819–15826.PubMedGoogle Scholar
  7. Chen, Y., & Swanson, R. A. (2003). Astrocytes and brain injury. Journal of Cerebral Blood Flow and Metabolism, 23(2), 137–149.PubMedGoogle Scholar
  8. Dong, Y., & Benveniste, E. N. (2001). Immune function of astrocytes. Glia, 36(2), 180–190.PubMedCrossRefGoogle Scholar
  9. Efficacy and Safety Study of Intravenous Progesterone in Patients with Severe Traumatic Brain Injury (SyNAPSe). (2010). BHR Pharma, LLC. Accessed July 9, 2012 from
  10. Erythropoietin in Traumatic Brain Injury (EPO-TBI). (2009). Australian and New Zealand Intensive Care Research Centre. Accessed July 9, 2012 from
  11. Fauriat, C., Long, E. O., Ljunggren, H. G., & Bryceson, Y. T. (2010). Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood, 115(11), 2167–2176.PubMedCrossRefGoogle Scholar
  12. Francis, J. S., Dragunow, M., & During, M. J. (2004). Over expression of ATF-3 protects rat hippocampal neurons from in vivo injection of kainic acid. Brain Research. Molecular Brain Research, 124(2), 199–203.PubMedCrossRefGoogle Scholar
  13. Gilchrist, M., Thorsson, V., Li, B., Rust, A. G., Korb, M., Roach, J. C., et al. (2006). Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature, 441(7090), 173–178.PubMedCrossRefGoogle Scholar
  14. Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S. Y., Gielen, A. W., Muhallab, S., et al. (2000). Neuroprotection by encephalomyelitis: Rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. Journal of Neuroscience, 20(14), 5283–5291.PubMedGoogle Scholar
  15. Harty, J. T., Tvinnereim, A. R., & White, D. W. (2000). CD8+ T cell effector mechanisms in resistance to infection. Annual Review of Immunology, 18, 275–308.PubMedCrossRefGoogle Scholar
  16. Israelsson, C., Bengtsson, H., Kylberg, A., Kullander, K., Lewen, A., Hillered, L., et al. (2008). Distinct cellular patterns of upregulated chemokine expression supporting a prominent inflammatory role in traumatic brain injury. Journal of Neurotrauma, 25(8), 959–974.PubMedCrossRefGoogle Scholar
  17. Lau, L. T., & Yu, A. C. (2001). Astrocytes produce and release interleukin-1, interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury. Journal of Neurotrauma, 18(3), 351–359.PubMedCrossRefGoogle Scholar
  18. Litvak, V., Ramsey, S. A., Rust, A. G., Zak, D. E., Kennedy, K. A., Lampano, A. E., et al. (2009). Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals. Nature Immunology, 10(4), 437–443.PubMedCrossRefGoogle Scholar
  19. Lu, X. C., Chen, R. W., Yao, C., Wei, H., Yang, X., Liao, Z., et al. (2009a). NNZ-2566, a glypromate analog, improves functional recovery and attenuates apoptosis and inflammation in a rat model of penetrating ballistic-type brain injury. Journal of Neurotrauma, 26(1), 141–154.PubMedCrossRefGoogle Scholar
  20. Lu, X. C., Hartings, J. A., Si, Y., Balbir, A., Cao, Y., & Tortella, F. C. (2011). Electrocortical pathology in a rat model of penetrating ballistic-like brain injury. Journal of Neurotrauma, 28(1), 71–83.Google Scholar
  21. Lu, X. C., Si, Y., Williams, A. J., Hartings, J. A., Gryder, D., & Tortella, F. C. (2009b). NNZ-2566, a glypromate analog, attenuates brain ischemia-induced non-convulsive seizures in rats. Journal of Cerebral Blood Flow and Metabolism, 29(12), 1924–1932.PubMedCrossRefGoogle Scholar
  22. Massi, L., Lagler, M., Hartwich, K., Borhegyi, Z., Somogyi, P., & Klausberger, T. (2012). Temporal dynamics of parvalbumin-expressing axo-axonic and basket cells in the rat medial prefrontal cortex in vivo. [Research Support, Non-U.S. Gov’t]. Journal of Neuroscience, 32(46), 16496–16502.PubMedCrossRefGoogle Scholar
  23. Matsumoto, Y., Kohyama, K., Aikawa, Y., Shin, T., Kawazoe, Y., Suzuki, Y., et al. (1998). Role of natural killer cells and TCR gamma delta T cells in acute autoimmune encephalomyelitis. European Journal of Immunology, 28(5), 1681–1688.PubMedCrossRefGoogle Scholar
  24. Mazzeo, A. T., Kunene, N. K., Gilman, C. B., Hamm, R. J., Hafez, N., & Bullock, M. R. (2006). Severe human traumatic brain injury, but not cyclosporin a treatment, depresses activated T lymphocytes early after injury. Journal of Neurotrauma, 23(6), 962–975.PubMedCrossRefGoogle Scholar
  25. Myer, D. J., Gurkoff, G. G., Lee, S. M., Hovda, D. A., & Sofroniew, M. V. (2006). Essential protective roles of reactive astrocytes in traumatic brain injury. Brain, 129(Pt 10), 2761–2772.PubMedCrossRefGoogle Scholar
  26. Natale, J. E., Ahmed, F., Cernak, I., Stoica, B., & Faden, A. I. (2003). Gene expression profile changes are commonly modulated across models and species after traumatic brain injury. Journal of Neurotrauma, 20(10), 907–927.PubMedCrossRefGoogle Scholar
  27. Perussia, B. (1996). The cytokine profile of resting and activated NK cells. Methods, 9(2), 370–378.PubMedCrossRefGoogle Scholar
  28. Raivich, G., Jones, L. L., Kloss, C. U., Werner, A., Neumann, H., & Kreutzberg, G. W. (1998). Immune surveillance in the injured nervous system: T-lymphocytes invade the axotomized mouse facial motor nucleus and aggregate around sites of neuronal degeneration. Journal of Neuroscience, 18(15), 5804–5816.PubMedGoogle Scholar
  29. Rosenberger, C. M., Clark, A. E., Treuting, P. M., Johnson, C. D., & Aderem, A. (2008). ATF3 regulates MCMV infection in mice by modulating IFN-gamma expression in natural killer cells. Proceedings of the National Academy of Sciences of the United States of America, 105(7), 2544–2549.PubMedCrossRefGoogle Scholar
  30. Saura, J., Curatolo, L., Williams, C. E., Gatti, S., Benatti, L., Peeters, C., et al. (1999). Neuroprotective effects of Gly-Pro-Glu, the N-terminal tripeptide of IGF-1, in the hippocampus in vitro. Neuroreport, 10(1), 161–164.PubMedCrossRefGoogle Scholar
  31. Schousboe, A., & Waagepetersen, H. S. (2005). Role of astrocytes in glutamate homeostasis: Implications for excitotoxicity. Neurotoxicity Research, 8(3–4), 221–225.PubMedCrossRefGoogle Scholar
  32. Schroeter, M., & Jander, S. (2005). T-cell cytokines in injury-induced neural damage and repair. Neuromolecular Medicine, 7(3), 183–195.PubMedCrossRefGoogle Scholar
  33. Seijffers, R., Allchorne, A. J., & Woolf, C. J. (2006). The transcription factor ATF-3 promotes neurite outgrowth. Molecular and Cellular Neuroscience, 32(1–2), 143–154.PubMedCrossRefGoogle Scholar
  34. Stoica, B., Byrnes, K., & Faden, A. I. (2009). Multifunctional drug treatment in neurotrauma. Neurotherapeutics, 6(1), 14–27.PubMedCrossRefGoogle Scholar
  35. Stow, J. L., Low, P. C., Offenhauser, C., & Sangermani, D. (2009). Cytokine secretion in macrophages and other cells: Pathways and mediators. Immunobiology, 214(7), 601–612.PubMedCrossRefGoogle Scholar
  36. Study of NNZ-2566 in Patients with Traumatic Brain Injury (INTREPID2566). (2008). Neuren Pharmaceuticals. Accessed July 9, 2012 from
  37. Tambuyzer, B. R., Ponsaerts, P., & Nouwen, E. J. (2009). Microglia: Gatekeepers of central nervous system immunology. Journal of Leukocyte Biology, 85(3), 352–370.PubMedCrossRefGoogle Scholar
  38. Thompson, M. R., Xu, D., & Williams, B. R. (2009). ATF3 transcription factor and its emerging roles in immunity and cancer. Journal of Molecular Medicine (Berlin, Germany), 87(11), 1053–1060.CrossRefGoogle Scholar
  39. Vianney-Rodrigues, P., Iancu, O. D., & Welsh, J. P. (2011). Gamma oscillations in the auditory cortex of awake rats. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov’t]. European Journal of Neuroscience, 33(1), 119–129.PubMedCrossRefGoogle Scholar
  40. Wei, H. H., Lu, X. C., Shear, D. A., Waghray, A., Yao, C., Tortella, F. C., et al. (2009). NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats. Journal of Neuroinflammation, 6, 19.PubMedCrossRefGoogle Scholar
  41. Whitmore, M. M., Iparraguirre, A., Kubelka, L., Weninger, W., Hai, T., & Williams, B. R. (2007). Negative regulation of TLR-signaling pathways by activating transcription factor-3. Journal of Immunology, 179(6), 3622–3630.Google Scholar
  42. Williams, A. J., Hartings, J. A., Lu, X. C., Rolli, M. L., Dave, J. R., & Tortella, F. C. (2005). Characterization of a new rat model of penetrating ballistic brain injury. Journal of Neurotrauma, 22(2), 313–331.PubMedCrossRefGoogle Scholar
  43. Williams, A. J., Hartings, J. A., Lu, X. C., Rolli, M. L., & Tortella, F. C. (2006a). Penetrating ballistic-like brain injury in the rat: Differential time courses of hemorrhage, cell death, inflammation, and remote degeneration. Journal of Neurotrauma, 23(12), 1828–1846.PubMedCrossRefGoogle Scholar
  44. Williams, A. J., Ling, G. S., & Tortella, F. C. (2006b). Severity level and injury track determine outcome following a penetrating ballistic-like brain injury in the rat. Neuroscience Letters, 408(3), 183–188.PubMedCrossRefGoogle Scholar
  45. Williams, A. J., Wei, H. H., Dave, J. R., & Tortella, F. C. (2007). Acute and delayed neuroinflammatory response following experimental penetrating ballistic brain injury in the rat. Journal of Neuroinflammation, 4, 17.PubMedCrossRefGoogle Scholar
  46. Zhang, S. J., Buchthal, B., Lau, D., Hayer, S., Dick, O., Schwaninger, M., et al. (2011). A signaling cascade of nuclear calcium-CREB-ATF3 activated by synaptic NMDA receptors defines a gene repression module that protects against extrasynaptic NMDA receptor-induced neuronal cell death and ischemic brain damage. Journal of Neuroscience, 31(13), 4978–4990.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York (outside the USA) 2013

Authors and Affiliations

  • Casandra M. Cartagena
    • 1
    Email author
  • Katie L. Phillips
    • 1
  • Garry L. Williams
    • 1
  • Melissa Konopko
    • 1
  • Frank C. Tortella
    • 1
  • Jitendra R. Dave
    • 1
  • Kara E. Schmid
    • 1
  1. 1.Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and NeuroscienceWalter Reed Army Institute of ResearchSilver SpringUSA

Personalised recommendations