title: 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.
authors.affiliation: Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. casandra.cartagena@us.army.mil
authors.forename: Casandra M
authors.initials: CM
authors.lastname: Cartagena
authors.forename: Katie L
authors.initials: KL
authors.lastname: Phillips
authors.forename: Garry L
authors.initials: GL
authors.lastname: Williams
authors.forename: Melissa
authors.initials: M
authors.lastname: Konopko
authors.forename: Frank C
authors.initials: FC
authors.lastname: Tortella
authors.forename: Jitendra R
authors.initials: JR
authors.lastname: Dave
authors.forename: Kara E
authors.initials: KE
authors.lastname: Schmid
mesh_terms: D000818:Animals; D008297:Male; D051381:Rats; D051700:Activating Transcription Factor 3 / Q000096:biosynthesis / Q000235:genetics; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology / Q000627:therapeutic use; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects; D020197:Head Injuries, Penetrating / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use; D009842:Oligopeptides / Q000494:pharmacology / Q000627:therapeutic use; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D005971:Glutamates
chemical_list: D051700:Activating Transcription Factor 3; D000894:Anti-Inflammatory Agents, Non-Steroidal; C497108:Atf3 protein, rat; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D009842:Oligopeptides; D012333:RNA, Messenger; C000656362:trofinetide; D005971:Glutamates