NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats.

BACKGROUND: Inflammatory cytokines play a crucial role in the pathophysiology of traumatic brain injury (TBI), exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. NNZ-2566, a synthetic analogue of the neuroprotective tripeptide Glypromate, has been shown to be neuroprotective in animal models of brain injury. The goal of this study was to determine the effects of NNZ-2566 on inflammatory cytokine expression and neuroinflammation induced by penetrating ballistic-like brain injury (PBBI) in rats. METHODS: NNZ-2566 or vehicle (saline) was administered intravenously as a bolus injection (10 mg/kg) at 30 min post-injury, immediately followed by a continuous infusion of NNZ-2566 (3 mg/kg/h), or equal volume of vehicle, for various durations. Inflammatory cytokine gene expression from the brain tissue of rats exposed to PBBI was evaluated using microarray, quantitative real time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA) array. Histopathology of the injured brains was examined using hematoxylin and eosin (H&E) and immunocytochemistry of inflammatory cytokine IL-1beta. RESULTS: NNZ-2566 treatment significantly reduced injury-mediated up-regulation of IL-1beta, TNF-alpha, E-selectin and IL-6 mRNA during the acute injury phase. ELISA cytokine array showed that NZ-2566 treatment significantly reduced levels of the pro-inflammatory cytokines IL-1beta, TNF-alpha and IFN-gamma in the injured brain, but did not affect anti-inflammatory cytokine IL-6 levels. CONCLUSION: Collectively, these results suggest that the neuroprotective effects of NNZ-2566 may, in part, be functionally attributed to the compound's ability to modulate expression of multiple neuroinflammatory mediators in the injured brain.

The role of tissue transglutaminase in cell-matrix interactions.

Numerous studies over the last two decades revealed a complexity and multiple functions of tissue transglutaminase (tTG or TG2, EC 2.3.2.13). Besides the ability to catalyze Ca2+-dependent transamidation of proteins and formation of protein polymers via protease-resistant covalent isopeptide bonds, tTG also possesses GTPase enzymatic activity which links this protein to certain intracellular signaling pathways. Moreover, in addition to cytoplasmic and nuclear localization, a significant part of the protein pool is present on the cell surface. A number of recent findings indicate that surface tTG is involved in the interactions of cells with the surrounding extracellular matrix (ECM). In this review we will focus on the newly defined non-enzymatic adhesive function of tTG in cell-matrix interactions and discuss contributions of previously characterized enzymatic activities of tTG to cell-matrix adhesion and adhesion-dependent processes. Understanding molecular interactions and enzymatic activities of tTG will gain further insights into the role of this protein in normal human physiology and various pathological conditions.

Amino acid starvation induced autophagic cell death in PC-12 cells: evidence for activation of caspase-3 but not calpain-1.

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis.