Studies on the Mechanism of Polyamine-and Prothrombin kringle-2-induced Degeneration of Neuron and Microglia

Abstract

Neural cell death is the defining feature of all neurodegenerative diseases and is the underlying cause of many functional deficits. Clarifying the mechanism of cell death in the nervous system is essential to understand disease pathology and to devise effective treatment strategies. Increasing evidence has linked chronic inflammation to a number of neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD). In the central nervous system, microglia, the resident innate immune cells play major role in inflammatory process. Microglia may directly toxic to neurons by releasing various substances such as inflammatory cytokines. Thus, it is pathophysiologically important to understand how the extent and duration of brain inflammation is controlled in vivo. In addition to down-regulation of inflammatory mediators, the extent and duration of inflammation in the CNS may be controlled by the removal of activated microglia. Therefore, this study shows the degeneration mechanism of dopaminergic neuron and the rolls of activated microglia to provocation or prevention of neuronal cell death.

Recently polyamines have been also implicated in cell death. I examined whether polyamines, endogenous ligand for transient receptor potential vanilloid 1 (TRPV1), could mediate 6-hydroxydopamine (6-OHDA)-induced Mesencephalic Dopaminergic (DA) Neurons In vivo and In Vitro. Intranigral injection of the 6-OHDA into the rat brain, or treatment of rat mesencephalic cultures with 6-OHDA, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was prevented by the -difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesizing enzyme ornithine decarboxylase (ODC), suggesting the direct involvement of Polyamines in neurotoxicity. TRPV1 antagonist capsazepine (CZP) reversed 6-OHDA-or polyamines-induced loss of DA neurons in vivo and in vitro, indicating TRPV1-mediated neurotoxicity. Western blot analysis also showed that pretreatment with DFMO reversed 6-OHDA-induced downregulation of phosphorylated Akt and upregulation of cleaved caspase-3 in mesencephalic cultures. This result is the first to show that 6-OHDA-polyamines-TRPV1 signaling pathway exerts neurotoxicity on dopaminergic neurons in vivo and in vitro. Prothrombin kringle-2 acts as an proinflammtory agent that activates microglia to release pro-inflammatory mediators. Intracortical injection of pKr-2 caused significant loss of cortical neurons in vivo after seven days, as evident from Nissl staining and immunohistochemical analysis using the neuronal-specific nuclear protein (NeuN) antibody. In parallel, pKr-2-activated microglia and ROS production were observed in rat cortex displaying degeneration of cortical neurons. Reverse transcription-PCR at various time points after pKr-2 administration disclosed early and transient expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, such as interleukin 1β (IL-1β). Co-localization of iNOS, IL-1β, and TNF-α within microglia was evident with double-label immunohistochemistry. Additionally, pKr-2 induced upregulation of cytosolic components of NADPH oxidase (p67phox), translocation of cytosolic p67phox protein to the membrane, and p67phox expression in microglia in the cortex in vivo, signifying NADPH oxidase activation. The pKr-2-induced oxidation of proteins and loss of cortical neurons were partially inhibited by DPI, an NADPH oxidase inhibitor, and trolox, an antioxidant. Consistent with my hypothesis, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of cortical neurons and microglia, but not in microglia-free neuron-enriched cortical cultures, indicating that microglia are required for pKr-2 neurotoxicity. My results strongly suggest that pKr-2 as an endogenous compound participates in cortical neuron death through microglial NADPH oxidase-mediated oxidative stress. Therefore, inhibition of microglial NADPH oxidase activation may offer prospective clinical therapeutic benefit for neuroinflammation-related neurodegenerative disorders. How to minimize brain inflammation is pathophysiologically important, since inflammation induced by microglial activation can exacerbate brain damage. Thus, Death of activated microglia could act as a critical mechanism for the resolution of brain inflammation. Microglia cell death was detected at eight days after co-treatment of pKr-2 with IL-13/IL-4 in vitro. This cell death assessed by live and dead assay, TUNEL and MTT assay. Interestingly, superoxide assay, WST-1 show significantly increased reactive oxygen species (ROS) in combination of pKr-2 and IL-13 or IL-4 treated microglia. Additionally, the IL-13/IL-4-enhanced ROS were partially inhibited by an NADPH oxidase inhibitor. The IL-13/IL-4 induced cell death of microglia were partially inhibited by an NADPH oxidase inhibitor and by an antioxidant. Therefore, I hypothesized that the effects of IL-13/IL-4 and NADPH oxidase-derived ROS production may be linked to death of activated microglia. Moreover, Western blot analysis show significantly increased Cyclooxygenase-2 (COX-2) expression in combination of pKr-2 and IL-13 or IL-4 treated microglia. The IL-13/IL-4-enhanced COX-2 expression were partially inhibited by an NADPH oxidase inhibitor and an antioxidant. Additional studies demonstrated that microglia cell death was reversed by treatment with NADPH oxidase inhibitor, antioxidant, and COX-2 inhibitor. To my knowledge, This result is the first to demonstrate that IL-13/IL-4 induced cell death of pKr-2 activated microglia is mediated by oxidative stress and COX-2 through NADPH oxidase.