Over the past year, we have completed several components of the specific aims in the original. A) Demonstrated an effective model of inflammation-induced HIV replication in microglia. In order to begin to test these hypotheses in microglia, we had to first establish an effective model of HIV transcription (LTR activity) in microglia. The pLTR luc construct was made by subcloning the 720 bp Xho I-HindIII containing the HIV LTR from the pNL4-3 HIV-1 proviral DNA plasmid into pGL3-Luc basic. BV2 cells, a murine microglia cell line, were transiently transfected with pLTR-luc and pRL-null using lipofectamine and treated 24 hours later. Results showed that BV2 cells transfected with pLTR-luc had a dose dependent increase in LTR activity in response to LPS, as determined by firefly luciferase assay. This data suggests that while difficult to transfect, microglia can be used to discern the effects of neuroprotective compounds on HIV-LTR activity. B) Showed that dynorphin inhibits HIV-LTR activity in a human monocyte cell line. U38 cells are a human monocyte cell line that has been stably transfected with the HIV-LTR promoter. HIV-LTR activity in this transfected cell line can be detected with the chloramphenicol acetyltransferase (CAT) ELISA. Our results show that femtomolar concentrations of dynorphin can inhibit LPS-induced LTR activity in human monocytes, as determined by CAT assay. These data suggest that the properties of dynorphin that provide the neuroprotective, anti-inflammatory and anti-HIV-LTR activity extend to other species, with emphasis on humans. C) Discerned components of the signaling and the transcription factors required for LPS- induced HIV-LTR activity in microglia. In order to understand how dynorphin and other neuroprotective compounds are inhibiting HIV-LTR activity, the basic mechanisms of LPS-induced activation of HIV-LTR activity in the microglia needed to be defined. We determined that in microglia transfected with the pLTR luc construct, p38 MAP kinase was critical for LPS-induced activation of the HIV LTR. Specifically, the p38 kinase inhibitor SB202190 significantly inhibited LPS-induced HIV-LTR activity, as determined by luciferase activity assay. Further, using mutated HIV-LTR constructs obtained from the Aids Research and Reference Reagent Program, we were able to discern what domain of the LTR was critical of LPS-induced HIV-LTR activity. Results showed that the mutation -105/-81, which abrogates the NF(kappa)B domain, was the only construct to show any significance difference in HIV-LTR activity in response to LPS, when compared to the full length construct. With the NF(kappa)B domain absent, the LTR-HIV response to LPS was abolished. D) Identified the smallest peptide fragment of dynorphin required for anti-inflammatory and neuroprotective effects. After testing several peptide fragments of dynorphin, it was determined that any sequence containing gly-gly-phe (GGF) was both anti-inflammatory and neuroprotective. We show that both gly-gly-phe (GGF), a tri-peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS-induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Previously, we have identified that NADPH oxidase contributes to neurotoxicity through two mechanisms. First, activation of NADPH oxidase results in the production of neurotoxic ROS. Second, NADPH oxidase activation increases intracellular ROS in the phagocyte, resulting in amplification of pro-inflammatory factors. However, in the case of HAD therapy, attenuating NADPH oxidase may provide an additional benefit of reducing the activation of the LTR promoter, helping to attenuate the vicious cycle oof microglial activation. Further, the targeting of NADPH oxidase may allow a selective inhibition of the activation of phagocytic cells, a characteristic of great importance to an immune system that is already comprised with T cell immunodeficiency.
| Li, Guorong; Cui, Gang; Tzeng, Nian-Ssheng et al. (2005) Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage. FASEB J 19:489-96 |
| Qin, Liya; Liu, Yuxin; Qian, Xun et al. (2005) Microglial NADPH oxidase mediates leucine enkephalin dopaminergic neuroprotection. Ann N Y Acad Sci 1053:107-20 |
| Qin, Liya; Block, Michelle L; Liu, Yuxin et al. (2005) Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress. FASEB J 19:550-7 |
