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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Poly(ADP-ribose) polymerase-1 promotes microglial activation, proliferation, and matrix metalloproteinase-9-mediated
Tiina M Kauppinen1, Raymond A Swanson
1Department of Neurology, University of California, and Veterans Affairs Medical Center, San Francisco, CA 94121, USA.
Abstract:
Activated microglia contribute to cell death in ischemic and neurodegenerative disorders of the CNS. Microglial activation is regulated in part by NF-kappaB, and the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) enhances NF-kappaB binding to DNA. In this study, the role of PARP-1 in microglia-mediated neurotoxicity was assessed using microglia from wild-type (wt) and PARP-1-/- mice. Cultured microglia were incubated with TNF-alpha, a cytokine that is up-regulated in many neurological disorders. When stimulated with TNF-alpha, wt microglia proliferated, underwent morphological changes characteristic of activation, and killed neurons placed in coculture. The effects of TNF-alpha were markedly attenuated both in PARP-1-/- microglia and in wt microglia treated with the PARP enzymatic inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2h)-isoquinolinone. These effects were also blocked by (E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile, which inhibits translocation of NF-kappaB to the nucleus. TNF-alpha also up-regulated microglial release of matrix metalloproteinase-9 (MMP-9), an enzyme with potential neurotoxic properties that is transcriptionally regulated by NF-kappaB. This up-regulation was blocked in PARP-1-/- microglia and in wt microglia by the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2h)-isoquinolinone. Microglia from MMP-9-/- mice were used to evaluate the contribution of MMP-9 to microglial neurotoxicity. MMP-9-/- microglia treated with TNF-alpha showed substantially reduced neurotoxicity relative to the wt microglia. TNF-alpha-stimulated wt microglia treated with the MMP inhibitor ilomastat also showed reduced neurotoxicity. These findings suggest that PARP-1 activation is required for both TNF-alpha-induced microglial activation and the neurotoxicity resulting from TNF-alpha-induced MMP-9 release.
Insights
Poly(ADP-ribose) polymerase-1 (PARP-1) activation drives microglial neurotoxicity by increasing NF-kappaB and matrix metalloproteinase-9 (MMP-9) release. Inhibiting PARP-1 or MMP-9 reduces this cell death in neurological disorders.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Activated microglia contribute to central nervous system (CNS) cell death in neurodegenerative and ischemic disorders.
- Microglial activation is regulated by nuclear factor-kappa B (NF-kappaB), with poly(ADP-ribose) polymerase-1 (PARP-1) enhancing NF-kappaB DNA binding.
Purpose of the Study:
- To investigate the role of PARP-1 in microglia-mediated neurotoxicity.
- To determine the involvement of matrix metalloproteinase-9 (MMP-9) in PARP-1-driven microglial activation and neurotoxicity.
Main Methods:
- Utilized microglia from wild-type (wt), PARP-1-/-, and MMP-9-/- mice.
- Stimulated microglia with tumor necrosis factor-alpha (TNF-alpha) and assessed cell proliferation, morphology, and neuronal toxicity.
- Administered PARP inhibitors (3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2h)-isoquinolinone) and NF-kappaB inhibitors ((E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile).
- Measured MMP-9 release and used MMP inhibitors (ilomastat).
Main Results:
- TNF-alpha-induced wt microglial activation and neurotoxicity were significantly attenuated in PARP-1-/- microglia and when treated with PARP or NF-kappaB inhibitors.
- TNF-alpha-stimulated MMP-9 release was blocked in PARP-1-/- microglia and by PARP inhibition.
- Mice lacking MMP-9 or treated with an MMP inhibitor exhibited reduced microglial neurotoxicity.
Conclusions:
- PARP-1 activation is essential for TNF-alpha-induced microglial activation.
- PARP-1 mediates neurotoxicity through the induction of MMP-9 release.
- Targeting PARP-1 or MMP-9 may offer therapeutic strategies for neurological disorders involving microglial activation.
