Received for publication February 7, 2006.
Revised August 3, 2006.
Accepted for publication August 3, 2006.

PACAP38 AND PACAP4-6 ARE NEUROPROTECTIVE THROUGH INHIBITION OF NADPH OXIDASE: POTENT REGULATORS OF MICROGLIA-MEDIATED OXIDATIVE STRESS

Abstract

Microglial activation is implicated in the progressive nature of numerous neurodegenerative diseases, including Parkinson's disease. Using primary rat mesencephalic neuron-glia cultures, we found that pituitary adenylate cyclase activating polypeptide (PACAP38), PACAP27, and its internal peptide Gly-Ile-Phe (GIF, PACAP4-6) are neuroprotective at 10^-13 M against lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity, as determined by [³H] DA uptake and the number of tyrosine hydroxylase-immunoreactive neurons. PACAP38 and GIF also protected against MPP⁺-induced neurotoxicity, but only in cultures containing microglia. PACAP38 and GIF ameliorated the production of microglia-derived reactive oxygen species (ROS), where both LPS- and phorbol 12-myristate 13-acetate-induced superoxide and intracellular ROS were inhibited. The critical role of NADPH oxidase for GIF and PACAP38 neuroprotection against LPS-induced DA neurotoxicity was demonstrated using neuron-glia cultures from mice deficient in NADPH oxidase (PHOX^-/-), where PACAP38 and GIF reduced TNF production and were neuroprotective in only in PHOX^+/+ cultures and not in PHOX^-/- cultures. Pre-treatment with PACAP6-38 (3µM; PAC1 receptor antagonist) was unable to attenuate PACAP38, PACAP27, or GIF (10^-13M) neuroprotection. PACAP38 and GIF (10^-13M) failed to induce cAMP in neuron-glia cultures, supporting that the neuroprotective effect was independent of traditional high affinity PACAP receptors. Pharmacophore analysis revealed that GIF shares common chemical properties (hydrogen bond acceptor, positive ionizable, and hydrophobic regions) with other sub-picomolar acting compounds known to inhibit NADPH oxidase: naloxone, dextromethorphan, and gly-gly-phe. These results indicate a common high-affinity site of action across numerous diverse peptides and compounds, revealing a basic neuropeptide regulatory mechanism that inhibits microglia-derived oxidative stress and promotes neuron survival.

Key words: Dopaminergic Neuron, Microglia, NADPH oxidase, Neuroprotection, PACAP, Tripeptide