Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP4-6 are neuroprotective through inhibition of NADPH oxidase: Potent regulators of microglia-mediated oxidative stress

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 (PACAP) 38, 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 1-methyl-4-phenylpyridinium⁺-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 tumor necrosis factor α production and were neuroprotective only in PHOX^+/+ cultures and not in PHOX^-/- cultures. Pretreatment with PACAP6-38 (3 μM; PACAP-specific receptor antagonist) was unable to attenuate PACAP38, PACAP27, or GIF (10^-13 M) neuroprotection. PACAP38 and GIF (10^-13 M) failed to induce cAMP in neuronglia 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 subpicomolar-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.