Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 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 (PACAP) 38, PACAP27, and its internal peptide, Gly-IlePhe (GIF; PACAP4 -6), are neuroprotective at 10 Ϫ13 M against lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity, as determined by [ 3 H]DA uptake and the number of tyrosine hydroxylase-immunoreactive neurons. PACAP38 and GIF also protected against 1-methyl-4-phenylpyridinium ϩ -induced neurotoxicity but only in cultures containing microglia. PACAP38 and GIF ameliorated the production of microgliaderived 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 Ϫ/Ϫ ), where PACAP38 and GIF reduced tumor necrosis factor ␣ production and were neuroprotective only in PHOX ϩ/ϩ cultures and not in PHOX Ϫ/Ϫ cultures. Pretreatment with PACAP6 -38 (3 M; PACAP-specific receptor antagonist) was unable to attenuate PACAP38, PACAP27, or GIF (10 Ϫ13 M) neuroprotection. PACAP38 and GIF (10 Ϫ13 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.Parkinson's disease (PD) is characterized by the progressive and selective degeneration of dopaminergic (DA) neurons in the substantia nigra, but the etiology and the precise mechanisms underlying the selective destruction of the nigrostriatal dopaminergic pathway are unknown. Recently, increasing evidence from clinical (McGeer et al., 1988) and animal studies (Castano et al., 1998;Sriram et al., 2002) suggests the involvement of inflammation in the pathogenesis of PD. Inflammation in the brain is characterized by the activation of microglia (Aloisi, 1999), the resident innate immune cells in the central nervous system. Microglia are activated by a diverse list of environmental (particulate matter, pesticides, heavy metals) and endogenous triggers (␣-synuclein, ␤-amyloid) to produce reactive oxygen species (ROS) and/or proinflammatory factors, which are toxic to

Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

“…The reason for the cotransfection of p22 phox with gp91 phox is that p22 phox facilitates the stable expression of gp91 phox in the membrane (Yu et al, 1997). In the empty vector-transfected COS-7 cells, no significant specific binding was observed, as the total binding and the nonspecific binding displayed very similar linear curves (Fig.…”

PACAP38 increases vesicular monoamine transporter 2 (VMAT2) expression and attenuates methamphetamine toxicity

“…Among its roles as a neurotransmitter and determinant of neuronal phenotype, it has several general neuroprotective actions: decreased oxidative stress, increased output of neurotrophic compounds by astrocytes, prevention of inflammation, and the attenuation of apoptosis (Delgado, et al 2002, Vaudry, et al 2002, Delgado, et al 2003, Vaudry, et al 2003, Reglodi, et al 2004a, Yang, et al 2006, Masmoudi-Kouki, et al 2007). In reference to midbrain dopamine pathways, PACAP38 has demonstrated neuroprotective properties in the 6-hydroxydopamine model of Parkinson's disease and PAC1, the receptor with the highest affinity for PACAP38, is present in high concentration in the nigrostriatal cell bodies and terminals (Vaudry, et al 2000, Reglodi, et al 2004b).…”

“…PACAP38 and a related peptide, PACAP27, along with VIP have been shown to activate three G protein coupled receptors: PAC1, which has much higher affinity for PACAP variants, and VPAC1/VPAC2 which bind PACAP variants and VIP (Vaudry, et al 2000, Fahrenkrug 2006. PACAP38 has been shown to have several roles in vertebrates such as a neurotransmitter, neuroprotectant, enzyme activity regulator and activator of neural stem cells (Arimura 1998, Hamelink, et al 2002, Mercer, et al 2004, Somogyvari-Vigh and Reglodi 2004, Dejda, et al 2005, Shioda, et al 2006, Yang, et al 2006, Bobrovskaya, et al 2007.…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…The antiapoptotic effect of PACAP38 after ischemia is indirect and involves IL-6 release . Some of the neuroprotective effects of PACAP38 may also result from an inhibition of microglial activation (Delgado, 2002;Delgado et al, 2002a;Lee and Suk, 2004;Suk et al, 2004;Yang et al, 2006). After focal cerebral ischemia, the tumor suppressor protein p53 and the zinc finger protein Zac-1 (two genes controlling growth arrest and apoptosis) are up-regulated (Gillardon et al, 1998;Ciani et al, 1999).…”