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NEUROPHARMACOLOGY
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (S.Y., J.Y., Z.Y., A.F., W.Z., H.P., X.G., B.W., J.-S.H., M.L.B.); and Institute of Basic Medical Sciences and Department of Psychiatry, College of Medicine, National Cheng Kung University, Tainan, Taiwan (P.C.)
Received February 6, 2006; accepted August 3, 2006.
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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.
) 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 neurons (Block and Hong, 2005). DA neurons are more vulnerable to oxidative damage compared with other cell types (Jenner, 1996), and microglia are the predominant source of inflammation and oxidative stress. Thus, inhibition of microglial activation and production of extracellular superoxide are critical to DA neuron survival. There is increasing support that neuropeptides are components of a critical homeostatic mechanism regulating the inflammatory milieu in the brain (Block et al., 2006). However, the details of the homeostatic mechanisms regulating microglial function are poorly defined.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is widely distributed in the peripheral and central nervous system, where PACAP release is reported to serve as a neuronal survival factor (Arimura et al., 1994; Arimura, 1998). PACAP is a member of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) family and has the highest sequence similarity to VIP. However, PACAP is 1000 to 10,000 times more potent than VIP in pituitary cells, neurons, and astrocyte cultures (Arimura et al., 1994). PACAP exists in two amidated forms with 38 and 27 amino acid residues, designated PACAP38 and PACAP27, respectively (Miyata et al., 1989, 1990). In tissues, PACAP38 is the pre-dominant form, whereas PACAP27 constitutes less than 10% of the total PACAP (Arimura et al., 1991). PACAP is reported to have diverse functions because PACAP38 has been shown to act as a neurotransmitter/neuromodulator (Kozicz et al., 1997), a neuroprotectant (Uchida et al., 1996; Brenneman et al., 2002), and a neurotrophic factor (Arimura et al., 1994). Brenneman et al. (2002) reported that PACAP38 (10–12 to 10–15 M) is neuroprotective against human immunodeficiency virus envelope protein-induced neurotoxicity by reducing chemokine release. Previous studies from our laboratory also indicate that PACAP38 (10–12 to 10–14 M) can prevent lipopolysaccharide (LPS)-induced cortical neuron death through anti-inflammatory properties (Kong et al., 1999). Although PACAP is often reported to exert its effects through the PAC1 and VPAC2 receptors (Vaudry et al., 2000; Yamada et al., 2004), whether these traditional G-protein-coupled receptors regulate all of the multiple effects of PACAP at diverse concentrations remains unclear.
The potential therapeutic benefits of biologically active compounds at subpicomolar concentrations (Gozes et al., 1999; Wilkemeyer et al., 2003) is receiving wide attention. Recently, we identified a class of compounds that are neuroprotective at subpicomolar concentrations through the inhibition of NADPH oxidase (Qin et al., 2005a) and the consequent reduction of extracellular superoxide production. Previously, we reported that Gly-Gly-Phe (GGF), a tripeptide from dynorphin, is chemically and functionally similar to other subpicomolar-acting compounds, such as dextromethorphan (Li et al., 2005) and naloxone (Qin et al., 2005a). Although the detailed mechanisms are still being investigated, the high-affinity receptor mechanism is likely to be independent of traditional opiate receptors because both opiate agonists (dynorphin) and antagonists (naloxone) elicit anti-inflammatory and neuroprotective effects. However, other opiate peptides containing the GGF peptide sequence, such as leucine enkephalin and des-tyrosine leucine enkephalin, are also neuroprotective at subpicomolar concentrations through the inhibition of NADPH oxidase (Qin et al., 2005b), suggesting that this high-affinity site of action may be a common mechanism of microglia regulation.
In the current study, we tested whether subpicomolar concentrations of PACAP and the internal PACAP4–6 tripeptide Gly-Ile-Phe (GIF) fall into the class of neuropeptides that inhibit microglial activation at subpicomolar concentrations through NADPH oxidase. Here, we report for the first time that PACAP and its peptide fragment GIF are neuroprotective through inhibition of NADPH oxidase and consequent reduction of microglia-derived ROS. Furthermore, using pharmacophore analysis, we show that GIF has chemical similarities to other previously reported subpicomolar-acting compounds, suggesting a common high-affinity site of action and mechanism of microglia regulation for several neuropeptides.
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Materials and Methods |
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Reagents. PACAP38 was purchased from American Peptide Company (Sunnyvale, CA). PACAP27, PACAP6–38, and GIF were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Cell culture materials were obtained from Invitrogen (Grand Island, NY). The polyclonal anti-tyrosine hydroxylase (TH) antibody was a generous gift from Dr. John Reinhard (GlaxoSmithKline, Research Triangle Park, NC). The Vecta stain ABC kit and biotinylated secondary antibodies were purchased from Vector Laboratories (Burlingame, CA). WST-1 was purchased from Dojindo Laboratories (Gaithersburg, MD). Lipopolysaccharide (strain O111:B4) and 2',7'-dichlorofluorescein diacetate (DCF-DA) were obtained from Calbiochem (La Jolla, CA). [3H]DA (28 Ci/mmol) was purchased from NEN Life Science (Boston, MA). All other reagents came from Sigma (Minneapolis, MN).
Primary Mesencephalic Neuron-Glia Cultures. Neuron-glia cultures were prepared from the ventral mesencephalic tissues of embryonic days 13 to 14 rat and mice, as described previously (Liu and Hong, 2003). In brief, midbrain tissues were dissected from day 14 Fisher 344 rat or mouse embryos (PHOX+/+ or PHOX–/–). Cells were dissociated via gentle mechanical trituration in minimum essential medium and immediately seeded (5 x 105/well) in poly D-lysine (20 µg/ml)-precoated 24-well plates. Cells were seeded in maintenance media and exposed to the treatment medium described previously (Liu et al., 2001). Three days after seeding, the cells were replenished with 500 µl of fresh maintenance medium. Cultures were exposed 7 days after seeding. At the time of treatment, cells were pretreated for 30 min with vehicle or indicated concentration of PACAP/GIF in treatment media, followed by exposure to 2.5 ng/ml LPS.
Microglia-Enriched Cultures. Primary microglia-enriched cultures were prepared from the whole brain of 1-day-old Fisher 344 rat pups, using a previously described procedure (Liu et al., 2001). Cells were treated 24 h after seeding the microglia.
Microglia-Depleted Cultures. Primary microglia-depleted cultures were prepared as described previously (Wang et al., 2006) by adding 1 µM leucine methyl ester into the primary neuron-glia cultures 24 h after the initial seeding. The medium was changed 6 days later. Seven-day-old cultures were used for treatment. At the time of treatment, ICC analysis indicated that the microglial composition was <0.1%.
Dopamine Uptake Assay. DA uptake assays were performed as previously described (Liu et al., 2000b). In brief, after washing twice with warm Krebs-Ringer buffer (16 mM NaH2PO4, 119 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, and 1.3 mM EDTA, pH 7.4), cultures were incubated for 20 min at 37°C with 1 µM [3H]DA in Krebs-Ringer buffer for DA uptake. Afterward, cultures were washed three times with ice-cold Krebs-Ringer buffer, and cells were then dissolved in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific [3H]DA uptake determined in the presence of 10 µM mazindol was subtracted.
Immunostaining. DA neurons were recognized with the anti-TH antibody, as described previously (Liu et al., 2000a). In brief, formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogen peroxide followed by sequential incubation with a blocking solution (30 min), the primary antibody (overnight, 4°C), a biotinylated secondary antibody (2 h), and ABC reagents (40 min). Color was developed with 3,3'-diaminobenzidine. For morphological analysis, images were acquired using an inverted microscope (Nikon, Tokyo, Japan) connected to a camera (DAGE-MTI, Michigan City, IN) operated with MetaMorph software (Universal Imaging Corporation, Downingtown, PA). To quantify cell numbers, nine representative areas per well in the 24-well plate were counted under the microscope at 100x magnification by two individuals. The average of these two scores was reported.
Tumor Necrosis Factor Assay. Culture supernatants were collected after 3 h of stimulation with LPS. The concentration of tumor necrosis factor (TNF) was measured with a mouse TNF enzyme-linked immunosorbent assay kit from Genzyme (Cambridge, MA). The threshold for detection with this assay was approximately 15 pg/ml. The antiserum did not have detectable cross-reactivity with any other cytokines.
cAMP Assay. At 30 min after treatment with PACAP peptides, cells were lysed, and cAMP was measured, as described in the cAMP Parameter Assay Kit (R&D Systems, Minneapolis, MN).
Nitrite Oxide Assay. The production of nitric oxide (NO) was assessed as the accumulation of nitrite in the culture supernatants, using a colorimetric reaction with the Griess reagent. The culture supernatants were collected after 24 h of stimulation with LPS and mixed with equal volumes of the Griess reagent (0.1% N-[1-naphthyl] ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4). The absorbance at 540 nm was measured with a UV MAX kinetic microplate reader (Molecular Devices, Sunnyvale, CA). The nitrite concentration was determined from a sodium nitrite standard curve. The threshold for detection with this assay was approximately 0.5 µM.
Superoxide Assay. Extracellular superoxide production from microglia was determined as reported (Gao et al., 2003a; Qin et al., 2004) by measuring the superoxide dismutase (SOD)-inhibitable reduction of a tetrazolium salt, WST-1 (Qin et al., 2002). Primary rat microglia-enriched cultures were plated at 5 x 104/well in 200 µl of culture medium in 96-well plates and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were washed twice with Hanks' balanced salt solution (HBSS) and pretreated with PACAP or GIF for 30 min before adding 10 ng/ml LPS. To each well, 50 µl of peptide (final concentration, 10–13 M), 50 µl of HBSS with or without SOD (600 U/ml), 50 µl of vehicle or LPS (final concentration, 10 ng/ml), and 50 µl of WST-1 (final concentration, 1 mM) in HBSS were added. The cultures were incubated for 30 min at 37°C and in 5% CO2. The absorbance at 450 nm was read with a Spectra Max Plus microtiter plate spectrophotometer (Molecular Devices).
Intracellular Reactive Oxygen Species Assay. The production of intracellular reactive oxygen species was measured by DCFH oxidation. The DCF-DA reagent passively enters cells, where it is deacetylated by esterases to nonfluorescent DCFH. Inside the cell, DCFH reacts with ROS to form DCF, the fluorescent product. For this assay, 10 mM DCF-DA was dissolved in methanol and diluted 500-fold in HBSS to give 20 µM DCF-DA. Microglia-enriched cultures seeded (5 x 104/well) in 96-well plates were then exposed to DCFH-DA for 1 h, followed by treatment with HBSS containing several concentrations of LPS, GIF, PACAP, and LPS pretreated with either GIF or PACAP38. After incubation, the fluorescence was read at the 485-nm excitation and 530-nm emission on a fluorescence plate reader. Cell-free experiments with and without GIF and PACAP38 were conducted to determine that the reagents themselves did not alter fluorescence. To calculate the amount of intracellular ROS produced, the mean of the control treatment was subtracted from the mean of the experimental treatment.
Pharmacophore Analysis. The Catalyst version 4.9.1 software (Accelrys, San Diego, CA) was used for conformational and pharmacophore analysis. All molecules were built within the Catalyst Modeling software, and peptides were constructed as linear chains. The maximum number of conformers generated for each molecule was 250. Conformers with relative energy >20 kcal/mol were removed. The best (most rigorous and time-consuming) conformer generation method was used. The following chemical features were included in the development of the pharmacophore model: hydrogen bond acceptor, hydrogen bond donor, hydrophobic, negative charge or negative ionizable, positive ionizable, aromatic ring, and hydrophobic aromatic.
Small molecules and peptides previously determined to be neuro-protective through inhibition of NADPH oxidase at subpicomolar concentrations (naloxone, GGF, and dextromethorphan) were used to generate the 10 best pharmacophores. Each of the 10 best pharmacophores was evaluated based on the fit for naloxone, GGF, and dextromethorphan. The pharmacophore with the highest score and best fit for all three molecules was used as the reference for comparison to GIF.
Statistical Analysis. The data are expressed as the mean ± S.E.M., and statistical significance was assessed with an analysis of variance followed by Bonferroni's multiple comparisons. A value of P < 0.05 was considered statistically significant.
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), the lower concentrations of PACAP (10–13 and 10–14 M) showed greater neuroprotection, compared with the higher concentrations of 10–7 and 10–10 M (Fig. 1B). Thus, further analysis of neuroprotection and the associated mechanisms focused on the most effective concentration of 10–13 M.
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To identify whether GIF is an active neuroprotective peptide sequence of its parent peptide PACAP38, we determined the ability of subpicomolar concentrations of both peptides to protect against LPS-induced reduction of DA uptake and loss of DA neurons. Figure 2A shows that GIF (10–13 M) has similar neuroprotective effects on DA neurons against LPS-induced neurotoxicity in neuron-glia cultures compared with PACAP38 (10–13 M). GIF and PACAP38 (10–13 M) also showed a similar neuroprotection against the LPS-induced loss of TH-immunoreactive neurons (Fig. 2B). Morphological analysis revealed that both PACAP38 and GIF also protected DA neurons against LPS-induced morphological damage (Fig. 2C), such as the loss of dendrites, axon disintegration, and loss of DA neurons. However, IGF, a scrambled PACAP4–6 tripeptide, failed to protect DA neurons against LPS-induced neurotoxicity in neuron-glia cultures for either DA uptake or morphological damage compared with 10–13 M PACAP38 (data not shown). Taken together, these data indicate that the GIF tripeptide possesses the same neuroprotective efficacy as its parent peptide, PACAP38, at 10–13 M.
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), where a component of MPP+-induced DA neuron damage can be attributed to microglial activation (reactive microgliosis) (Gao et al., 2003b). We compared the ability of PACAP38 and GIF (10–13 M) to protect DA neurons against MPP+-induced neurotoxicity in mixed neuron-glia cultures and in microglia-depleted cultures (containing only neurons and astrocytes). PACAP38 and GIF were only able to protect against MPP+ in the presence of microglia (Fig. 2D), supporting that these peptides attenuate microglial activation and reactive microgliosis.
PACAP38 and GIF Inhibit Microglial Activation. LPS-induced microglial activation and the consequent production of proinflammatory factors has been linked to DA neurotoxicity (Gao et al., 2002). To discern the effect of PACAP and GIF on the release of proinflammatory factors from microglia, the LPS-induced production of TNF (3 h) and NO (24 h) was measured in mesencephalic neuron-glia cultures pretreated for 30 min with GIF or PACAP38. The results show that pretreatment with GIF or PACAP38 at 10–13 M reduces both TNF and NO production in response to LPS, compared with LPS (Fig. 3, A and B). Together, these results suggest that subpicomolar concentrations of PACAP and GIF are neuroprotective through inhibition of microglial activation.
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). To test the effect of PACAP38 and GIF on the microglial generation of ROS, microglia-enriched cultures were pretreated with PACAP38 or GIF and then exposed to LPS or PMA. Both 10–13 M PACAP38 and 10–13 M GIF reduced PMA-induced intracellular ROS production by approximately 30% (Fig. 4C) and reduced LPS-induced intracellular ROS production by approximately 60% to nearly control levels (Fig. 4D). Both PACAP and GIF were shown to have no effect on intracellular ROS alone (data not shown). Here, we are the first to report that PACAP attenuates microglial intracellular and extracellular ROS and demonstrates a similar efficacy of PACAP38 and its internal peptide GIF in reducing microglial-derived ROS from different immunological stimuli.
PACAP38 and GIF Are Neuroprotective through Inhibition of NADPH Oxidase. Although the effect of PACAP38 and GIF on intracellular ROS and extracellular superoxide was evident, it was unclear how these subpicomolar concentrations of peptides attenuated microglial activation and whether the neuroprotective effects were due to the anti-ROS qualities. To identify the mechanism through which subpicomolar concentrations of PACAP38 and GIF are inhibiting the microglial inflammatory response, the ability of PACAP38 and GIF to protect DA neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures from NADPH oxidase-deficient mice (PHOX–/–) was determined. PHOX–/– mice are unable to produce extracellular superoxide in response to LPS due to the lack of functional NADPH oxidase. Compounds that protect through reduction of NADPH oxidase activation are ineffective in PHOX–/– cultures. Both PACAP and GIF failed to show neuroprotection in PHOX–/– cultures as determined by DA uptake (Fig. 5A), supporting that inhibition of this enzyme is critical for their neuroprotective mechanism of action.
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The production of TNF was also measured in response to LPS in PHOX–/– and PHOX+/+ mesencephalic neuron-glia cultures pretreated for 30 min with GIF or PACAP38. Again, pretreatment with either neuroprotective peptide failed to show any reduction of TNF in response to LPS in PHOX–/– cultures, whereas pretreatment with GIF or PACAP38 (10–13 M) attenuated the LPS-induced TNF production in PHOX+/+ cultures (Fig. 5B), demonstrating that these subpicomolar-acting compounds also inhibit the ROS-induced amplification of TNF production. Together, these results support the conclusion that subpicomolar concentrations of PACAP and GIF afford neuroprotection through inhibition of microglial NADPH oxidase.
PACAP6–38 Fails to Attenuate PACAP38, PACAP27, and GIF Neuroprotection. In an effort to understand how PACAP38, PACAP27, and GIF were exerting their effects, we pretreated neuron-glia cultures with PACAP6–38 (PAC1 receptor antagonist) before exposing the cells to PACAP38, PACAP27, or GIF, followed by LPS treatment. PACAP6–38 (3 µM) by itself did not protect against LPS-induced loss of DA uptake (data not shown) and failed to attenuate the neuroprotective effects of PACAP38, PACAP27, or GIF (10–13 M) (Fig. 6).
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), but not all effects of VIP or PACAP are mediated through these traditional pathways (Hill et al., 1999; Brenneman et al., 2002, 2003). Consistent with previous reports (Brenneman et al., 2003), we show that the addition of PACAP27 (5 µM) to neuron-glia cultures resulted in the production of cAMP, expressed as picomolar per milliliter of cell lysate (mean = 211; S.D. = 21; P < 0.01). However, subpicomolar concentrations of PACAP38, PACAP27, and GIF (10–13 M) showed no significant effect on cAMP, compared with control. The data are the mean of cAMP levels from three separate experiments. Together, this suggests that PACAP38, PACAP27, and GIF are neuroprotective through a receptor mechanism independent of traditional PACAP receptors. Chemical Similarities of Subpicomolar-Acting Neuroprotective Compounds. Pharmacophore modeling and analysis were performed on the compounds experimentally identified as exhibiting neuroprotective activity: naloxone; dextromethorphan; and dynorphin A (2–4), GGF. This reference pharmacophore aligned with GIF is shown in Fig. 7. We found that the highest scoring pharmacophore contained a hydrogen bond acceptor (3.21/3.11), a hydrophobic region (2.11), and a positive ionizable region (1.11). This pharmacophore illustrates the common chemical properties that are shared among naloxone, dextromethorphan, GGF, and GIF with a common relationship in three-dimensional space. The shared common properties identified by pharmacophore analysis that are necessary to elicit neuroprotection at subpicomolar concentrations were: a hydrogen bond acceptor, a hydrophobic region, and a positive ionizable region.
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Discussion |
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Several biological effects of PACAP have been reported previously at a wide dose range, where the detailed mechanisms and the receptors responsible are often unclear. It has been traditionally accepted that there are two classes of PACAP binding sites that are characterized by their relative affinities for PACAP and VIP. Type 1 PACAP receptors are predominantly located in the pituitary and the hypothalamus with a reported Kd of approximately 0.5 nM for PACAP (Laburthe and Couvineau, 2002). Type 2 receptors were reported to be localized in other tissues, such as the lung, duodenum, and thymus with a reported Kd of approximately 0.5 nM for both PACAP and VIP (Laburthe and Couvineau, 2002). However, the reported Kd values for both the type 1 and type 2 receptors are unable to explain the effects of PACAP at lower concentrations, such as 10–13 M, suggesting the existence of an additional high-affinity site of action that is independent of these receptors. Furthermore, PACAP (14– 38), which lacks the GIF sequence, is still able to stimulate PAC1 receptors, suggesting that GIF is not necessary to stimulate cyclic adenylate cyclase activity of PACAP (Vandermeers et al., 1992). In fact, several domains are required for PACAP to interact with its traditional receptors (Vaudry et al., 2000).
In the current study, we demonstrate that GIF, an internal sequence of PACAP (from a region that is not necessary for the activation of PAC1 or VIP receptors), is able to attenuate microglial activation and associated neurotoxicity with an efficacy similar to its parent compound, PACAP38. The activation of PAC1/VPAC receptors is well documented to cause an increase in cAMP levels (Nowak et al., 2003), and we could find no evidence of cAMP production with subpicomolar concentrations of the PACAP peptides (10–13 M). Furthermore, the PAC1 antagonist was unable to attenuate PACAP38, PACAP27, or GIF (10–13 M) neuroprotection. Although it is clear that many of the effects exerted by PACAP work through traditional receptor-mediated mechanisms, the current data support that 10–13 M GIF and PACAP exert their low-dose neuroprotective and anti-inflammatory effects through a mechanism independent of traditional PACAP receptor mechanisms. Although the detailed mechanism of this alternative site of action for PACAP is under investigation, there are striking similarities among PACAP, GIF, and other neuroprotective compounds studied in our laboratory that may provide valuable insight.
Previously, we identified a subset of compounds (GGF, dynorphin, leucine enkephalin, des-tyrosine leucine enkephalin, dextromethorphan, and naloxone) that were neuroprotective through NADPH oxidase inhibition (Li et al., 2005; Qin et al., 2005a,b). These subpicomolar-acting compounds share chemical, functional, and mechanistic similarities, indicating that these ligands exert their similar effects through a common high-affinity sight of action. Specifically, this group of subpicomolar-acting compounds was shown to be neuroprotective against LPS-induced DA neurotoxicity through inhibition of NADPH oxidase and shared structural and chemical similarities, as determined by pharmacophore analysis (Qin et al., 2005a). The pharmacophore generated from this analysis is a graphical representation of the common three-dimensional chemical features of ligands that are neuroprotective through NADPH oxidase inhibition at subpicomolar concentrations. Noting that the primary structure of GIF is similar to the neuroprotective peptide GGF discovered in our laboratory, we sought to determine whether PACAP38 and GIF were neuroprotective through the same mechanism and chemically similar to the previously defined pharmacophore for subpicomolar-acting molecules. In the current study, we demonstrate that at subpicomolar concentrations, both GIF and PACAP are neuroprotective through the attenuation of microglial activation and NADPH oxidase inhibition. In addition, pharmacophore analysis revealed that PACAP and GIF fit the subpicomolar-acting pharmacophore, whereas the ineffective scrambled peptide (IGF) did not. Together, these data demonstrate that PACAP and GIF join a class of subpicomolar-acting compounds that are neuroprotective through a high-affinity site of action common to many subpicomolar-acting compounds.
These findings have broad implications because the potential therapeutic utility of this class of subpicomolar-acting neuroprotective compounds is extensive and provides great hope for the treatment of neurodegenerative disease. First, compounds exerting their effects at low concentrations through high-affinity receptors offer a low potential for unwanted side effects. Second, inflammation-mediated neuro-degeneration is a characteristic of multiple neurodegenerative diseases, where microglial NADPH oxidase has been implicated as a common mechanism of microglia-mediated neurotoxicity (Block and Hong, 2005). Interestingly, previous reports from our laboratory have indicated that microglial activation is regulated by neuropeptide control of NADPH oxidase at subpicomolar concentrations (Block et al., 2006). The attenuation of microglial NADPH oxidase both inhibits the production of neurotoxic ROS and reduces microglial signaling leading to the production of proinflammatory factors (Qin et al., 2005a), making the inhibition of this enzyme an effective and ideal therapeutic target.
In summary, here we demonstrate that PACAP4–6 (GIF) shares a similar neuroprotective dose-response curve and mechanism of action with its parent peptide PACAP38 that is independent of traditional receptor pathways. In addition, we show that PACAP joins a growing list of subpicomolar-acting neuropeptides with mechanistic, chemical, and neuroprotective similarities, suggesting that attenuation of microglial activation may be regulated by multiple neuropeptides at subpicomolar concentrations.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PD, Parkinson's disease; DA, dopaminergic; ROS, reactive oxygen species; PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal peptide; LPS, lipopolysaccharide; GGF, Gly-Gly-Phe; GIF, Gly-Ile-Phe; TH, tyrosine hydroxylase; DCF-DA, 2',7'-dichlorofluorescin diacetate; DA, dopamine; TNF, tumor necrosis factor; NO, nitric oxide; SOD, superoxide dismutase; HBSS, Hanks' balanced salt solution; MPP+, 1-methyl-4-phenylpyridinium; PMA, phorbol-12-myristate 13-acetate; PAC1, PACAP receptor; VPAC, VIP/PACAP receptor; WST, 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; DCFH, 2',7'-dichlorodihydrofluorescein.
Address correspondence to: Dr. Michelle L. Block, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, MD F1-01, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: block{at}niehs.nih.gov
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