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* Neuropharmacology Section, Laboratory of Pharmacology and Chemistry and
the National Center for Toxicogenomics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA;
Pathology & Lab Medicine, University of North Carolina at Chapel Hill, North Carolina, USA; and
Departments of Psychiatry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
1Correspondence: Neuropharmacology Section NIEHS/NIH, PO BOX 12233, MD: F1-01 111 Alexander Dr., Research Triangle Park, NC 27709, USA. E-mail: hong3@niehs.nih.gov; guorongl{at}med.unc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, prostaglandin E2 and superoxide from microglia in comparable degrees. The important role of superoxide was demonstrated by DM’s failure to show a neuroprotective effect in neuron-glia cultures from NADPH oxidase-deficient mice. These results suggest that the neuroprotective effect elicited by femtomolar concentrations of DM is mediated through the inhibition of LPS-induced proinflammatory factors, especially superoxide. These findings suggest a novel therapeutic concept of using "ultra-low" drug concentrations for the intervention of inflammation-related neurodegenerative diseases.—Li, G., Cui, G., Tzeng, N.-S., Wei, S,.-J., Wang, T., Block, M. L., Hong, J.-S. Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage.
Key Words: DM • femtomolar • inflammation • microglia • neuroprotection • Parkinson’s disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. The activated microglia secrete various cytokines and free radicals, such as superoxide and nitric oxide (NO), resulting in cerebral inflammation and subsequent neuron death. Accumulation and/or overproduction of these factors impact on neurons to induce their degeneration (5
6
7)
. In fact, cerebral inflammation sustained by microglia activation that is triggered by the inflammagen lipopolysaccharide (LPS) results in a delayed and progressive degenerative feature of nigra dopaminergic neurons (8)
. Dopaminergic neurons are especially vulnerable to oxidative damage (9
, 10)
. In addition, the density of microglia in the substantia nigra region is 4- to 5-fold higher than that in other brain regions (11)
. Thus, for these two reasons, the over-activation of nigral microglia and release of neurotoxic factors are a crucial component associated with the degenerative process of dopaminergic neurons in PD.
Dextromethorphan (DM), a d-isomer of the codeine analog levorphanol, is one of the most widely used over-the-counter antitussives. Studies using animal models of cerebral ischemia and hypoglycemic neural injuries have demonstrated that DM showed neuroprotective effects through the inhibition of glutamate receptors (12
13
14
15
16
17)
. However, a novel mechanism was proposed by a recent report from our laboratory, where we have demonstrated that the neuroprotective effect of DM is through the inhibition of microglia over-activation (18)
.
Using neuron-glia cultures prepared from the rodent embryo (E-14/15) mesencephalon, which encompasses the substantia nigra region of the brain, we have demonstrated that activation of microglia induced by the inflammagen LPS, results in the degeneration of dopaminergic neurons (7
,11
). We have recently reported potent neuroprotective effects of DM at micromolar concentrations on LPS-induced dopaminergic neurodegeneration (18)
. Mechanistic studies indicated that the neuroprotective effect of DM is mediated through the inhibition of microglia over-activation (18)
. In the course of attempting to determine DM’s action site, we realized that the inhibitory effect of DM on the production of superoxide mimicked the effect of dynorphins at femtomolar concentrations (19)
. This comparison led us to study the neuroprotective effect of DM at femtomolar concentrations. In this paper, we report the extremely unusual and exciting finding that femtomolar concentrations of DM exert an effect as efficient as micromolar concentrations of DM in protecting dopaminergic neurons against LPS-induced damage. We have elucidated the underlying mechanism for this femtomolar acting compound’s neuroprotective effect. DM has been used clinically for decades with a proven safety record and it is a small molecule that can be administered orally, suggesting that DM is an ideal remedy for long-term usage for neurodegenerative diseases. In view of the lack of therapeutic agents that can halt the progression of PD, our findings may provide a novel therapy for these neurodegenerative diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
NADPH oxidase-deficient (gp91phox–/–) and wild-type C57BL/6J (gp91phox+/+) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Breeding of the mice was performed to achieve timed pregnancy with the accuracy of ± 0.5 d. Timed-pregnant Fisher F344 rats were obtained from Charles River Laboratories (Raleigh, NC, USA). Housing and breeding of the animals were performed in strict accordance with the National Institutes of Health guidelines.
Primary mesencephalic neuron-glia cultures
Neuron-glia cultures were prepared from the ventral mesencephalic tissues of embryonic day 13–14 rats or day 12–13 mice, as described previously (20)
. Briefly, dissociated cells were seeded at 1 x 105/well and 5 x 105/well to poly-D-lysine-coated 96-well and 24-well plates, respectively. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, in minimal essential medium (MEM) containing 10% fetal bovine serum (FBS), 10% horse serum (HS), 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/mL penicillin, and 50 µg/mL streptomycin. Seven-day-old cultures were used for treatment. At the time of treatment, immunocytochemical analysis indicated that the rat neuron-glia cultures were made up of 11% microglia (OX42 immunostained), 48% astrocytes (GFAP immunostained), 41% neurons (NeuN immunostained), and 1% tyrosine hydroxylase-immunoractive (TH-IR) neurons. The composition of the neuron-glia cultures of NADPH oxidase-deficient mice was very similar to that of the wild-type mice in that there were 12% microglia (F4/80 immunostained), 48% astrocytes (GFAP immunostained), 40% neurons (NeuN immunostained), and 1% TH-IR neurons.
Primary rat mesencephalic neuron-enriched cultures
Neuron-enriched culture were prepared from the ventral mesencephalic tissues of embryonic day 13 or 14 rats as described (20)
. Briefly, dissociated cells were seeded at 1 x 105/well and 5 x 105/well to poly-D-lysine-coated 96-well and 24-well plates, respectively. Glial proliferation was suppressed by the inclusion of cytosine ß-D-arabinocide (5–10 µM). Used for treatment were 7-d-old cultures, which were composed of 91% neurons, 9% astrocytes, and <0.1% microglia.
Treatment
DM was freshly prepared as stock solutions (1 mM) in ddH2O and sterile-filtered right before use. For treatment of cultures, DM was serially diluted (10x) with fresh culture medium containing 2% of fetal bovine and horse serum. Cultures were pretreated with DM for 30 min before treatment with LPS.
Uptake assays
[3H]-DA uptake assays were performed as described (7)
. Briefly, after washing twice with warm Krebs-Ringer buffer (KRB, 16 mM sodium phosphate, 119 mM Nacl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH7.4), cultures were incubated for 20 min at 37°C with 1 µM [3H]-DA in KRB for DA uptake. Afterward, cultures were washed (three times) with ice-cold KRB and cells were collected in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA uptake observed in the presence of mazindol (10 µM) was subtracted.
Immunostaining
Dopaminergic neurons were recognized with the anti-TH antibody and microglia were detected with the OX-42 antibody, which recognizes the CR3 receptor as described (7)
. Briefly, formaldehyde (3.7%) -fixed cultures were treated with 1% hydrogen peroxide (10 min) followed by sequential incubation with blocking solution (30 min), primary antibody (overnight, 4°C), biotinylated secondary antibody (2 h), and ABC reagents (40 min). Color was developed with 3,3'-diaminobenzidine. For morphological analysis, the images were recorded with an inverted microscope (Nikon, Tokyo, Japan) connected to a charge-coupled device camera (DAGE-MTI, Michigan City, IN, USA) operated with the MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA). For visual counting of TH-IR neurons, three wells with same treatment in the 24-well plate were counted under the microscope at 100x magnification by three individuals. The average of these scores was reported.
Measurement of superoxide release
The release of superoxide was determined by measuring the superoxide dismutase (SOD) -inhibitable reduction of cytochrome c as described (7)
. To measure the immediate release of superoxide from microglia-enriched or neuron-glia after stimulation, cultures grown in 96-well plates were switched to phenol red-free HBSS (50 µL/well). To each well was added 50 µL of HBSS containing vehicle or DM. The cultures were then incubated at 37°C for 30 min followed by 50 µL of ferricytochrome c (100 µM) in HBSS, with and without 600 U/mL SOD, 50 µL of vehicle or LPS in HBSS. The absorbance at 550 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Assay of intracellular ROS
The production of intracellular reactive oxygen species was measured by DCFH oxidation. The DCFH-DA (Molecular Probes, Eugene, OR, USA) reagent passively diffuses into cells in which it is hydrolyzed by intracellular esterase to liberate 2'-7'-dichlorofluoressein, which, during reaction with oxidizing species, yields a highly fluorescent compound 2'-7'-dichlorofluorescein (DCF) that is trapped inside the cell (21)
. For each measurement, a fresh stock solution of CM-H2-DCFDA (5 mM) was prepared in DMSO. CM-H2-DCFDA, diluted to a final concentration of 1 µM in phenol red-free HBSS containing 2% FBS and 2% HS, was added to cultures and incubated for 30 min at 37°C. After washing twice with warm HBSS, vehicle or stimulators in HBSS were added to cultures. After incubation for 30 min at 37°C, fluorescence intensity was measured at 485 nm for excitation and 530 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices).
Nitrite and TNF- assays
The production of NO was determined by measuring the accumulated levels of nitrite in the supernatant with the Griess reagent, and release of tumor necrosis factor (TNF-) was measured with a rat TNF- enzyme-linked immunosorbent assay kit from R and D System (Minneapolis, MN, USA), as described (22)
.
PGE2 production
PGE2 in supernatant was measured with a prostaglandin E2 EIA kit from Cayman (Ann Arbor, MI, USA) according to the manufacturer’s instructions.
Statistical analysis
The data were presented as the mean ± SE. For multiple comparisons of groups, ANOVA was used. Statistical significance between groups was assessed by paired or unpaired Student’s t test, with Bonferroni’s correction. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, DM at femtomolar concentrations is neuroprotective against inflammation-mediated dopaminergic neuron degeneration, a wide range of concentrations of DM (10–5 to 10–17 M) were tested. The neuron-glia cultures were exposed to LPS 10 ng/mL 30 min after DM pretreatment. Seven days later, the degeneration of dopaminergic neurons was assessed by [3H]-dopamine (DA) uptake. As shown in Fig. 1
a, consistent with our previous report, DM at micromolar (10–5 to 10–7 M) concentrations attenuated LPS-induced decrease in [3H]-dopmaine uptake. The most striking finding was that femtomolar (10–13 and 10–14 M) concentrations of DM showed equal efficacy neuroprotective effect as that at micromolar concentrations. It is interesting to note that nanomolar and picomolar of DM (10–8 to 10–12 M) showed no protective effects. It appears that this dose-response curve can be divided into three regions: 1) the micromolar responsive region, 2) nonresponse region, and 3) femtomolar responsive region. Thus, we selected 10–5M, 10–10 M and 10–14 M as representative concentrations of each of the three regions for further studies.
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The degeneration of dopaminergic neurons was assessed by counting the number of tyrosine hydroxylase-immunoreactivity (TH-IR) neurons (counting was performed in a double-blind manner by three individuals). As shown in Fig. 1b
, treatment with LPS 10 ng/mL alone caused a significant reduction in the loss of TH-IR neurons compared with vehicle-treated control cultures. Thirty minute pretreatment with DM 10–5 and 10–14 M significantly attenuated the LPS-induced reduction in the number of TH-IR neurons from 60% to 23% and 32%, respectively. DM at 10–10 M had no protective effects on dopaminergic neuron degeneration. Results from cell counts (Fig. 1b
) were comparable to that of the [3H]-dopamine uptake study (Fig. 1a
). The neuroprotective effects of DM at micro- and femtomolar concentrations against Aß-induced neurotoxicity were observed in a similar degree as the LPS-induced neurotoxicity (Fig. 2
).
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Glia are essential for femtomolar DM neuroprotection
It is known that microglia play major roles in inflammation-mediated dopaminergic neurodegeneration. To investigate whether the presence of glial cells is critical for the neuroprotective activity of DM, we determined the protective effect of DM in ß-amyloid peptide (Aß, 1-42)- or 1-methyl-4-phenylpyridinium (MPP+) -induced dopaminergic neurotoxicity using neuron-enriched cultures. Nine days after treatment with 4 µM Aß 1–42, DA uptake was reduced by 50% compared with the control cultures. Pretreatment on the neuronal cultures with DM (10–5, 10–10, and 10–14 M) before Aß 1–42 treatment did not significantly alter the magnitude of the Aß 1–42-induced reduction of DA uptake in the cultures. Similar effect was observed in the MPP+ study (data not shown). These results suggested that the presence of glial cells is necessary for DM to express its neuroprotective effect.
Femtomolar DM inhibits LPS-induced microglia activation
LPS can activate microglia to overproduce proinflammatory cytokines and free radicals, such as NO, PGE2, TNF-, superoxide, and other reactive oxygen species (ROS), which in turn cause neurodegeneration (24)
. In this study (Fig. 3
), we demonstrated that femtomolar concentrations of DM inhibited LPS-induced microglia over-activation and the subsequent release of proinflammatory factors from microglia. Neuron-glia cultures were treated with vehicle, LPS, or LPS plus DM respectively. Pretreatment with DM at 10–5 and 10–14 M significantly decreased the LPS-induced increase in the release of NO, PGE2, and TNF-, whereas DM at 10–10 M showed no significant reduction of NO, PGE2, and TNF- production (Fig. 3a-c
). Similarly, DM at 10–5 and 10–14 M significantly reduced LPS-induced superoxide and intracellular ROS (iROS) production, while DM at 10–10 M showed no effect (Fig. 3d, e
).
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ROS in mediating DM-elicited neuroprotective effect
Among the various proinflammatory factors released from microglia, femtomolar DM showed the most potent effect on the ROS production. To further study the role of ROS in DM-elicited neuroprotection, neuron-glia cultures were prepared from NADPH oxidase-deficient (PHOX–/–) and wild-type (PHOX+/+) mice. Previous report from our laboratory indicates that NADPH oxidase is the major enzyme in microglia that produces extracellular superoxide anions and a major contributor of the increase in the iROS, which in turn enhances TNF- production (25)
. As shown in Fig. 4
a, in neuron-glia cultures prepared from PHOX+/+ mice, LPS treatment reduced [3H]-dopamine uptake by 46%; DM (10–14 M) significantly attenuated the decrease. In contrast, LPS treatment reduced the uptake capacity by only 25% in PHOX–/– mice and DM (10–14 M) failed to show protective effect. Similar to DA uptake result, LPS-induced iROS production in PHOX–/– mice is only half of that in PHOX+/+ mice, and DM at 10–14 M significantly inhibited iROS production in PHOX+/+ mice, while failed to show significant effect in PHOX–/– mice (Fig. 4c
). Consistently, LPS-induced TNF- production in PHOX–/– mice is two thirds of that in PHOX+/+ mice, and DM at 10–14 M was able to significantly reduce TNF- production, which was not seen in PHOX–/– mice (Fig. 4b
). These results strongly support the possibility that inhibition of ROS production and subsequently TNF- production is associated with the neuroprotective effect of DM at femtomolar concentrations.
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DM post-treatment is still neuroprotection
As inflammation may already be well developed in most PD patients with clinical symptoms, it is desirable to have a therapeutic agent for PD patients, which can suppress the ongoing inflammatory process and halt the progression of disease states. For this purpose, we tested the possibility if post-treatment with DM is still neuroprotective in LPS-induced neurotoxicity. Neuron-glia cocultures were first treated with LPS (20 ng/mL) for 12 h, then LPS was removed by removing media from cultures and washed twice with fresh media. Different concentrations of DM (10–5, 10–10,10–14 M) were added to the cultures and incubation continued for another 6 or 7 days. The presence of LPS in the media for only 12 h was capable of reducing the dopamine uptake capacity by 70% as shown in Fig. 5
a. Post-treatment with DM showed protective effect at concentrations of 10–5 and 10–14 M, but not at 10–10 M. In the same experiment, superoxide levels were measured in companion cultures 24 h after LPS treatment. Consistent with pretreatment studies, post-treatment with DM at 10–5 and 10–14 M concentrations significantly inhibited LPS-induced increase in superoxide production. In contrast, neither pretreatment nor post-treatment with DM at 10–10 M significantly affected the production of this free radical (Fig. 5b
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous reports from our laboratory showed that the neuroprotective effect of DM at micromolar concentrations is due to inhibition of LPS-induced over-activation of microglia and the subsequent suppression in the release of microglial proinflammatory factors, such as superoxide, NO, or TNF- (26)
. Among the array of factors released from activated microglia, inhibition of the production of ROS is the major action site for the protective effect of DM. Parallel experiments of micromolar vs. femtomolar concentrations of DM showed great similarity in their efficacy in neuroprotection and in the patterns of inhibition of different proinflammatory factors. These similarities lend important insights into the possible mechanism underlying the neuroprotective effect of femtomolar concentrations of DM.
Similar to micromolar concentrations of DM, the neuroprotective effect of femtomolar DM was clearly demonstrated in LPS-induced dopaminergic neurotoxicity shown by [3H]dopamine uptake (Fig. 1a
), cell count of TH-IR neurons (Fig. 1b
), and morphologic observation (Fig. 1c
). In addition, the protective effect was only observed in neuron-glia cultures, but not in neuron-enriched cultures, since DM failed to show protective effect against Aß- or MPP+-induced dopaminergic neurotoxicity in neuron-enriched culture regardless concentration of DM. The comparison studies indicate that femtomolar DM-elicited protection against inflammation-mediated dopaminergic neurotoxicity is dependent on the presence of microglia.
Analyses of the various proinflammatory factors affected by DM show both micromolar and femtomolar concentrations of DM are equal efficacy in inhibiting the release of nitrite (40%), TNF- (30%), PGE2 (37%), superoxide (50%) and intra-cellular ROS (more than 70%) (Fig. 3)
. The potent inhibition in the production of ROS by DM at 10–14 M led us to examine these factors in greater details by using PHOX-deficient mutant mice. Our previous reports indicate that PHOX is the major superoxide-producing enzyme in microglia and the major contributor to the increase in iROS concentrations in response to a variety of immune stimulants such as LPS, ß-amyloid peptides (Aß) (25
, 27)
. For example, through the activation of PHOX, Aß at low concentrations increases the production of neurotoxic superoxide, but not the other factors, such as nitrite and TNF-. The findings that micromolar and femtomolar concentrations of DM could protect Aß-induced dopaminergic neurotoxicity (Fig. 2)
suggest that DM affords its neuroprotection by inhibiting PHOX activity. Femtomolar DM, while significantly lessening the LPS-induced DA uptake reduction in wild-type mice, has no significant protective effect in PHOX–/– mice (Fig. 4a
). These observations strongly support the contention that the protective effect of femtomolar DM is most likely mediated through the inhibition of PHOX activity. Activation of PHOX in microglia not only increases the production of superoxide, but indirectly increases the intracellular ROS concentration, possibly through the conversion of superoxide to H2O2, which is membrane permeable. Increase of iROS can intensify the activation of NF-
B, which leads to higher TNF-, PGE2 production (24
, 28)
. The result that femtomolar DM inhibited TNF- production in wild type while not in PHOX–/– mice further supports the notion that the major site of action for femtomolar DM is on PHOX.
An interesting finding in this study that needs to be addressed is the phenomenon of bimodal reversed W-shape dose-response relationship (Fig. 1a
). Our data indicate that both micromolar and femtomolar concentrations of DM were neuroprotective, whereas picomolar concentrations of DM failed to show neuroprotection. The micromolar concentration range of DM exhibited a typical sigmoid dose-response; the femtomolar concentration range exhibited the same sigmoid dose-response when the effects of DM at concentrations below 10–14 M are taken into consideration. This bimodal phenomenon resembles previous reports from our laboratory (23)
and others (29
30
31
32
33
34
35
36
37
38)
showing a similar reversed W-shape dose response of a variety of peptides in their neuroprotective effects. However, this is the first report demonstrating that a small molecule, like DM, is capable of exerting potent neuroprotection at femtomolar concentrations.
We speculate that DM may bind to PHOX in two distinct fashions: one with micromolar affinity and the other with femtomolar affinity. The former could be the site reported for the widely used inhibitor diphylene iodonium (DPI) with an affinity at micromolar concentrations (39)
. The nature of the binding site with femtomolar affinity has not been reported, however, the existence of this high-affinity site was strongly suggested by our data on neuroprotective and inhibitory effect of femtomolar DM in this study. Our preliminary experiments aimed to elucidate the binding sites of DM showed that DM was capable of competing with [3H]-labeled naloxone in the binding to gp91 protein of PHOX (unpublished data) at micromolar concentrations and this binding was likely associated with the flavo moiety of the molecule (40
, 41)
. An interesting finding in our laboratory showed that naloxone shared high similarity with tripeptide GGF (0.85), and both act on gp91 of PHOX in femtomolar concentrations (Qin et al., unpublished results). Unfortunately, similar experiments using femtomolar concentrations were not technically feasible due to the limitation of specific activity of [3H]-labeling. Nevertheless, in view of the similar effects produced by these two different concentrations over a 109-fold range, it is likely that femtomolar DM inhibits PHOX in a similar fashion as that of micromolar DM. Finally, the question regarding the inability of DM to show any protective effect in the picomolar concentration range (10–8 to 10–12 M) remains unanswered. One interpretation could be "substrate inhibition" for the high-affinity binding site; alternatively, a hypothetical third binding site may exist to mask the protective effect of DM at this range of concentrations.
Findings from our research not only reveal an extremely unusual and interesting femtomolar acting compound, but also raise a new concept of using "ultra-low" concentrations of drugs for future therapeutic interventions for inflammation-related diseases. Although whether these in vitro findings can be substantiated in animal studies and eventually tested in clinical trials remain to be studied, the obvious advantages of this low-dose therapy, including the much reduced side effects warrants serious consideration of this approach. To obtain a preliminary glimpse, we have recently observed in an animal study that 100 million-fold lower than the regular dose of DM was effective in reducing the plasma level of alanine aminotransferas (ALT) in LPS/D-galactosamine-induced liver damage (unpublished observations). It is very surprising to learn that DM in such low concentrations is still effective to possess anti-inflammatory effect. However, in view of its potent inhibitory effect on the flavo-containing enzyme (PHOX) we have studied, which is one of the contributors of proinflammatory factors, may explain its potent anti-inflammatory potency and justify further studies of DM or its analogs using different animal models of inflammation.
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ACKNOWLEDGMENTS |
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Received for publication June 12, 2004. Accepted for publication September 23, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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