Background
Clozapine, an atypical antipsychotic medication, is widely used for treatment-resistant schizophrenia patients, demonstrating greater efficacy against both positive and negative symptoms, compared with typical antipsychotics [
1]. Other benefits of clozapine application include the lack of extrapyramidal adverse effects [
2] and the ability to improve cognitive function [
3]. Despite these advantages, the usage of clozapine is rather restricted due to a relatively high risk of neutropenia or agranulocytosis [
4‐
6]. Currently, the mechanism responsible for neutrophil toxicity of clozapine is not fully understood.
N-Desmethylclozapine (NDC) and clozapine
N-oxide (CNO), as well as reactive nitrenium intermediate, are the known major metabolites of clozapine in humans [
7,
8]. In vitro studies demonstrated that reactive nitrenium intermediates but not clozapine itself or NDC/CNO is toxic to neutrophils [
9,
10]. In addition, it is generally accepted that NDC and CNO are different in terms of receptor activity as CNO has been proved to be pharmacologically inert [
11,
12] to the receptors for which clozapine and NDC function as agonists or antagonists [
11,
13,
14].
Microglia, the resident innate immune cells in the central nervous system (CNS), serve immune surveillance function in physiological conditions. In response to certain cues, such as brain injury or immunological stimuli, microglia are readily activated and play a central role in the process called “neuroinflammation.” It is now widely accepted that dysregulated neuroinflammation featured by microglial over-activation has significant impacts on the pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD) [
15,
16] and Alzheimer’s disease (AD) [
17‐
21]. Furthermore, increasing evidence also suggests an association of neuroinflammation with several psychiatric disorders, including schizophrenia (SCZ), autism, depression, and anxiety disorders [
22‐
24].
We have previously reported that clozapine protects dopaminergic neurons from inflammation-induced damage by inhibiting microglial NADPH oxidase (NOX2, a superoxide-producing enzyme) in primary cell cultures [
25]. The main purpose of this study was to determine whether the two different metabolites could be less toxic than clozapine and preserve the same neuroprotective and anti-inflammatory effects of the parent drug. In this study, we found that both CNO and NDC protected DA neurons through suppression of microglia-mediated neuroinflammation both in vitro and in vivo, where inactivation of microglial NOX2 played a central role. More importantly, unlike clozapine, CNO displayed no effects on blood neutrophils. These effects revealed a novel bioactivity of CNO and NDC without the propensity of producing neutropenia.
Methods
Animals
Time-pregnant Fisher F344 rats were provided by the Charles River Laboratories (Raleigh, NC). Wild-type C57BL/6J (gp91
phox+/+) and phagocytic NADPH oxidase (NOX2)-deficient (gp91
phox−/−) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Breeding of time-pregnant mice was performed with accuracy of 0.5 day. All the mice were euthanized at the desired time points. Housing, breeding, and experimental use of the animals were performed in strict accordance with the National Institutes of Health guidelines. All procedures were approved by the NIEHS animal care and use committee.
Reagents
N-Desmethylclozapine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), MPP+, and Leu methyl ester (LME) were purchased from Sigma-Aldrich (St. Louis, MO). Clozapine and clozapine N-oxide were obtained from the NIMH Chemical Synthesis and Drug Supply Program (Research Triangle Institute, RTP NC). Lipopolysaccharide (LPS strain O111:B4) was purchased from Calbiochem (San Diego, CA). WST-1 was purchased from Dojindo Laboratories (Gaithersburg, MD). Cell culture ingredients were obtained from Invitrogen (Carlsbad, CA). [3H]DA was purchased from PerkinElmer Life Sciences (Boston, MA). The polyclonal anti-tyrosine hydroxylase (TH) antibody was purchased from CHEMICON International (Temecula, CA). The polyclonal ionized calcium-binding adaptor molecule 1 (Iba-1) antibody was purchased from Wako Chemicals USA (Richmond, VA). The monoclonal CD11b antibody was purchased from AbDSerotec (Raleigh, NC). The biotinylated secondary antibodies were purchased from Vector Laboratories (Burlingame, CA).
Animal treatment
Eight-week-old male C57BL/6J mice received daily MPTP injections (20 mg/kg, s.c.) for six consecutive days. One day prior to MPTP injection, clozapine or CNO (1 mg/kg, s.c.) was administered twice daily for 21 consecutive days. Eight days after initial MPTP injection, five mice from each group were sacrificed for the detection of microglial activation in substantia nigra (SN). Fourteen days after initial MPTP injection, the protective effects of clozapine and CNO against MPTP-induced motor deficits were measured by the accelerating rotarod test. At 8 and 21 days after the first injection of MPTP, respectively, mice were euthanized, and brains were removed and postfixed in 4 % paraformaldehyde overnight at 4 °C. Brains were then placed into 30 % sucrose/PBS solution at 4 °C until the brains sank to the bottom of the container. Coronal sections including SN pars compacta (SNpc) were cut on a −20 °C frozen sliding microtome (Thermo Scientific, microm HM525) into 40-μm transverse free-floating sections.
Blood analysis
Twenty-one days after the first injection of MPTP, the mice were euthanized and eyeball blood was collected in a 1.5-ml heparin tube. After a fully vortex, the number of different types of cells in the blood was counted by an Automatic Blood cell Counter (Model: CA-800, SANKYO Inc.).
Rotarod test
The rotarod behavior was measured as described previously using a Rota-Rod (ZS-RDM R03-1, Zhong-Shi Inc., Beijing, China). The parameters of the rotarod system were set as accelerating speed from 4 to 40 rpm in 300 s [
26,
27]. Mice received three consecutive trials. The rest period between each trial was 30 min. The mean latency for the last two trails was used for the analysis.
Immunostaining
The free-floating brain sections or fixed cells in 24-well culture plate were immune-blocked with 4–10 % goat serum and then incubated with polyclonal rabbit anti-TH antibody (1:5000 dilution), or Iba-1 antibody (1:5000 dilution), or rat anti-CD11b antibody (1:800 dilution) for 24 h at 4 °C, respectively. Antibody binding was visualized using a Vectastain ABC Kit (Vector Laboratories, Inc) and diaminobenzidine (or with cobalt) tablet as substrate.
Images were recorded with a CCD camera and the MetaMorph software (Molecular Devices). TH immunostaining-reactive (THir) neuron or Iba-1ir microglia number were counted according to published protocol [
28] and was carried out by at least two investigators without knowledge of the treatment. For immunocytochemistry staining in 24-well cell culture, three to six wells per treatment condition were used, and results from three to five independent experiments were obtained.
Primary cell cultures
Primary neuron-glia cultures were prepared as described previously [
29]. In brief, dissociated cells from the ventral mesencephalon of embryonic day 14 ± 0.5 Fischer 334 rats, gp91
phox+/+
or gp91
phox−/−
mice were seeded at 5.5 × 10
5 cells/well (rat) or 6.5 × 10
5 cells/well (mice) in poly-
d-lysine-coated 24-well plates, respectively. The cultures were maintained at 37 °C in the incubator with 5 % CO
2 and 95 % air in minimum essential medium. The cultures were ready for experiments 7 days later, when the cultures became mature and stable of each cell component (astrocytes ~50 %, neurons ~40 %, and microglia ~10 %) as described previously [
29,
30]. Microglia-depleted neuron-glia cultures were obtained by depleting microglia in neuron-glia cultures with 1.5 mM of LME 48 h after seeding (~45 % neurons and ~55 % astrocyte), as described previously [
31].
Mixed-glia cultures were prepared from whole brains of postnatal day 1 rats as reported before [
32]. Briefly, disassociated cells were seeded into 24-well (1 × 10
5/well) or 96-well (5 × 10
4/well) culture plates and maintained in 1 ml/well or 0.2 ml/well of Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium. The medium was changed every 3 days. On 11–12 days after plating, the cultures were mature and stable with different cell components [
29,
30] (astrocytes ~80 %, GFAP immunopositive cells; microglia ~20 % OX-42 immunopositive cells) ready for drug treatment or superoxide assay.
Culture treatment
Rat mesencephalic neuron-glia cultures or microglia-depleted neuron-glia cultures were maintained in the maintenance medium (10 % fetal bovine serum, 10 % horse serum, 0.1 % d-glucose, 1 % none essential AA, Na pyruvate 1 %, l-glutamine 1 %, Pen/Strep1% in MEM) for 7 days untile the cultures became mature (astrocytes ~50 %, neurons ~40 %, and microglia ~10 %). Then, the cultures were pretreated with vehicle or indicated concentrations of CNO, NDC, or clozapine prepared in the serum-reduced treatment medium (2 % fetal bovine serum, 2 % horse serum, Na pyruvate 1 %, l-glutamine 1 %, Pen/Strep1% in MEM) for 30 min before the addition of LPS (15 ng/ml) or MPTP (0.25 μM), which also prepared in the serum-reduced treatment medium. At indicated time points after treatment, the culture supernatant was collected for the detection of inflammatory factors. And 7 days after LPS or MPTP treatment, the protective effects of CNO, NDC, or clozapine against inflammation-elicited damage of DA neurons were determined by quantifying functional changes of [3H]DA uptake capacity and counts of THir neurons.
Cell lines
The rat microglia HAPI cell line was a gift from Dr. J. R. Connor (Pennsylvania State University, Hershey, PA) [
33] and maintained as described previously [
34]. Briefly, HAPI cell line were maintained at 37 °C in DMEM (Sigma) supplemented with 10 % fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified incubator with 5 % CO
2 and 95 % air. The cells were split or harvested every 3−5 days.
[3H]DA uptake assay
[
3H]DA uptake assays were performed as described previously [
35]. Briefly, cells were incubated for 21 min at 37 °C with 1 μM [
3H]DA (PerkinElmer Life Sciences) in Krebs-Ringer buffer. Cells were washed with ice-cold Krebs-Ringer buffer three times and then 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.
NO and TNF-α assays
The production of nitric oxide (NO) was determined by measuring accumulated levels of nitrite in the supernatant with Griess reagent, and the release of tumor necrosis factor-α (TNF-α) was measured with a TNF-α ELISA kit from R&D Systems (Minneapolis, MN) following manufacture’s protocol.
Measurement of superoxide
The production of superoxide was assessed by measuring the SOD-inhibitable reduction of the tetrazolium salt WST-1 [
36]. Briefly, primary neuron-glia cultures were washed twice with HBSS balanced salt solution and then pretreated with indicated concentrations of clozapine metabolites dissolved in HBSS for 15 min. Immediately after the addition of LPS, 50 μl of HBSS with or without SOD (50 U/ml) was added to each well along with 50 μl of WST-1 (1 mM) in HBSS. The absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices Sunnyvale). The amount of SOD-inhibitable superoxide was calculated and expressed as percentage of vehicle-treated control cultures.
Whole cell lyses from HAPI microglia were prepared with lysis buffer (Cell Signaling, Danvers, MA). Subcellular fractionation was performed as described previously [
37]. For subcellular fractions, HAPI microglia were lysed in hypotonic lysis buffer (1 mM Tris, 1 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF, and 10 μg/ml cocktail protease inhibitor) and then subjected to Dounce homogenization (20–25 St, tight pestle A). The lysates were centrifuged at 1600×
g for 15 min; the supernatant was centrifuged at 100,000×
g for 30 min. The pellets solubilized in 1 % nonidet P-40 hypotonic lysis buffer were used as membranous fraction.
Western blot analysis
For western blot analysis, equal amounts of protein were separated by 4−12 % Bis-Tris Nu-PAGE gel and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5 % nonfat milk and incubated with rabbit antibodies (1:1000) against Iba-1, p47
phox
, gp91
phox
(BD Transduction Laboratories), GAPDH, or rat CD11b antibody overnight at 4 °C. The next day, membranes were incubated with HRP-linked secondary anti-rabbit or rat IgG (1:3000) for 2 h at room temperature. ECL reagents (Amersham Biosciences) were used as a detection system.
Statistical analysis
All group data are expressed as mean ± standard error of mean (SEM). Group means were compared using one-way analysis of variance (ANOVA) with treatment as the independent variable. When ANOVA showed a significant difference, pairwise comparisons between group means were examined by Dunnett’s post hoc test. Differences were considered significant at p value <0.05. Statistical analysis was performed using GraphPad Prism version 6.00 for Windows.
Discussion
In this study, we demonstrated that CNO and NDC, two major metabolites of clozapine, exert potent anti-inflammatory and neuroprotective effects in MPTP- and inflammation-generated dopaminergic neurotoxicity. CNO displayed the same potent efficacy as clozapine in protecting DA neurons in MPTP-mediated PD mouse model. Further, in vitro studies revealed a dose-related correlation of anti-inflammatory effect and their potency in neuroprotection by both CNO and NDC. Mechanistic studies indicated that inhibition of microglial NOX2-generated superoxide is the major target for the anti-inflammatory actions: a novel pharmacological property shared by both clozapine and its two metabolites. This study clearly demonstrates that CNO and NDC exhibit potent central effects and may contribute to some of the neuropsychopharmacological actions of clozapine.
Neuroinflammation mediated by microglia is known to be accompanied by oxidative stress, a common pathogenic pathway for neurodegenerative process and psychiatric syndromes [
50,
51]. Either overproduction of oxidants such as ROS, or deficiencies in antioxidant defense, or some combination thereof, can perturb the redox homeostasis, resulting in oxidative damage [
52]. Though in the process of neuroinflammation, both microglia and astrocytes play critical roles in counterbalancing the neurotoxicity or neuroprotective effect to a variety of cytotoxic insults [
53]; results from this study and our previous report provided a novel anti-inflammatory mechanism of actions of clozapine and its metabolites as we found that microglia were essential for the neuroprotective effect of CNO or NDC (shown in Fig.
2, protective effect disappeared as microglia were depleted). Among the proinflammatory factors released by activated microglia, superoxide production was the most severely inhibited by CNO and NDC. Superoxide is one of the prominent factors released by activated microglia, and NOX2 has been identified as a major source [
54]. In addition to extracellular ROS, NOX2 also contributes to increase intracellular ROS that is a crucial secondary messenger for microglial signaling and proinflammatory properties [
46,
55]. We found that both CNO and NDC inhibited NOX2-derived superoxide production. Moreover, the anti-inflammatory and neuroprotective effects of CNO and NDC were abolished once the gene of
NOX2 was ablated, suggesting that NOX2 is the action target of CNO and NDC. Our results suggest that the inhibitory effects of CNO and NDC are independent of neurotransmitter receptors but directly through acting on NOX2. This possibility is consistent with the report showing that clozapine is able to bind to neutrophil NOX2 in vitro [
56]. Binding experiments for CNO/NDC to microglial NOX2 should be guaranteed in our future studies.
Clozapine is an atypical antipsychotic medicine used to treat schizophrenia in patients whose symptoms are not controlled with standard antipsychotic treatment. Despite intensive studies on various G protein-coupled receptors (GPCR), such as monoaminergic receptors and muscarinic receptors [
11,
12], mechanisms underlying the antipsychotic action of clozapine remain unclear. Recent advance in the research of neuroinflammation has provided a new avenue to uncover mechanisms underlying the pathogenesis of neurodegenerative diseases and mental disorders [
57,
58]. There has been an increasing recognition of the pathogenic role of microglia-mediated neuroinflammation in psychiatric diseases such as depression, bipolar, schizophrenia, and obsessive-compulsive disorder [
22‐
24]. For instance, both postmortem and PET studies have provided evidence for the existence of microglial activation in the brains of schizophrenia patients [
59‐
61]. Some clinical trial studies have demonstrated a beneficial effect of adjunctive minocycline, a well-known anti-inflammatory compound, on the negative symptoms of schizophrenia [
62,
63] and on cognitive function [
63]. Indeed, a wide variety of psychoactive drugs, such as antidepressants and antipsychotics, have been shown to suppress microglial activation [
23,
47,
64,
65].
Earlier report from our laboratory illustrating the anti-inflammatory and neuroprotective action of clozapine lends further support to the notion that neuroinflammation may play a role in schizophrenia [
25]. Current study further extends our previous report indicating that CNO and NDC, which are not shown to be centrally active metabolites, possess both anti-inflammatory and neuroprotective actions with equal or even higher potency than that of clozapine. These findings raise a possibility that these two metabolites may be used as anti-inflammatory and neuroprotective agents for treating neurodegenerative diseases or even as adjunctive drugs for treatment of various mental diseases. Given that a relatively high risk of neutropenia or agranulocytosis greatly hampers the long-term usage of clozapine, if future studies find CNO or NDC effective clinically in treating certain CNS diseases, these two metabolites would possess a great advantage over clozapine in terms of potential toxicities. It has been reported that NDC is much less toxic to neutrophils in comparison to clozapine and CNO shows no toxicity at all at concentrations up to 100 μM [
8,
66]. For this reason, exploring the potential therapeutic use of CNO may be of great interest for two reasons: (1) CNO shows higher potency in both anti-inflammatory and neuroprotective effects than that of NDC and clozapine in LPS-treated neuron-glial cultures; (2) Consistent with previous reported in vitro study, our study showed that CNO had no significant toxicity on neutrophils in vivo even combined with MPTP for up to 21 consecutive days of treatment. Currently, testing the antipsychotic efficacy of CNO using schizophrenia animal models is underway in our laboratory to determine the possibility that CNO can be effective as a substitute of clozapine in treating this mental disease.
On the other hand, currently, the role of astrocyte in PD remains controversial. We recently demonstrated that in LPS-treated neuron-glia cultures, astrocytes tend to protect DA neurons against neuroinflammation-mediated degeneration through secretion of neurotrophic factor GDNF [
53]. We further revealed that astroglia may not possess the capability to directly response to the innate immune stimuli LPS, but rather depend on crosstalk with microglia. However, the protective effects of astrocyte against MPP
+-induced DA degeneration in vitro were not observed in our previous study, although the reason still remains unclear [
67]. Saijo et al. [
68] reported that astrocyte is not only non-neuroprotective, but even amplified the proinflammatory response in microglia, resulting in exacerbated DA degeneration. Despite of these conflicting results, targeting astrocyte, such as cystine/glutamate exchange transporter [
69], glutathione synthesis, [
70] and 5-HT(1A) receptor [
71], exhibited potent neuroprotective effects in multiple models of PD. Therefore, future studies focusing on the role astrocyte in the neuroprotective effects elicited by CNO and NDC should be guaranteed, which may provide new opportunities for developing novel strategy for PD therapy.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JSH, QW, and KQ designed the experiments and supervised the project. LJ and QW carried out the cellular and molecular analyses. LJ and SW carried out the in vivo experiments. LJ and XW analyzed the data and wrote the manuscript. HZ took part in the pilot studies. BW gave the technical training and support. SHC, CYJ, and RBL revised the manuscript. All authors have read and approved the final version of the manuscript.