Figures
Abstract
Background
Desipramine is known principally as a tricyclic antidepressant drug used to promote recovery of depressed patients. It has also been used in a number of other psychiatric and medical conditions. The present study is the first to investigate the neuroprotective effect of desipramine.
Methodology/Principal Findings
Mes23.5 dopaminergic cells were used to examine neuroprotective effect of desipramine. Western blot, reverse transcription-PCR, MTT assay, siRNA transfection and electrophoretic mobility shift assay (EMSA) were carried out to assess the effects of desipramine. Desipramine induces endogenous anti-oxidative enzyme, heme oxygenase-1 (HO-1) protein and mRNA expression in concentration- and time-dependent manners. A different type of antidepressant SSRI (selective serotonin reuptake inhibitor), fluoxetine also shows similar effects of desipramine on HO-1 expression. Moreover, desipramine induces HO-1 expression through activation of ERK and JNK signaling pathways. Desipramine also increases NF-E2-related factor-2 (Nrf2) accumulation in the nucleus and enhances Nrf2-DNA binding activity. Moreover, desipramine-mediated increase of HO-1 expression is reduced by transfection with siRNA against Nrf2. On the other hand, pretreatment of desipramine protects neuronal cells against rotenone- and 6-hydroxydopamine (6-OHDA)-induced neuronal death. Furthermore, inhibition of HO-1 activity by a HO-1 pharmacological inhibitor, ZnPP IX, attenuates the neuroprotective effect of desipramine. Otherwise, activation of HO-1 activity by HO-1 activator and inducer protect 6-OHDA-induced neuronal death.
Conclusions/Significance
These findings suggest that desipramine-increased HO-1 expression is mediated by Nrf2 activation through the ERK and JNK signaling pathways. Our results also suggest that desipramine provides a novel effect of neuroprotection, and neurodegenerative process might play an important role in depression disorder.
Citation: Lin H-Y, Yeh W-L, Huang B-R, Lin C, Lai C-H, Lin H, et al. (2012) Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons. PLoS ONE 7(11): e50138. https://doi.org/10.1371/journal.pone.0050138
Editor: Malú G. Tansey, Emory University, United States of America
Received: April 9, 2012; Accepted: October 17, 2012; Published: November 27, 2012
Copyright: © 2012 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Science Council (NSC 101-2320-B-039-048-MY2), and Taichung Tzu Chi General Hospital (TTCRD 101-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons of the substantia nigra (SN), giving rise to dopamine depletion in the striatum [1]. The resulting loss of dopaminergic neurons leads to debilitating motor dysfunction including rigidity, resting tremor, mask face and bradykinesia. Although PD is well characterized by motor symptoms, clinical depression is the most common neuropsychiatric disorder in PD patients [2]–[4]. More than 40% of PD patients are observed in depression [5], [6]. Importantly, numerous studies have shown that there is an increased incidence of depression before the onset of PD, and motor fluctuations may greatly affect the occurrence [7], [8]. Depression has been classified as a disorder of the brain and CNS, and is manifested by a combination of symptoms that interferes with the ability to work, study, sleep, eat, and enjoy once pleasurable activities [9]–[12].
Desipramine is a tricyclic antidepressant (TCA), one of an antidepressant drug used to promote recovery of depressed patients. Desipramine does not affect mood or arousal but may cause sedation in non-depressed individuals. However, desipramine exerts a positive effect on mood in depressed individuals. TCAs are potent inhibitors of serotonin and norepinephrine reuptake [13], [14]. It has been reported that desipramine significantly increases anti-apoptotic protein Bcl-2 expression, repairs serotonin and noradrenaline production [15], and prevents stress-induced depressive-like behavioral changes [16]. Interestingly, it has been reported that desipramine is able to reduce MPP+-induced cell toxicity in SH-SY5Y, however, despite many studies of those links, the detail molecular mechanisms of antidepressants on neuroprotective effect remain unknown.
HO-1 (also referred to heat-shock protein 32) is a rate-limiting enzyme that catalyzes the degradation of heme, produces biliverdin, iron, and carbon monoxide [17], [18]. In normal brain, the level of HO-1 is rather low [19], but can be strongly induced in response to diverse stress-related cellular stimuli [20], [21], oxidative stress and neuroinflammation [22]–[24]. Therefore, induction of HO-1 contributes to cytoprotection and anti-inflammation [25]–[27], and HO-1 may be a therapeutic target in neurodegenerative diseases and brain inflammation [28]–[30]. In this study, we investigated the neuroprotective effect of antidepressant desipraimine on rotenone- and 6-OHDA-induced neuronal cell death. Our results suggest that desipramine protects rat dopaminergic neurons against cell death through heme oxygenase-1 expression.
Materials and Methods
Materials
Desipramine was purchased from Fluka (Buchs, Switzerland). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), and OPTI-MEM were purchased from Invitrogen-Gibco (Carlsbad, CA). Primary antibodies against β-actin, JNK, ERK2, phospho-ERK1/2, Nrf2 and PCNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against JNK phosphorylated at Thr 183/185 was purchased from Cell Signaling and Neuroscience (Danvers, MA). ON-TARGET smart pool Nrf2 siRNA, and Control nontargeting pool siRNA were purchased from Dharmacon (Lafayette, CO). 6-OHDA, rotenone, SB203580 and PD98059 were obtained from Sigma–Aldrich (St. Louis, MO). SP600125 was obtained from Tocris Bioscience (Ellisville, MO).
Cell Cultures
Mes23.5 cell line is a dopaminergic cell line hybridized from murine neuroblastoma-glioma N18TG2 cells with rat mesencephalic neurons, which shows several properties similar to those of primary neurons originated in the substantia nigra [31]. The culture protocol of Mes23.5 was followed our previous report [32]. Briefly, cells were cultured in DMEM/F12 containing Sato’s components growth medium supplemented with 5% FBS at 37°C in a humidified incubator in an atmosphere of 5% CO2. Confluent cultures were passaged by trypsinization.
Western Blot Analysis
The rat Mes23.5 cell line was treated with desipramine for indicated time periods, and whole cell extracts were lysed on ice with radioimmunoprecipitation assay buffer. The nuclear extracts were prepared as described previously [33]. Cells were rinsed with PBS and suspended in hypotonic buffer A for 10 min on ice, and vortexed for 10 s. The lysates were separated into cytosolic and nuclear fractions by centrifugation at 12,000 g for 10 min. The supernatants containing cytosolic proteins were collected. Pellet containing nuclear fraction was re-suspended in buffer C for 30 min on ice.
The supernatants containing nuclear proteins were collected by centrifugation at 13,000 g for 20 min and stored at −80°C. Protein samples were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) and transferred to PVDF membranes which were then blocked with 5% nonfat milk, and then probed overnight with primary antibody at 4°C. After undergoing PBS washes, the membranes were incubated with secondary antibodies. The blots were visualized by enhanced chemiluminescence using Kodak XOMAT LS film (Eastman Kodak, Rochester, NY). The blots were subsequently stripped through incubation in stripping buffer [34] and reprobed for β-actin as a loading control. Quantitative data were obtained using a computing densitometer and a shareware Image J.
Reverse Transcription-PCR (RT-PCR)
Total RNA was extracted from Mes23.5 cell line using a TRIzol kit (MDBio Inc., Taipei, Taiwan). The reverse transcription reaction was performed using 2 µg of total RNA that was reverse transcribed into cDNA with the oligo(dT) primer. After preincubation at 50°C for 2 min and 95°C for 10 min, the PCR was performed as 30 cycles of 95°C for 10 s and 60°C for 1 min. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as CT). The oligonucleotide primers were
HO-1:
5′- TCTAT CGTGC TCGCA TGAAC-3′ and
5′-CAGCT CCTCA AACAG CTCAA -3′
GAPDH:
5′-CTCAA CTACA TGGTC TACAT GTTCC A-3′ and
5′-CTTCC CATTC TCAGC CTTGA CT-3′
Measurement of Cell Viability
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells cultured in 96-well plates were pre-treated with various inhibitors before treated with desipramine for 8 h followed by treatment with rotenone or 6-OHDA. After treatment, MTT (0.5 µg/ml) was added for 60 min, the culture medium was then removed, cells were dissolved in dimethyl sulfoxide and shaken for 10 min. OD values at 550 nm were measured by microplate reader. The absorbance indicates the enzymatic activity of mitochondria and provides information of cell viability.
Sulphorhodamine B Assay
Cell viability was assessed by the sulphorhodamine B (SRB) assay. Cells cultured in 96-well plates were treated with various inhibitors before treated with desipramine for 8 h followed by stimulation with rotenone or 6-OHDA. After treatment, the culture medium was then removed and cells were fixed in situ by gentle addition of 50 µl per well of 10% TCA and plates were incubated for 1 h at 4°C. After discarding supernatant, plates were then washed for 5 times with PBS, and then added 50 µl of SRB solution to each well, and plates were incubated for 15 min at room temperature. After staining, cells were washed 5 times with 1% acetic acid and dissolved in tris-base. After 10 mins’ shaking, OD values at 515 nm were measured by microplate reader. The absorbance provides information of cell viability.
Trypan Blue Exclusion Assay
The trypan blue exclusion assay was used to assess cell viability after 6-OHDA or rotenone exposure. Culture medium was removed and 0.2% trypan blue in TBS was added to the cells for 10 min. After the removal of the trypan blue solution, cells were fixed with 4% paraformaldehyde in PBS for 5 min, then replaced by TBS. Cells were then observed under bright field microscope.
Immunocytofluorescent Staining
Cells were seeded onto glass coverslips and exposed to desipramine for 2 h, then washed with PBS and fixed with 4% paraformaldehyde for 15 min, after which they were permeabilized with Triton X-100 for 30 min. After blocking with 5% nonfat milk in PBS buffer, cells were incubated with rabbit anti-Nrf2 antibody for 1 h at room temperature. After a brief wash, cells were incubated with a secondary antibody conjugated with Alexa 488-Flour (1∶200; Invitrogen Life Technologies, Carlsbad, CA). Finally, cells were washed again, mounted, and visualized with fluorescence microscope. Quantitative data were obtained using a computing densitometer and Image J.
Transfection
Mes23.5 cells were grown to 80% confluent in 6-well plates and transfected with control siRNA or Nrf2 siRNA on the following day by Lipofectamine™ 2000 (LF2000; Invitrogen). Control siRNA or Nrf2 siRNA and LF2000 were premixed in OPTI-medium for 20 min and applied to the cells (0.8 ml/well). Medium containing 5% FBS (0.8 ml) was added 4∼6 h later. After 24 hrs’ transfection, LF2000-containing medium was replaced with DMEM/F12 medium containing 2% FBS and treated with desipramine for another 24 h.
Electrophoretic Mobility Shift Assay (EMSA)
The electrophoretic mobility shift assay gel shift kit (Panomics, Redwood City, CA) was used according to our previous report [35]. Nuclear extract (4 µg) of Mes23.5 was incubated with poly d(I-C) at room temperature for 5 min. The nuclear extract was then incubated with biotin-labeled probes at room temperature for 30 min. After electrophoresis on an 8% polyacrylamide gel, the samples on gel were transferred onto a presoaked Immobilon-Nyt membrane (Millipore, Billerica, MA). The membrane was cross-linked in an oven for 1 min and then developed with blocking buffer and streptavidin–horseradish peroxidase conjugate before being subjected to Western blot analysis.
Statistics
Statistical analysis was performed using software Graphpad Prism 4.01 (Graph Pad Software Inc., San Diego, CA). The values given are means ± S.E.M. Statistical analysis between two samples was performed using Student’s t-test. Statistical comparisons of more than two groups were performed using one-way ANOVA with Bonferroni’s post hoc test. In all cases, a value of p less than 0.05 was considered significant.
Results
Desipramine and Fluoxetine Induce HO-1 Expression in Mes23.5 Cells
We recently reported that Omega-3 polyunsaturated fatty acids [32] and berberine [15] increase HO-1 expression and exert anti-neuroinflammatory responses in glial cells. Firstly, we identified the effect of desipramine on the regulation of HO-1 in Mes23.5 dopaminergic neurons. Stimulation of desipramine with various concentrations (5, 10, or 20 µM for 24 h) and for indicated time periods (0, 4, 8 or 24 h) increased HO-1 protein expression (Fig. 1A and 1B). Moreover, desipramine also increased HO-1 mRNA expression in time-dependent and concentration-dependent manners (Fig. 1E and 1F). We further analyzed whether a different type of antidepressant SSRI fluoxetine regulates HO-1 expression. Stimulation of fluoxetine with various concentrations (5, 10, or 20 µM for 24 h) and for indicated time periods (0, 4, 8 or 24 h) also increased HO-1 protein expression (Fig. 1C and 1D). In addition, fluoxetine also increased HO-1 mRNA up-regulation (Fig. 1G and 1H).
Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (A) or fluoxetine (C) for 24 h or with desipramine (20 µM; B) or fluoxetine (20 µM; D) for the indicated time periods (4, 8 and 24 h). Whole cell lysates were extracted and subjected to Western blot for detection of HO-1 expression. The HO-1 expression is significantly different between control group and desipramine or fluoxetine treatment groups (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, p<0.05 as compared with the vehicle control group. Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (E) or fluoxetine (G) for 8 h or with desipramine (20 µM; F) or fluoxetine (20 µM; H) for indicated time periods (4, 8 and 24 h). The quantitative data are shown in below. HO-1 mRNA expression was determined by RT-PCR. Results are the representatives of three independent experiments.
ERK, JNK Signaling Pathways are Involved in Desipramine-mediated HO-1 up-regulation
MAP kinase pathways are the most important signaling pathways that participate in transducing a variety of biological responses. Numerous evidences reported that activation of MAP kinase pathways contribute to regulation of HO-1 expression [36]–[38]. Therefore, we examined the effect of desipramine on the activation of MAP kinases in Mes23.5 dopaminergic neurons. As shown in Figure 2A, desipramine-increased HO-1 expression could be inhibited by ERK (PD98059) and JNK (SP600125) inhibitors, but not inhibitor of p38 (SB203580). Desipramine also resulted in the JNK and ERK phosphorylation initiated at 10 min and sustained to 120 min (Fig. 2B and 2C). Similarly, treatment of JNK inhibitor SP600125 also effectively reduced fluoxetine-induced HO-1 expression (Fig. 2D). Stimulation of cells with fluoxetine also increased JNK and ERK phosphorylation time-dependently (Fig. 2E and 2F).
Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.
Involvement of Nrf2 Activation in Desipramine-induced HO-1 Expression in Mes23.5 Dopaminergic Neurons
Numerous evidences reported that stress response element (StRE)/Nrf2 transcription factor pathway is an important transcriptional factor for HO-1 expression [39]–[41]. We therefore examined whether the Nrf2 signaling is involved in desipramine-induced HO-1 expression. Treatment of Mes23.5 dopaminergic neurons with desipramine enhanced Nrf2 expression time-dependently (Fig. 3A), and desipramine also resulted in an accumulation of Nrf2 in nucleus (Fig. 3B). Immunofluorescence staining of Nrf2 localization also showed that Nrf2 translocated from cytoplasm to nucleus in response to desipramine treatment for 2 h (Fig. 3C). Moreover, to evaluate desipramine-mediated Nrf2 transcription factor binding activity in Mes23.5 cells, EMSA were performed to test DNA binding activity. Stimulation of cells with desipramine for 1 or 2 h increased the DNA binding activity of Nrf2 in nuclear extracts, treatment with PD98059 or SP600125 reduced desipramine-increased DNA binding activity of Nrf2 (Fig. 4A). There was no detectable DNA binding complex without loading nuclear protein. To investigate whether the desipramine-induced HO-1 expression is mediated through Nrf2 activation, cells were transfected with Nrf2 siRNA for 24 h followed by stimulation with desipramine. Transfection with Nrf2 siRNA for 24 h in Mes23.5 dopaminergic neurons reduced Nrf2 expression. Moreover, tranfection with Nrf2 siRNA also significantly reduced desipramine-induced HO-1 expression in a concentration-dependent manner (Fig. 4B). These results suggest that desipramine-increased HO-1 expression was mediated through ERK, JNK and Nrf2 pathways in Mes23.5 dopaminergic neurons.
Cells were incubated with desipramine (20 µM) for indicated time periods (4 or 8 h), and Nrf2 expression levels in whole cell lysates (A) and nuclear extracts (B) were determined by immunoblotting with Nrf2-specific antibody. PCNA was used as the loading control for nuclear fraction. The quantitative data are shown in below. The Nrf2 expression is significantly different between desipramine treatment group and control group in nuclear extract (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with the vehicle control group. Cells were treated with or without desipramine (20 µM) for 2 h, and the levels of Nrf2 were determined by immunoflourescence (C). Note that the Nrf2 translocates from cytoplasm to nucleus in response to desipramine stimulation. Results are the representatives of three independent experiments.
(A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, p<0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, p<0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.
Desipramine-induced HO-1 Expression Protects Mes23.5 Cells from 6-OHDA or Rotenone-induced Neurotoxicity
Rotenone and 6-OHDA are useful neurotoxins as resulting in dopaminergic neuron degeneration [42]–[45]. Studies using neurotoxins have provided insights into the molecular mechanisms of dopaminergic neuronal death. To ensure the actual cell loss was correlated with the cell death assay values, a cell count assay had been evaluated. The Mes23.5 dopaminergic neurons’ cell death induced by rotenone and 6-OHDA were correlated with MTT assay value in a concentration-dependent manner (Figure S1). Surprisingly, pretreatment of desipramine at 20 µM followed by rotenone (Fig. 5A and 5C) and 6-OHDA (Fig. 5B and 5D) dramatically protected cells from neurotoxicity. The neuroprotective effect of desipramine exerts only by pretreatment, but not co-treatment or posttreatment with the neurotoxins (Figure S2). Moreover, pretreatment with desipramine also reversed rotenone- and 6-OHDA-induced tyrosine hydroxylase (TH) loss in Mes23.5 dopaminergic neurons (Fig. 5E and 5F). To further confirm the role of desipramine-induced HO-1 expression on neuroprotective effect, we examined whether desipramine protects neuronal cell death through HO-1 up-regulation. Inhibition of HO-1 activity by a HO-1 pharmacological inhibitor Znpp IX attenuated desipramine-protected neuronal cell death determined by MTT (Fig. 6A and 6B) and SRB (Fig. 6C and 6D) assay. Otherwise, HO-1 activators cobalt protoporphyrin IX (CoPP IX) (Fig. 7A and 7C) and hemin (Fig. 7B and 7D) also protected Mes23.5 cells from 6-OHDA-induced neurotoxicity.
Mes23.5 dopaminergic neurons were pretreated with various concentrations of desipramine for 8 h and followed by treatment with rotenone (3 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (A and C, respectively). The neuroprotective effects are significantly different between rotenone alone group and rotenone treated with desipramine group in both MTT and SRB assays (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with the vehicle group. #, p<0.05 as compared with the desipramine-treated group. Cells were pretreated with various concentrations of desipramine for 8 h and followed by treatment with 6-OHDA (50 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (B and D, respectively). The neuroprotective effect is significantly different between 6-OHDA alone group and 6-OHDA treated with desipramine group in SRB assay (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with the vehicle group. #, p<0.05 as compared with the desipramine-treated group. Whole cell lysates were subjected to Western blot for detection of tyrosine hydroxylase (TH). Results are the representatives of at least three independent experiments.
Cells were treated with ZnPP IX (0.1 or 0.3 µM) for 30 min and follow by treatment with desipramine for 8 h, and then treated with rotenone (A and C) or 6-OHDA (B and D) for another 16 h. Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with vehicle group.
Cells were treated with Copp IX (1 µM) (A and C) or hemin (B and D) for 8 h followed by treatment with 6-OHDA (50 µM) for another 16 h. Cell viability was determined by MTT assay and SRB assay. Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with vehicle group. #, p<0.05 as compared with desipramine-treated group.
Discussion
Clinical studies have suggested that depression and PD are closely related [46]–[48], and depression is a negative impact on the quality of life of PD patients and their families. Dopamine replacement therapy, especially with higher doses of levodopa, is sometimes accompanied by depression [49]. Moreover, the presence of levodopa-induced dyskinesia is correlated with an increased incidence of depression [50]. Furthermore, a number of PD patients who had subthalamic nucleus deep brain stimulation suffered from behavioral side effects including cognitive impairments and depression [51]–[53]. Treatment with TCAs is restricted by adverse effects, however, TCAs like desipramine are effective in treating depression in PD patients and may even reduce motor symptoms [54]. In this study, we used neurotoxins rotenone and 6-OHDA to induce dopaminergic neuron toxicity that mimics human neurodegenerative disease and neuropathological representative of PD. Previous report has reported the antioxidant effects of antidepressant agents [55]. Furthermore, it has also been reported that desipramine and fluoxetine protect neurons against microglial activation-mediated neurotoxicity [56]. However, the mechanism of antidepressants on neuroprotective effect remains unknown.
Desipramine is one of the most common tricyclic antidepressant (TCA) used to promote recovery of depressed patients. For adults, the therapeutic concentration of desipramine is approximately 1 µM [57]. Recently, it has been reported that a higher concentration of desipramine (up to 390 µM) exerts neuroprotective effect in primary culture [58]. Importantly, fluoxetine accumulates in the human brain relative to plasma, with brain concentrations ranging up to 30 µM [59]. Here, we showed that desipramine and fluoxetine at concentrations up to 20 µM increase HO-1 expression and protect neuronal cell death. Hence, explore the therapeutic potential of antidepressants may help us to identify target molecules for drug development and therapy against neurodegeneration. To ascertain the significance of their neuroprotective effects in humans, further clinical trials with antidepressants are required as well as retrospective epidemiological studies assessing the prevalence of neurodegenerative diseases. The observation that antidepressants have neuroprotective properties might have important clinical implications since these drugs are heavily prescribed worldwide and chronic treatment often lasts several months.
It has been reported that HO-1 plays a significant role in anti-apoptosis, anti-oxidant and neuroprotection [50], [60]–[63]. Numerous evidences have showed that neuronal and non-neuronal cells increase the synthesis of HO-1 under oxidative injury and inflammation conditions, which plays an important role in neuroprotective function [64], [65]. We have also reported that increased HO-1 expression in glial cells may contribute to anti-neuroinflammatory responses and exert neuroprotection [15], [32]. Importantly, the expression of anti-oxidative enzyme HO-1 exerts neuroprotective effect by protecting dopaminergic neurons [66]–[69] and might characterize the antidepressant mechanisms [32], [49], [70]–[72]. Previous study also reveal that fluoxetine, an antidepressant of the SSRIs class, attenuates brain injury via enhancement of HO-1 expression [73]. ERK and JNK pathways are also associated with various plasticity processes including neurogenesis, axonal growth, and regulation of BDNF levels [74]. Alteration of the MAPK pathways such as ERK expression has been studied in post-mortem samples of depression patients and animal models [75]–[77]. Furthermore, previous report also shown that there is different potency of ERK activation between desipramine and fluoxetine [78]. In present study, the enhancement of HO-1 expression by desipramine could be regulated by ERK and JNK, but the effect of fluoxetine was only regulated by JNK. Moreover, it has also been reported that the difference in potency between desipramine and fluoxetine exerts in many functions, such as inhibition of chemotaxis on polymorphonuclear leukocytes [79], acute hypnotic/sedative effect of ethanol [80] and development of ethanol-induced behavioral sensitization [81]. Our results reveal that desipramine-enhanced HO-1 expression sustains to 24 h, however, fluoxetine-increased HO-1 expression reaches a peak at 8 h. Our results and previous reports indicate that different types of antidepressant exert different molecular mechanisms which could provide a novel view for clinical drug choice.
It has been reported that Nrf2 may be a new drug target of treating depression [82]. Nrf2 is known as an important transcription factor involves in antioxidant response, binding to antioxidant response elements (ARE) encoding detoxification enzymes such as HO-1 [83]. Moreover, it has also been reported that activation of Nrf2 provides the insight of treatment of depression and neurodegenerative disease [84], [85]. Previous reports have shown that ERK and JNK pathways are involved in the Nrf2 activation and HO-1 up-regulation in various cell types [86], [87]. Here, our study also showed that desipramine increases HO-1 expression through ERK and JNK pathways, leading to Nrf2 activation. In line with our results, we also found that fluoxetine-induced HO-1 up-regulation is mediated by ERK and JNK pathways in Mes23.5 dopaminergic neurons. Our results demonstrated that pretreatment of desipramine protects neuronal cell death in dopaminergic neurons through HO-1 up-regulation. The neuroprotective effect of desipramine exerts only by pretreatment, but not co-treatment or posttreatment with the neurotoxin. It suggests that desipramine-increased HO-1 expression and protecting neuronal cell death might require a de novo synthesis pathway. Moreover, our study also showed that HO-1 inducer and activator effectively protect dopaminergic neurons from cell death. These findings indicate that when neuronal cells have adequate or enhanced levels of anti-oxidative agents, they can protect themselves from oxidative damage. Neuroprotection of dopaminergic neurons by the activation of Nrf2 in PD is also regarded as a promising therapeutic approach.
In conclusion, our study elucidates the neuroprotection effects of desipramine and the regulatory molecular mechanisms of desipramine-induced HO-1 expression through the ERK and JNK signaling pathways in Mes23.5 dopaminergic neurons. This is the first study reporting antidepressant against neuronal cell death. Connecting the evidence pointing to depression in PD and clinical studies will support exploring the novel therapeutic potential of desipramine and fluoxetine in treating neurodegeneration.
Supporting Information
Figure S1.
Effect of rotenone- and 6-OHDA-induced neurotoxicity in Mes23.5 dopaminergic neurons. Cells were treated with various concentrations of 6-OHDA (25, 50, 75, or 100 µM; A) or rotenone (1, 3, 5, or 10 µM; B) for 24 h. The cell viability was determined by MTT assay and cell number count. Results are expressed as the means ± S.E.M. from four independent experiments. Note that the cell viability do not have significant difference between MTT assay and cell number count in 6-OHDA treatment (up to 50 µM) and rotenone treatment (up to 10 µM).
https://doi.org/10.1371/journal.pone.0050138.s001
(TIF)
Figure S2.
Protective effect of desipramine on rotenone- and 6-OHDA-induced neurotoxicity. Mes23.5 cells were treated with desipramine before (0 or 5 h) or after (5 h) rotenone (3 µM; A) or 6-OHDA (50 µM; B) for another 16 h. The cell viability was determined by MTT assay. Results are expressed as the means ± S.E.M. from four independent experiments. *, p<0.05 as compared with the vehicle control group. #, p<0.05 as compared with the rotenone- or 6-OHDA-treated group.
https://doi.org/10.1371/journal.pone.0050138.s002
(TIF)
Author Contributions
Conceived and designed the experiments: H-YL HL D-YL. Performed the experiments: H-YL W-LY B-RH C-HL CL. Analyzed the data: H-YL W-LY HL D-YL. Contributed reagents/materials/analysis tools: B-RH HL D-YL. Wrote the paper: H-YL W-LY HL D-YL.
References
- 1. D’Amelio M, Ragonese P, Sconzo G, Aridon P, Savettieri G (2009) Parkinson’s disease and cancer: insights for pathogenesis from epidemiology. Ann N Y Acad Sci 1155: 324–334.
- 2. Livingston G, Watkin V, Milne B, Manela MV, Katona C (1997) The natural history of depression and the anxiety disorders in older people: the Islington community study. J Affect Disord 46: 255–262.
- 3. Schrag A, Jahanshahi M, Quinn N (2000) What contributes to quality of life in patients with Parkinson’s disease? J Neurol Neurosurg Psychiatry 69: 308–312.
- 4. Shulman LM, Taback RL, Rabinstein AA, Weiner WJ (2002) Non-recognition of depression and other non-motor symptoms in Parkinson’s disease. Parkinsonism Relat Disord 8: 193–197.
- 5. Cummings JL (1992) Depression and Parkinson’s disease: a review. Am J Psychiatry 149: 443–454.
- 6.
Reijnders JS, Ehrt U, Weber WE, Aarsland D, Leentjens AF (2008) A systematic review of prevalence studies of depression in Parkinson’s disease. Mov Disord 23: 183–189; quiz 313.
- 7. Leentjens AF, Van den Akker M, Metsemakers JF, Lousberg R, Verhey FR (2003) Higher incidence of depression preceding the onset of Parkinson’s disease: a register study. Mov Disord 18: 414–418.
- 8. Shiba M, Bower JH, Maraganore DM, McDonnell SK, Peterson BJ, et al. (2000) Anxiety disorders and depressive disorders preceding Parkinson’s disease: a case-control study. Mov Disord 15: 669–677.
- 9. Levkovitz Y, Caftori R, Avital A, Richter-Levin G (2002) The SSRIs drug Fluoxetine, but not the noradrenergic tricyclic drug Desipramine, improves memory performance during acute major depression. Brain Res Bull 58: 345–350.
- 10.
Larsson B (2002) Cognitive outcome of childhood depression using cognitive behavior therapy. Lakartidningen 99: 1810–1812, 1815–1819.
- 11. Alexopoulos GS, Bruce ML, Hull J, Sirey JA, Kakuma T (1999) Clinical determinants of suicidal ideation and behavior in geriatric depression. Arch Gen Psychiatry 56: 1048–1053.
- 12. Lester D, Hoffman S (1992) Self-defeating behavior, depression, and suicidal preoccupation. Psychol Rep 70: 1106.
- 13. Turker RK, Khairallah PA (1967) Demethylimipramine (desipramine), an alpha-adrenergic blocking agent. Experientia 23: 252.
- 14. Stewart JW, Quitkin F, Fyer A, Rifkin A, McGrath P, et al. (1980) Efficacy of desipramine in endogenomorphically depressed patients. J Affect Disord 2: 165–176.
- 15. Chen JH, Huang SM, Tan TW, Lin HY, Chen PY, et al. (2012) Berberine induces heme oxygenase-1 up-regulation through phosphatidylinositol 3-kinase/AKT and NF-E2-related factor-2 signaling pathway in astrocytes. Int Immunopharmacol 12: 94–100.
- 16. Bravo JA, Diaz-Veliz G, Mora S, Ulloa JL, Berthoud VM, et al. (2009) Desipramine prevents stress-induced changes in depressive-like behavior and hippocampal markers of neuroprotection. Behav Pharmacol 20: 273–285.
- 17. Maines MD, Mark JA, Ewing JF (1993) Heme Oxygenase, a Likely Regulator of cGMP Production in the Brain: Induction in Vivo of HO-1 Compensates for Depression in NO Synthase Activity. Mol Cell Neurosci 4: 396–405.
- 18. Trakshel GM, Kutty RK, Maines MD (1988) Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1). Arch Biochem Biophys 260: 732–739.
- 19. Chopra VS, Chalifour LE, Schipper HM (1995) Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells. Brain Res Mol Brain Res 31: 173–184.
- 20. Fukuda K, Panter SS, Sharp FR, Noble LJ (1995) Induction of heme oxygenase-1 (HO-1) after traumatic brain injury in the rat. Neurosci Lett 199: 127–130.
- 21. Ewing JF, Maines MD (2006) Regulation and expression of heme oxygenase enzymes in aged-rat brain: age related depression in HO-1 and HO-2 expression and altered stress-response. J Neural Transm 113: 439–454.
- 22. Ghattas MH, Chuang LT, Kappas A, Abraham NG (2002) Protective effect of HO-1 against oxidative stress in human hepatoma cell line (HepG2) is independent of telomerase enzyme activity. Int J Biochem Cell Biol 34: 1619–1628.
- 23. Scapagnini G, Butterfield DA, Colombrita C, Sultana R, Pascale A, et al. (2004) Ethyl ferulate, a lipophilic polyphenol, induces HO-1 and protects rat neurons against oxidative stress. Antioxid Redox Signal 6: 811–818.
- 24. Guzman-Beltran S, Espada S, Orozco-Ibarra M, Pedraza-Chaverri J, Cuadrado A (2008) Nordihydroguaiaretic acid activates the antioxidant pathway Nrf2/HO-1 and protects cerebellar granule neurons against oxidative stress. Neurosci Lett 447: 167–171.
- 25. Hseu YC, Chou CW, Senthil Kumar KJ, Fu KT, Wang HM, et al. (2012) Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes. Food Chem Toxicol 50: 1245–1255.
- 26. Lee YM, Cheng PY, Chim LS, Kung CW, Ka SM, et al. (2011) Baicalein, an active component of Scutellaria baicalensis Georgi, improves cardiac contractile function in endotoxaemic rats via induction of heme oxygenase-1 and suppression of inflammatory responses. J Ethnopharmacol 135: 179–185.
- 27. Huang YN, Wu CH, Lin TC, Wang JY (2009) Methamphetamine induces heme oxygenase-1 expression in cortical neurons and glia to prevent its toxicity. Toxicol Appl Pharmacol 240: 315–326.
- 28. Schipper HM (1999) Glial HO-1 expression, iron deposition and oxidative stress in neurodegenerative diseases. Neurotox Res 1: 57–70.
- 29. Lee IT, Luo SF, Lee CW, Wang SW, Lin CC, et al. (2009) Overexpression of HO-1 protects against TNF-alpha-mediated airway inflammation by down-regulation of TNFR1-dependent oxidative stress. Am J Pathol 175: 519–532.
- 30. Li L, Hamilton RF Jr, Holian A (2000) Protection against ozone-induced pulmonary inflammation and cell death by endotoxin pretreatment in mice: role of HO-1. Inhal Toxicol 12: 1225–1238.
- 31. Crawford GD Jr, Le WD, Smith RG, Xie WJ, Stefani E, et al. (1992) A novel N18TG2 × mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin. J Neurosci 12: 3392–3398.
- 32. Lu DY, Tsao YY, Leung YM, Su KP (2010) Docosahexaenoic acid suppresses neuroinflammatory responses and induces heme oxygenase-1 expression in BV-2 microglia: implications of antidepressant effects for omega-3 fatty acids. Neuropsychopharmacology 35: 2238–2248.
- 33. Lin HY, Tang CH, Chen JH, Chuang JY, Huang SM, et al. (2011) Peptidoglycan induces interleukin-6 expression through the TLR2 receptor, JNK, c-Jun, and AP-1 pathways in microglia. J Cell Physiol 226: 1573–1582.
- 34. Lu DY, Tang CH, Chen YH, Wei IH (2010) Berberine suppresses neuroinflammatory responses through AMP-activated protein kinase activation in BV-2 microglia. J Cell Biochem 110: 697–705.
- 35. Lin HY, Tang CH, Chen JH, Chuang JY, Huang SM, et al. (2011) Peptidoglycan induces interleukin-6 expression through the TLR2 receptor, JNK, c-Jun, and AP-1 pathways in microglia. J Cell Physiol 226: 1573–1582.
- 36. De Backer O, Elinck E, Blanckaert B, Leybaert L, Motterlini R, et al. (2009) Water-soluble CO-releasing molecules reduce the development of postoperative ileus via modulation of MAPK/HO-1 signalling and reduction of oxidative stress. Gut 58: 347–356.
- 37. Hsu JT, Kan WH, Hsieh CH, Choudhry MA, Schwacha MG, et al. (2008) Mechanism of estrogen-mediated intestinal protection following trauma-hemorrhage: p38 MAPK-dependent upregulation of HO-1. Am J Physiol Regul Integr Comp Physiol 294: R1825–1831.
- 38. Li-ying Z, Zhong-yuan X, Fang X, Bang-chang C (2007) Effect of radix paeoniae rubra on expression of p38 MAPK/iNOS/HO-1 in rats with lipopolysaccharide-induced acute lung injury. Chin J Traumatol 10: 269–274.
- 39. Zhang Y, Guan L, Wang X, Wen T, Xing J, et al. (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362–371.
- 40. de Vries HE, Witte M, Hondius D, Rozemuller AJ, Drukarch B, et al. (2008) Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease? Free Radic Biol Med 45: 1375–1383.
- 41. Surh YJ, Na HK (2008) NF-kappaB and Nrf2 as prime molecular targets for chemoprevention and cytoprotection with anti-inflammatory and antioxidant phytochemicals. Genes Nutr 2: 313–317.
- 42. Antkiewicz-Michaluk L, Wardas J, Michaluk J, Romaska I, Bojarski A, et al. (2004) Protective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline against dopaminergic neurodegeneration in the extrapyramidal structures produced by intracerebral injection of rotenone. Int J Neuropsychopharmacol 7: 155–163.
- 43. Betarbet R, Canet-Aviles RM, Sherer TB, Mastroberardino PG, McLendon C, et al. (2006) Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system. Neurobiol Dis 22: 404–420.
- 44. Perez V, Marin C, Rubio A, Aguilar E, Barbanoj M, et al. (2009) Effect of the additional noradrenergic neurodegeneration to 6-OHDA-lesioned rats in levodopa-induced dyskinesias and in cognitive disturbances. J Neural Transm 116: 1257–1266.
- 45. Hurtado-Lorenzo A, Millan E, Gonzalez-Nicolini V, Suwelack D, Castro MG, et al. (2004) Differentiation and transcription factor gene therapy in experimental parkinson’s disease: sonic hedgehog and Gli-1, but not Nurr-1, protect nigrostriatal cell bodies from 6-OHDA-induced neurodegeneration. Mol Ther 10: 507–524.
- 46. Palhagen SE, Ekberg S, Walinder J, Granerus AK, Granerus G (2009) HMPAO SPECT in Parkinson’s disease (PD) with major depression (MD) before and after antidepressant treatment. J Neurol 256: 1510–1518.
- 47. Richard IH, LaDonna KA, Hartman R, Podgorski C, Kurlan R (2007) The patients’ perspective: Results of a survey assessing knowledge about and attitudes toward depression in PD. Neuropsychiatr Dis Treat 3: 903–906.
- 48. Keppel Hesselink JM (1993) Serotonin, depression, and PD. Neurology 43: 1624–1625.
- 49. Tai YH, Tsai RY, Lin SL, Yeh CC, Wang JJ, et al. (2009) Amitriptyline suppresses neuroinflammation-dependent interleukin-10-p38 mitogen-activated protein kinase-heme oxygenase-1 signaling pathway in chronic morphine-infused rats. Anesthesiology 110: 1379–1389.
- 50. Choi AM, Alam J (1996) Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15: 9–19.
- 51. Appleby BS, Duggan PS, Regenberg A, Rabins PV (2007) Psychiatric and neuropsychiatric adverse events associated with deep brain stimulation: A meta-analysis of ten years’ experience. Mov Disord 22: 1722–1728.
- 52.
Takeshita S, Kurisu K, Trop L, Arita K, Akimitsu T, et al.. (2005) Effect of subthalamic stimulation on mood state in Parkinson’s disease: evaluation of previous facts and problems. Neurosurg Rev 28: 179–186; discussion 187.
- 53. Temel Y, Blokland A, Ackermans L, Boon P, van Kranen-Mastenbroek VH, et al. (2006) Differential effects of subthalamic nucleus stimulation in advanced Parkinson disease on reaction time performance. Exp Brain Res 169: 389–399.
- 54. Richard IH (2000) Depression in Parkinson’s Disease. Curr Treat Options Neurol 2: 263–274.
- 55. Behr GA, Moreira JC, Frey BN (2012) Preclinical and clinical evidence of antioxidant effects of antidepressant agents: implications for the pathophysiology of major depressive disorder. Oxid Med Cell Longev 2012: 609421.
- 56. Zhang F, Zhou H, Wilson BC, Shi JS, Hong JS, et al. (2012) Fluoxetine protects neurons against microglial activation-mediated neurotoxicity. Parkinsonism Relat Disord 18 Suppl 1S213–217.
- 57. Wille SM, Cooreman SG, Neels HM, Lambert WE (2008) Relevant issues in the monitoring and the toxicology of antidepressants. Crit Rev Clin Lab Sci 45: 25–89.
- 58. Roth S, Kinne A, Schweizer U (2010) The tricyclic antidepressant desipramine inhibits T3 import into primary neurons. Neurosci Lett 478: 5–8.
- 59. Karson CN, Newton JE, Livingston R, Jolly JB, Cooper TB, et al. (1993) Human brain fluoxetine concentrations. J Neuropsychiatry Clin Neurosci 5: 322–329.
- 60. Le WD, Xie WJ, Appel SH (1999) Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury. J Neurosci Res 56: 652–658.
- 61. Lee TS, Chau LY (2002) Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med 8: 240–246.
- 62. Minamino T, Christou H, Hsieh CM, Liu Y, Dhawan V, et al. (2001) Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia. Proc Natl Acad Sci U S A 98: 8798–8803.
- 63. Terry CM, Clikeman JA, Hoidal JR, Callahan KS (1998) Effect of tumor necrosis factor-alpha and interleukin-1 alpha on heme oxygenase-1 expression in human endothelial cells. Am J Physiol 274: H883–891.
- 64. Suttner DM, Dennery PA (1999) Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J 13: 1800–1809.
- 65. Parfenova H, Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, et al. (2006) Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection. Am J Physiol Cell Physiol 290: C1399–1410.
- 66. Quesada A, Ogi J, Schultz J, Handforth A (2011) C-terminal mechano-growth factor induces heme oxygenase-1-mediated neuroprotection of SH-SY5Y cells via the protein kinase C/Nrf2 pathway. J Neurosci Res 89: 394–405.
- 67. Huang JY, Chuang JI (2010) Fibroblast growth factor 9 upregulates heme oxygenase-1 and gamma-glutamylcysteine synthetase expression to protect neurons from 1-methyl-4-phenylpyridinium toxicity. Free Radic Biol Med 49: 1099–1108.
- 68. Yamamoto N, Izumi Y, Matsuo T, Wakita S, Kume T, et al. (2010) Elevation of heme oxygenase-1 by proteasome inhibition affords dopaminergic neuroprotection. J Neurosci Res 88: 1934–1942.
- 69. Minelli A, Conte C, Grottelli S, Bellezza I, Emiliani C, et al. (2009) Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling. J Neurochem 111: 956–966.
- 70. Oh GS, Pae HO, Choi BM, Chae SC, Lee HS, et al. (2004) 3-Hydroxyanthranilic acid, one of metabolites of tryptophan via indoleamine 2,3-dioxygenase pathway, suppresses inducible nitric oxide synthase expression by enhancing heme oxygenase-1 expression. Biochem Biophys Res Commun 320: 1156–1162.
- 71. Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression. Nature 455: 894–902.
- 72. Chen Z, Xu H, Haimano S, Li X, Li XM (2005) Quetiapine and venlafaxine synergically regulate heme oxygenase-2 protein expression in the hippocampus of stressed rats. Neurosci Lett 389: 173–177.
- 73. Shin TK, Kang MS, Lee HY, Seo MS, Kim SG, et al. (2009) Fluoxetine and sertraline attenuate postischemic brain injury in mice. Korean J Physiol Pharmacol 13: 257–263.
- 74. Chen G, Manji HK (2006) The extracellular signal-regulated kinase pathway: an emerging promising target for mood stabilizers. Curr Opin Psychiatry 19: 313–323.
- 75. Dwivedi Y, Rizavi HS, Roberts RC, Conley RC, Tamminga CA, et al. (2001) Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects. J Neurochem 77: 916–928.
- 76. Dwivedi Y, Rizavi HS, Zhang H, Roberts RC, Conley RR, et al. (2009) Aberrant extracellular signal-regulated kinase (ERK)1/2 signalling in suicide brain: role of ERK kinase 1 (MEK1). Int J Neuropsychopharmacol 12: 1337–1354.
- 77. Feng P, Guan Z, Yang X, Fang J (2003) Impairments of ERK signal transduction in the brain in a rat model of depression induced by neonatal exposure of clomipramine. Brain Res 991: 195–205.
- 78. Valjent E, Pages C, Herve D, Girault JA, Caboche J (2004) Addictive and non-addictive drugs induce distinct and specific patterns of ERK activation in mouse brain. Eur J Neurosci 19: 1826–1836.
- 79. Sacerdote P, Bianchi M, Panerai AE (1994) Chlorimipramine and nortriptyline but not fluoxetine and fluvoxamine inhibit human polymorphonuclear cell chemotaxis in vitro. Gen Pharmacol 25: 409–412.
- 80. Boyce-Rustay JM, Palachick B, Hefner K, Chen YC, Karlsson RM, et al. (2008) Desipramine potentiation of the acute depressant effects of ethanol: modulation by alpha2-adrenoreceptors and stress. Neuropharmacology 55: 803–811.
- 81. Simon O’Brien E, Legastelois R, Houchi H, Vilpoux C, Alaux-Cantin S, et al. (2011) Fluoxetine, desipramine, and the dual antidepressant milnacipran reduce alcohol self-administration and/or relapse in dependent rats. Neuropsychopharmacology 36: 1518–1530.
- 82. Maes M, Fisar Z, Medina M, Scapagnini G, Nowak G, et al. (2012) New drug targets in depression: inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates–Nrf2 activators and GSK-3 inhibitors. Inflammopharmacology 20: 127–150.
- 83. Scapagnini G, Vasto S, Abraham NG, Caruso C, Zella D, et al. (2011) Modulation of Nrf2/ARE pathway by food polyphenols: a nutritional neuroprotective strategy for cognitive and neurodegenerative disorders. Mol Neurobiol 44: 192–201.
- 84. Barone MC, Sykiotis GP, Bohmann D (2011) Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson’s disease. Dis Model Mech 4: 701–707.
- 85. Tufekci KU, Civi Bayin E, Genc S, Genc K (2011) The Nrf2/ARE Pathway: A Promising Target to Counteract Mitochondrial Dysfunction in Parkinson’s Disease. Parkinsons Dis 2011: 314082.
- 86. Yuan X, Xu C, Pan Z, Keum YS, Kim JH, et al. (2006) Butylated hydroxyanisole regulates ARE-mediated gene expression via Nrf2 coupled with ERK and JNK signaling pathway in HepG2 cells. Mol Carcinog 45: 841–850.
- 87. Xu C, Yuan X, Pan Z, Shen G, Kim JH, et al. (2006) Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2. Mol Cancer Ther 5: 1918–1926.