Significant outcomes
• Methylphenidate (MPD) increases the level of phosphorylation of protein extracellular signal-regulated kinase 1 (ERK1) at a dose of >1 mg/mg.
• MPD decreases the level of phosphorylation of c-Jun amino-terminal kinase 1 (JNK1) at a dose of >2 mg/kg.
• MPD decreases the level of phosphorylation of JNK2 at 2 mg/kg.
Limitation
• This study could have used other brain structures like the hippocampus and cortex, which are also involved in attention deficit/hyperactivity (ADHD).
Introduction
ADHD disorder is the most commonly diagnosed neuropsychiatric disorder in childhood, characterised by excessive levels of inattentiveness, impulsivity, and hyperactivity (Reference Peterson, McDonagh and Fu1). Most ADHD patients benefit from treatment with MPD, irrespective of the aetiology of the disorder, which effectively reduces symptoms in up to 70% of paediatric patients (Reference Wilens2). However, recognition that ADHD may persist into adulthood has led to an increased use of MPD in adult patients; extended use of MPD can have chronic effects in the organism (Reference Quinn3). Studies on humans suggest that blockade of the dopamine transporter (DAT) produces the therapeutic effect of MPD by increasing the concentration of dopamine in the central nervous system (CNS) (Reference Davis, Vanhoutte, Pages, Caboche and Laroche4). However, it is not yet clear whether the effects on the CNS are due to prolonged use of MPD.
In this context, the CNS is well known for its involvement in the pathway of mitogen-activated protein kinases (MAPKs) (Reference Sweatt5). The three main MAPKs characterised in the brain of mammals are the protein kinase regulated by ERK1/2, proteins JNK1/2, and the protein p38. Activation of these proteins regulates several cellular events ranging from proliferation and cellular differentiation to apoptosis (Reference Wang and Bonner6). In addition, dopamine also positively regulates ERK by acting through multiple signalling mechanisms that involve D1 and/or D2-class dopamine receptors. Although ERK has been shown to participate in acute responses to cocaine, it appears to be mostly involved in the development of long-term changes in gene expression, synaptic plasticity, and locomotor responses following repeated exposure to this drug (Reference Beaulieu, Sotnikova, Gainetdinov and Caron7).
Because critical actions of psychostimulants appear to involve the dopaminergic inputs into the striatum and the nucleus accumbens, these structures should also be investigated. To understand the brain alterations caused by long-term exposure to MPD, the aim of this work was to investigate the influence of the medicament MPD on the expression and phosphorylation of MAPKs in the striatum of young rats subjected to continued treatment and to try establish the molecular basis of its mechanism of action and, consequently, possible processes in the physiopathology and neurochemistry responsible for the manifestation of ADHD.
Materials and methods
Animals
Young [postnatal day (PD) 25; 75–85 g] male Wistar rats obtained from our breeding colony were housed five to a cage with food and water available ad libitum and maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental procedures were in accordance with the recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) for animal care.
Experimental design
Young rats (n = 10) were administered MPD [1, 2, 5, or 10 mg/kg; Ritalin® (Novartis; East Hanover, NJ, USA) – racemic dextroand levo-MPD mixture dissolved in saline solution] once a day or an intraperitoneal injection of saline for 28 days (chronic treatment) or for 1 day (acute treatment). The acute treatment was at PD25 and the chronic treatment between PD25 and PD53 (Reference Andreazza, Frey and Valvassori8–Reference Gomes, Souza and Inácio12). Two hours after the last administration of MPD or saline, the animals were killed and their striatum was dissected and rapidly solubilised with SDS-stopping solution (4% SDS, 2 mM EDTA, 8% β-mercaptoethanol, and 50 mM Tris, pH 6.8). Aliquots of proteins were quantified and extracts were stored at −80°C until the preparation of samples for immunoblot analysis.
Immunoblot analysis
Equal amounts of protein (50 μg) were separated for SDS–PAGE using a 12% polyacrylamide gel, followed by their transfer to nitrocellulose membranes (Hybond ECL, GE Healthcare, Piscataway, NJ, USA). Protein loading and efficiency of blot transfer were monitored by staining with Ponceau S (0.5% ponceau/1% acetic acid; Sigma Chemical Co., St Louis, MO, USA). The membranes were blocked for 45 min with TBS-T (tris-buffered saline and Tween 20 0.1%, pH 7.4) plus 5% non-fat milk. Membrane blots were incubated with polyclonal anti-phospho-p44/42 MAPK or anti-phospho SAPK/JNK (both 1 : 1000 – Cell Signaling Technology, MA, USA) diluted in TBS-T and stored overnight at 4°C. After washing, the membranes were incubated for 1 h with goat anti-rabbit IgG (1 : 2000 – Cell Signaling Technology, MA, USA) conjugated with horseradish peroxidase. Immunocomplexes were visualised using the enhancing chemiluminescence detection system (LumiGLO™ – Cell Signaling Technology) as described by the manufacturer and visualised on ImageQuant. Densitometric analysis was performed using Scion Image Software, version Beta 4.0.2 (Scion Corporation, Frederick, MD, USA).
Statistical analysis
Results were analysed by STATISTICA, version 7.0 (StatSoft Inc., Tulsa, OK, USA). All data are presented as a percentage of control ± SEM (standard error of the mean), and each value reflects the mean of 4–5 animals per group for immunoblot analysis. In all cases, statistical analyses were performed using two-way analysis of variance (ANOVA) with chronic and acute treatments as the main factors, followed by the Duncan test for immunoblot analysis. The level of significant difference was accepted at p < 0.05.
Results
Figure 1 shows that chronic treatment with MPD is capable of modifying the levels of phosphorylation of proteins ERK1/2 (a) and JNK1/2 (b). Our results showed that MPD increased phosphorylation of the protein ERK1 at a dose of 1 mg/mg (p < 0.05) (Fig. 1a) and decreased phosphorylation of JNK1/2 at 2 mg/kg (p < 0.05), when compared as a percentage of control (Fig. 1b). However, Fig. 2 demonstrates that acute treatment with MPD did not alter the phosphorylation of proteins ERK1/2 (Fig. 2a) and JNK1/2 (Fig. 2b; p > 0.05).
Fig. 1 Phosphorylation of proteins JNK1/2 and ERK1/2 in the striatum as a result of chronic treatment with MPD (1, 2, 5, or 10 mg/kg) or saline. Immunodetections were determined as described in the ‘Materials and methods’ section. (a) A summary of optical density data expressed as a percentage of control of pERK1/2 contents ± SEM. (b) A summary of optical density data expressed as a percentage of control of pJNK1/2 contents ± SEM. *p < 0.05 versus the control group; two-way ANOVA and post hoc Duncan test. ERK1/2, extracellular signal-regulated kinases 1 and 2; JNK1/2, c-Jun amino-terminal kinases 1 and 2; MPD, methylphenidate; SAL, saline.
Fig. 2 Phosphorylation of proteins JNK1/2 and ERK1/2 in the striatum as a result of acute treatment with MPD (1, 2, 5, or 10 mg/kg) or saline. Immunodetections were determined as described in the ‘Materials and methods’ section. (a) A summary of optical density data expressed as a percentage of control of pERK1/2 contents ± SEM. (b) A summary of optical density data expressed as a percentage of control of pJNK1/2 contents ± SEM; two-way ANOVA and post hoc Duncan test. ERK1/2, extracellular signal-regulated kinases 1 and 2; JNK1/2, c-Jun amino-terminal kinases 1 and 2; MPD, methylphenidate; SAL, saline.
Discussion
The prolonged time course of the initiation and maintenance of psychostimulant dependence has raised the question of whether long-term drug-induced alterations in gene expression play a critical role. In this study, we observed that chronic treatment with MPD is capable of modifying the levels of phosphorylation of proteins ERK1 (at a dose of 1 mg/kg) and JNK1/2 (at a dose of 2 mg/kg). The alteration in the index of activation of MAPKs can be a molecular mechanism through which MPD exerts its therapeutic effect. Previous studies demonstrate that amphetamine increases the phosphorylation of cAMP-responsive element binding protein (CREB) in the striatum (Reference Konradi, Cole, Heckers and Hyman13). A recent study shows that both cocaine and methamphetamine upregulate pERK1/2 in the striatum (Reference Adams, Hanson and Keefe14). These findings suggest that both dopaminergic and glutamatergic inputs are capable of regulating the ERK1/2 signalling pathways leading to the phosphorylation of CREB and Elk-1 and thus to alterations in gene expression in striatal neurons.
In this context, ERK1 and ERK2 can also be activated by a variety of extracellular signalling molecules, including growth factors, hormones, and neurotransmitters. Calcium influx in particular has gained importance because of an increase in ERK activity as a result of N-methyl-d-aspartate (NMDA) glutamate receptor-stimulated calcium influx. Thus, it is attractive to speculate that NMDA receptor activation may be the initial trigger leading to activation of the ERK pathway; calcium influx may also lead to activation of the JNK pathway through other signalling mechanisms (Reference Shen, Tsimberg, Salvadore and Meller15). It is known that pERK is increased by high levels of reactive oxygen species. In this study, only 1 mg/kg increased the phosphorylation of ERK1 and 2 mg/kg decreased the phosphorylation of JNK1/2. Previous findings showed that MPD doses of <5 mg/kg administered intraperitoneally better reflect those used clinically in patients (Reference Bolaños, Barrot, Berton, Wallace-Black and Nestler16) and previous studies showed that a dose of 2 mg/kg is utilised in studies on animals to investigate alterations in the CNS (Reference Andreazza, Frey and Valvassori8–Reference Gomes, Souza and Inácio12).
In contrast to ERK and Akt, JNK is more commonly considered a pro-death signalling molecule. In addition to its well-established role in the execution of apoptosis, the JNK pathway is likely to be involved in the neurodegenerative cascade and has been implicated in the loss of dopamine neurons (Reference Saporito, Hudkins and Maroney17). Our study shows that chronic MPD treatment with 2 mg/kg decreases phosphorylation of JNK1/2 in the striatum. Inflammatory signals, changes in levels of reactive oxygen species, ultraviolet radiation, protein synthesis inhibitors, and a variety of stress stimuli can activate JNK. One way this activation may occur is through disruption of the conformation of sensitive protein phosphatase enzymes; specific phosphatases normally inhibit the activity of JNK itself and the activity of proteins linked to JNK activation. Downstream molecules that are activated by JNK include c-Jun, ATF2, ELK1, SMAD4, p53, and HSF1. The downstream molecules that are inhibited by JNK activation include NFAT4, NFATC1, and STAT3. By activating and inhibiting other small molecules in this manner, JNK activity regulates several important cellular functions including cell growth, differentiation, survival, and apoptosis [for a review, see Vlahopoulos and Zoumpourlis (Reference Vlahopoulos and Zoumpourlis18)]. Studies have shown that MPD is capable of modifying the expression of immediate genes c-fos and zif 268 (Reference Brandon and Steiner19), whose expression and activation are regulated by MAPKs (Reference Eriksson, Taskinen and Leppä20).
Although MPD has been used extensively, there is limited knowledge on its potential for producing molecular changes in the brain, especially in intracellular signalling events triggered by receptor activation, and their implications in functional effects. In other studies conducted by our group using MPD, chronic treatment led to the following changes in the striatum: (1) increase in TBARS content and protein carbonyl formation (1, 2, and 10 mg/kg) (Reference Martins, Reinke, Petronilho, Gomes, Dal-Pizzol and Quevedo21); (2) increase in mitochondrial respiratory chain enzyme activities (2 and 10 mg/kg) (Reference Fagundes, Rezin and Zanette22); (3) increase in DNA damage (10 mg/kg) (Reference Andreazza, Frey and Valvassori8); (4) increase in creatine kinase activity (2 and 10 mg/kg) (Reference Scaini, Fagundes and Rezin23); and (5) cognitive impairment (2 mg/kg) (Reference Gomes, Souza and Valvassori10–Reference Gomes, Souza and Inácio12). Recently, another research group observed that a low dose of MPD enhanced the learning and memory capacity of rats, rather than deteriorating it, supporting the use of MPD as a drug to treat attention deficit hyperactive disorder (Reference Veetil and Mukkadan24). In addition, an overdose of MPD was seen to upregulate adenosine A(1) receptors in the frontal cortex, but this receptor was not involved in anxiolytic effects (Reference Mioranzza, Costa and Botton25). A reduction in the immunocontent of brain-derived neurotrophic factor and increased acetylcholinesterase activity in the prefrontal cortex but not in the hippocampus of rats were also observed after treatment with MPD (Reference Scherer, da Cunha, Matté, Schmitz, Netto and Wyse26). As the phosphorylation of ERK1 leads to proliferation and differentiation and the phosphorylation of JNK1/2 leads to inflammation and apoptosis, we believe that chronic treatment with MPD at low doses is beneficial to the central nervous system during the course of development.
Acknowledgements
This research was supported by grants from CNPq (C.R.B. and J.Q.) and UNESC (C.R.B. and J.Q.), both from Brazil. C.R.B. and J.Q. are CNPq Research Fellows. C.M.C. is the holder of a CNPq Studentship.
