- Email: [email protected]
Apelin-13 attenuates motor impairments and prevents the changes in synaptic plasticity-related molecules in the striatum of Parkinsonism rats
Peptides 117 (2019) 170091
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Apelin-13 attenuates motor impairments and prevents the changes in synaptic plasticity-related molecules in the striatum of Parkinsonism rats Elham Haghparasta,b, Vahid Sheibania, Mehdi Abbasnejadb, Saeed Esmaeili-Mahania,b, a b
T
⁎
Laboratory of Molecular Neuroscience, Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences. Kerman, Iran Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman. Kerman, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Apelin-13 Parkinson’s disease 6-OHDA Motor impairment Striatum Synaptic plasticity
The striatum plays a critical role in motor control and also learning and memory of motor skills. It has been reported that striatal synaptic components are significantly decreased in dopaminergic-denervated striatum. In this study the effects of apelin-13 were investigated on motor disorders and striatal synaptosomal expression of PSD-95, neurexin1, neuroligin, metabotropic glutamate receptor (mGlu R1) and dopaminergic receptors (DR1 and DR2) in rat parkinsonism experimental model. 6-hydroxydopamine (6-OHDA) was injected into the substantia nigra. Apelin-13 (1, 2 and 3 μg/rat) was administered into the substantia nigra one week after the 6-OHDA injection. Accelerating rotarod, beam-balance, beam-walking and bar tests were performed one month after the apelin injection. Immunohistochemistry staining of dopaminergic neurons was performed. The levels of synaptic proteins were determined by immunoblotting. 6-OHDA-treated animals showed a significant impairment in motor-skill tasks and a dramatically change in the expression levels of mentioned proteins. Apelin-13 (3 μg/rat) significantly attenuates the motor impairments and prevents the changes in striatal synaptic elements in 6-OHDA-treated animals. In addition, it could rescue the dopaminergic neurons of the substantia nigra. The data will potentially extend the possible benefic aspect of apelin in neurodegenerative disorders.
1. Introduction Parkinson’s disease (PD) is defined by the dopamine (DA) neurons degeneration originating in the substantia nigra pars compacta (SNc) that leads to profound depletion of dopamine in the striatum and consequently severe movement abnormalities such as tremors, catalepsy and bradykinesia [1,2]. PD pathology is associated not only with dopaminergic neurodegeneration, but also with abnormal patterns of firing of neural pathways. Dysfunctional synaptic plasticity at the onset of PD is associated with the development of the motor complications, such as dyskinesia [3,4]. Projection pathways of DA neurons from the nigrostriatal bundle to the dorsal striatum have a central role in control of fine motor functions. More than 95% of striatal neurons are medium spiny neurons (MSNs) that form asymmetric synapses with glutamatergic inputs and symmetric connections at the DA synapses [5]. Therefore, the
degeneration of the nigrostriatal dopaminergic pathway causes a significant change in the striatal neurons and then resulting in loss of striatal synaptic plasticity [6]. Postsynaptic density protein 95 (PSD-95) is a main scaffolding protein in the dendritic spines of MSNs in the striatum [7], which interacts with dopamine receptor [8] and potentially regulates N-methylD-aspartate (NMDA) and dopamine D1 receptors trafficking and function in the synapses [9,10]. It has been reported that striatal PSD-95 levels significantly decrease in dopaminergic-denervation striatum which support its role in PD [11–13]. The neuroligin and β-neurexin complex is a major component of synaptic connection and acts as balancer of pre- and post-synaptic proteins. The ability of neuroligin and β-neurexin to form and maintain excitatory and inhibitory synapses indicates that they have potential roles in the induction of neurological disorders such as PD [14]. Striatal MSNs exhibit numerous ionic conductance that shape their firing properties, and such conductances are sensitive to some
Abbreviations: DA, dopamine; SNc, substantia nigra pars compacta; PD, Parkinson's disease; 6-OHDA, 6-hydroxydopamine; CNS, central nervous system; APJ, apelin receptor; MSNs, medium spiny neurons; PSD-95, postsynaptic density protein 95; mGluR1, metabotropic glutamate receptor 1; DR1, dopamine receotor1; DR2, dopamine receotor2 ⁎ Corresponding author at: Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, P.O. Box 76135-133, Kerman, Iran. E-mail addresses: [email protected], [email protected] (S. Esmaeili-Mahani). https://doi.org/10.1016/j.peptides.2019.05.003 Received 7 February 2019; Received in revised form 24 April 2019; Accepted 17 May 2019 Available online 21 May 2019 0196-9781/ © 2019 Elsevier Inc. All rights reserved.
Peptides 117 (2019) 170091
E. Haghparast, et al.
allowed to recover from the anesthesia and then placed in individual cages. After one weeks, as a recovery period, apelin (phoenix pharmaceuticals INC, USA; cat # :057-18) was dissolved in ACSF and delivered into the right SNc over 2 min. The injection needles were left in place for an additional 1 min before they were slowly withdrawn [24].
neuromodulators such as DA. The expression of D1 receptors in striatonigral MSNs regulates sodium, potassium, and calcium channels which results in modulation of MSN excitability [15]. In PD, the loss of the DAergic neurons in the striatum leads to loss of DA-mediated inhibition, possibly results in excess excitatory transmission [16]. Neurotransmission, during PD-like neurodegeneration, can be regulated by dopamine (DA) and metabotropic glutamate (mGlu) receptors [17]. The apelin peptide is the endogenous ligand for G protein-coupled receptor, APJ. Apelin-13 is more potent than other type of apelin in competitive binding to the APJs [18,19]. The distribution of apelin and its receptors in the amygdala, hypothalamus, hippocampus, cerebellum, striatum, substantia nigra, and hypothalamus [20,21], suggests that apelin might have critical and broad roles in physiological and pathophysiological conditions. Recently, it has been shown that the apelinAPJ system might play an important role in the neuronal survival and signaling in the central nervous system and therefore, apelin should be further investigated as a potential protective neuropeptide in various neurological disorders that might cause brain injury [22]. It has been documented that the plasma concentrations of apelin are reduced with age, so that plasma apelin concentrations in aged rats are just half of those in adults [23]. We have recently reported that apelin significantly improves cognitive performance in 6-OHDA-induced rat model of PD [24] and also, has a protective effect on 6-OHDA-induced toxicity in SH-SY5Y dopaminergic cells [25]. The present study was design to find the possible neuromodulatory effects of apelin-13 on the movement disorders and some of the striatal synaptosomal proteins levels (mGlu, D1 and D2 receptors, neuroligin, neurexin1 and PSD-95 which play important roles in the maintaining and integrity of neural connection) in 6-OHDA-induced hemi parkinsonian male rats.
2.3. Accelerating rotarod The rotarod apparatus (TSE, SER-NO; 040910-02, Germany) was used for Motor-skill learning and motor performance at different progressively higher speeds. Briefly, at the first day, the rats were trained on the rotating rod (with persistent 10 rpm for 3 min) to adapt with the apparatus. Next day, the animals were located on the rotating rod with an accelerating protocol (10–60 rpm in 5 min). The latency to fall was measured in seconds. Each animal was given three trials with a 30 min inter-trial rest interval and the mean of trials was calculated (Bellum et al., 2013). 2.4. Balance beam and Beam traversal test The balance beam is a test for the evaluation of motor coordination in rodents. The beam consists of 80 cm long horizontal wooden rod (1.5 cm diameter) which was placed at a height of 80 cm above a thick foam. Each animal was placed on the center of the rod and the time that each animal remained on the beam was measured. Cut-off time was 60 s. Motor coordination and balance of the animals were assessed by measuring the ability of the rats to traverse of a narrow beam (2.5 × 100 cm) from bright light to reach a dark goal box placed on the opposite end of the beam. The wooden beam was fixed 80 cm above a thick foam. The animal was placed behind the starting line (facing dark goal box) and released. The latency to traverse the beam (beam Freeze time) and the time of the hind feet were placed entirely inside the goal box at the opposite end of the beam were recorded. If an animal was incapable to complete the beam run, the test was finished at 2 min (cutoff time) [12].
2. Materials and methods 2.1. Animals Adult male Wistar rats weighing 250–300 g were used in this study. Before and during the study, the animals were kept in polypropylene cages, four per cage, under controlled temperature (24 ± 1 °C) and 12h light-dark cycle and had free access to food (standard rodent diet) and water. All experimental protocols and treatments were approved by the Ethics Committee of Kerman Neuroscience Research Center (Ethics Code: EC/97-54/KNRC). The animals were randomly divided into several experimental groups, each containing 7 rats: (I) Control; (II) Neurotoxin sham (sham N); (III) Lesion; (IV) Lesion + Vehicle; (V) Lesion animals which received 1 μg/rat of apelin (Lesion + apelin 1); (VI) Lesion animals which had 2 μg/rat of apelin (Lesion + apelin 2); and (VII) lesion animals which were treated with 3 μg/rat of apelin (Lesion + apelin 3).
2.5. Bar test Anterior limbs of the rats were placed on a bar (9 mm in diameter) fixed at the height of 9 cm away from the surface. The duration of maintaining this position was determined as the intensity of catalepsy. The end point of catalepsy was considered when any exploratory head movements or displacement of one or both forearms was performed. The cut off time of the test was 120 sec and the test was done in three consecutive times with 10 min interval and the average latencies were calculated (Nayebi et al., 2010).
2.2. Experimental design
2.6. Immunohistochemistry (IHC)
Under ketamine and xylazine (80 and 10 mg/kg, i.p, respectively) anesthesia, the rats were fixed in a stereotaxic frame in the flat position with the incisor bar positioned 3.3 mm below the interaural line. A guide cannula (22-gauge stainless steel) was implanted and fixed just above (1 mm) the right SNc according the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986); coordinates with respect to bregma: 3.7 mm antero-posterior; 2.2 mm lateral to the midline; 7.7 mm dorsoventral from the dura surface. 6-OHDA (Sigma-Aldrich Co., LLC, USA) was dissolved in saline with 0.2 % ascorbic acid and gradually injected into the right SNc (8 μg/2 μl) at a rate of 1 μL/min. Neurotoxin sham animals were submitted to the same procedure but 2 μl of vehicle was infused into the SNc. Lesion + vehicle animals were also submitted to the same procedure and received 6-OHDA as a neurotoxin plus artificial cerebrospinal fluid (ACSF, consisting of (in mM): NaCl, 124; NaHCO3, 25; D glucose, 10; KCl, 4.4; MgCl2, 2; KH2PO4, H2O, 1.25 and CaCl2, 6H20, 2.) as vehicle of apelin. Animals were
Under deep anesthesia using intramuscular injection of ketamine and xylazine, the brains were fixed by cardiac perfusion with 0.9% NaCl followed by 4% paraformaldehyde, pH 7.4. The brains were removed and post fixed in the same fixative overnight. Then, the striatum and the SN were cut and dehydrated in alcohol series, cleared in xylene, infiltrated with paraffin and embedded in paraffin. The paraffin embedded tissue sections (5 mm sized) were de-paraffinized with xylene and endogenous peroxidase activity was quenched with 3% H2O2 in methanol for 30 min in the dark. Tissue sections were dehydrated through graded alcohols and subjected to antigen retrieval using 10 mM sodium citrate. The sections were washed with TBST (Tris Borate Saline Tween-20) and then blocked with 5% bovine serum albumin (BSA) for one hour. The SN sections were incubated with the respective mouse monoclonal primary antibody against tyrosine hydroxylase and striatal sections were incubated with anti-Dopamine D2 receptor (PA2234). The Slides were then washed for 5 min in TBST and incubated for 1 h 2
Peptides 117 (2019) 170091
E. Haghparast, et al.
with the respective HRP (Horseraddish Peroxidase) conjugated antimouse secondary antibody diluted with TBS in a ratio of 1:200. After washing, the slides were incubated with DAB (3,39-diaminobenzidine tetrahydrochloride) (Sigma) and immediately washed under tap water just after color development. The sections were then counter with hematoxylin. and mounted with DPX (dibutyl phthalate xylene) and then observed under a light microscope.
2.7. Western blot analysis for synaptic proteins The right striatum was homogenized in 10% (w/v) of 0.32 M sucrose-HEPES buffer (HEPES-buffer solution: 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 5 mM HEPES, pH 7.4) along with protease inhibitor. Further, homogenate was centrifuged at 4 °C for 10 min at 600 g. Then supernatant was diluted 1:1 with 1.3 M HEPES sucrose to produce a suspension at a final concentration of 0.8 M HEPES sucrose. This suspension was further centrifuged at two to three times at 12,000 g for 15 min at 4 °C. The supernatant was discarded each time. The pellet that consisting of synaptosomes was suspended in RIPA buffer (mixed with protease inhibitors: 1 mM phenyl methyl sulfonyl fluoride, 2.5 mg/ml leupeptin, and 10 mg/ml aprotinin) along with 0.2% Triton X-100 and centrifuged at 20,000 g for 30 min. This step is responsible for the disruption of synaptic membranes and enriches the synapse proteins. Further, the resulting pellet was re-suspended only in RIPA buffer (mixed with protease inhibitors). Proteins contents were quantified by the Bradford assay method. Bovine serum albumin (BSA; 0.01–0.1 mg/ml) was used as standard. Equal amounts of protein per sample (40 μg) were added to sample buffer, heated at 100 °C for 5 min and were resolved electrophoretically on 10% SDS–PAGE and then proteins were transferred to PVDF membranes (Hybond ECL, GE Healthcare Bio-Sciences Corp. NJ, USA). After blocking (2 h at room temperature) with 5% non-fat dried milk in Trisbuffered saline with Tween 20 (TBS-T: 20 mM Tris–HCl, 150 mMNaCl, pH 7.5, 0.1% Tween 20), the membranes were probed with anti-PSD95, anti-neurexin1, anti-neuroligin, anti-D1 or anti-mGlu1 receptors antibodies (Santa Cruz Biotechnology, USA, 1:1000) for overnight at 4 °C. After washing with TBS-T (three times for 5 min), the membranes were exposed to secondary anti-species antibody (1:10,000, Santa Cruz Biotechnology) for 1 h at room temperature with a secondary antibody attached to horseradish peroxidase (1:15,000, GE Healthcare BioSciences Corp. NJ, USA). All antibodies were diluted in blocking buffer. Pre-stained protein ladder (SM7012, Cinagen Co, Iran) was used for monitoring protein separation and estimating the molecular weight and integrated intensity of blotting bands. The antibody-antigen complexes were detected using the ECL system and exposed to Lumi-Film chemiluminescent detection film (Roche Germany). Lab Works analyzing software (UVP, UK) was used to evaluate the intensity of the blotting bands. β-actin (1:10,000) was used as loading control. The expression values were presented as tested proteins/β-actin ratio for each sample (Kamat et al., 2014).
Fig. 1. Effect of apelin on rotarod test (A) and bar test (B) performance. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control and sham N groups. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion and lesion + vehicle groups. † p < 0.05 versus apelin-13 (1 μg/rat).
3. Results 3.1. Effects of apelin on rotarod and bar test performance in 6-OHDAinjected rats There was no significant difference in rotarod performance between control and neurotoxin sham groups (P = 0.995; Fig. 1A). Injection of 6-OHDA caused a significant reduction in the latency to fall in lesion and drug sham groups [F (6,48) = 13.739; P = 0.000]. However, apelin-treated animals (3 μg/rat) significantly attenuated the decreasing effect of 6-OHDA on rotarod motor performance (P < 0.01). Apelin in doses of 1 and 2 μg/rat had no significant effect on 6-OHDAinduced motor impairment in rotarod test. Results of the bar test in different groups showed that control and surgical sham had no sign of catalepsy [F (6,48) = 53.880; P = 0.000; Fig. 1B]. 6-OHDA induced a significant catalepsy in the lesion and drug sham groups (P = 0.000). In contrast, all apelin-treated groups showed a significant and dose dependent (P < 0.0001) recovery in catalepsy time.
2.8. Statistical analysis 3.2. Effects of apelin on balance beam and beam traversal test in 6-OHDAinjected rats
All of the statistical analyses were performed using IBM SPSS Statistics 22 software (Chicago, IL, United States). All data were expressed as mean ± S.E.M. Analyses of variances were followed by the Tukey's post hoc multiple comparison test to assign points of significant difference. The blotting values were expressed as tested proteins/βactin ratio for each sample. The averages for different groups were compared by ANOVA, followed by the Tukey's post hoc. P < 0.05 was considered significant.
Control and neurotoxin sham animals were able to balance themselves on the beam for the maximum 60 s, while lesion and drug sham groups were able to stay on the beam for significantly (p < 0.0001) shorter times than control or surgical sham animals [F (6,48) = 124.968; P = 0.000]. All of the apelin-treated groups (1, 2 and 3 μg/rat) took significantly and dose dependently longer time on the balance beam as compared with 6-OHDA-treated animals (P < 0.0001, Fig. 2A). Results of the beam traversal task showed that 6-OHDA-treated and 3
Peptides 117 (2019) 170091
E. Haghparast, et al.
(P < 0.001) TH immunoreactive neurons in the SN region as compared to 6-OHDA treated group [F (2,11) = 608.149; P = 0.000; Fig. 3B]. 3.4. Apelin improve D2R proteins expression in the striatum IHC with anti-D2R antibody revealed a severe downregulation of striatal dopamine D2 receptor in 6-OHDA-treated animals (P < 0.001; Fig. 4A). Quantitative measurements revealed a 59.7% reduction in positive reaction to D2 receptor antibody in the striatum of the lesion group as compared to the control animals. Apelin-13 (3 μg/rat) treatment increased 91% immunoreactivity in rat striatum [F (2,11) = 41.112; P = 0.000; Fig. 4B]. 3.5. Effects of 6-OHDA and 6-OHDA plus apelin treatment on PSD-95, nurexin1, neuroligin, mGluR1 and DR1 proteins expression in the striatum The data showed that 6-OHDA-treated animals had a significant decrease in the level of PSD-95 (P = 0.001; Fig. 5A), neurexin 1 (P < 0.05; Fig. 5B) and neuroligin (P < 0.05; Fig. 5C). Apelin-treated animals (specially 3 μg/rat) showed a significant and higher level for PSD-95 (P < 0.001), neurexin (P < 0.01) and neuroligin (P < 0.05) than the lesion animals. Tukey's test following one-way ANOVA indicated that the animals in 6-OHDA group had a significant decrease in the level of D1 receptor (P = 0.001; Fig. 6A) and a significant increase in the level of mGluR1 (P = 0.000; Fig. 6B). Apelin-13 (2 and 3 μg/rat) significantly prevented the effect of 6-OHDA on D1 (P < 0.05) and mGlu1 (P < 0.05) receptors density. 4. Discussion It has been demonstrated that motor dysfunctions in PD are due to striatum and basal ganglia circuit dysfunction as well as morphological changes in the striatal synapses [26]. The data showed that intra-SNc injection of 6-OHDA resulted in motor impairments (increased catalepsy, freezing or traveling time and decreased motor-balance and motor coordination) in rats. Furthermore, the results showed that apelin-13 significantly improved motor impairments in 6-OHDA-treated rats. This result is also consistent with apelin-13 ability for alleviating motor function in mouse model of intracerebral hemorrhage [27]. In addition, it has been reported that spinal cord apelin/APJ system in amyotrophic lateral sclerosis has a neuroprotective effect against selective death of motor neurons [28]. Furthermore, Jaszberenyi and colleagues demonstrated that apelin-13 cause a markedly activation of locomotion in a dosedependent manner [29]. In contrast, it has been shown that apelin-13 has no influence in the rotarod test and wire hanging task in depressionlike behavior of mice [30]. However, apomorphine-induced rotational test showed that 6OHDA-induced substantia nigra damage can be attenuated by apelin-13 [24]. It has been demonstrated that there is a straight correlation between apomorphine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesion rats [31]. It seems that the nigrostriatal dopaminergic content was reserved or restored. Furthermore, IHC study showed the histological changes in lesion- and apelintreated rats. Overall, 6-OHDA administration caused a significant depletion of dopaminergic (TH positive) cells in the SNc of the lesion animals, but treatment with apelin-13 significantly preserved SN dopaminergic neurons. The destruction of the nigrostriatal dopaminergic pathway leads to main morphological and functional alterations in the striatal neuronal circuitry, and therefore, loss of striatal synaptic plasticity [6]. It has been demonstrated that the membrane-associated proteins, localized at synapses, are critical for synapse specification and integrity [32,33] and they act as balancer of inhibitory and excitatory synapses in CNS. A balance of excitation and inhibition is essential for almost all
Fig. 2. Effect of apelin on time to fall in balance test (A), the average of time to cross on the beam (B) and the average of the latency to begin (freeze time) the task (C). Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control and sham N groups. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion and lesion + vehicle groups. † p < 0.05 versus apelin-13 (1 μg/rat).
drug sham animals had a significant increase in the duration to cross beam (P < 0.001; Fig. 2B) and freezing time (P < 0.001; Fig. 2C) as compared to the control or surgical sham groups. There was a significant improvement in performance on the beam traversal test in the apelin-treated animals (2 and 3 μg/rat) [F (6,48) = 15.875; P = 0.000]. 3.3. Apelin attenuates dopaminergic neuron death in 6-OHDA-injected rats Immunohistochemistry was used to observe the changes in the number of tyrosine hydroxylase (TH)-positive neurons (Fig. 3). The TH immunoreactive neurons of the SN region of the rats in the control group displayed clear boundaries and morphology, strong immunoactivity, higher TH expression and clear neurites (Fig. 3A, upper pannel). 6-OHDA administration caused 85.44% reduction of the number of TH+ terminals and cell bodies in the SN, (P < 0.001), as well as reduced immunoactivity, lighter-stained cytoplasm and unclear cell boundaries (Fig. 3A, middle panel). Interestingly, treatment with apelin-13 (3 μg/rat) reduced the dopaminergic cell loss, and rescued 4
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 3. Comparative photomicrographs of tyrosine hydroxylase (TH) immunoreactivity which were taken from the substantia nigra (SN) of control, lesion and lesion plus apelin-treated group (A). Statistical comparison of the number of TH positive cells in the SN sections of the experimental groups (B). Data are shown as mean ± SEM (n = 4). *** p < 0.001 versus control group. ### P < 0.001 as compared to lesion group.
significantly decreased the levels of striatal PSD 95, neuroligin and neurexin and confirmed that the protective effects of apelin are accompanied with preserving the synaptic proteins in the striatum. It has been reported that striatal neurexin and PSD-95 levels significantly decrease in dopaminergic-denervation striatum which support its pathophysiological role in PD [11,12,4]. The data showed that apelin-13 treatment (3 μg/rats) significantly increased the striatal levels of PSD95, neurexin1 and neuroligin in SNc lesioned animals. It has also been reported that apelin can induce some mechanisms for the regulation of synaptic plasticity [41]. It seems apelin can also prevent molecular
physiological functions, including cognitive processes and motor control [34]. For instance, neurexins and PSD-95 can regulate excitatory and inhibitory synapses [35,36]. PSD-95 manages synaptic activity, arranges glutamate receptors and their related signaling proteins and intracellular signaling [37–39]. In addition to its role in the localization and anchoring of receptors and signaling proteins, PSD-95 plays an important role in stabilization, localization and trafficking of glutamate and DA receptors in the synapse [10,40]. Furthermore, presynaptic and postsynaptic differentiation are mediated by neurexins and neuroligins in the CNS excitatory synapses. The data indicated that SN lesion 5
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 4. The effects of 6-OHDA and 6-OHDA plus apelin-13 on striatal DA2 receptors (A), and percentage of positive reaction (B). Data are shown as mean ± SEM (n = 4). *p < 0.05 and *** p < 0.001 versus control group. ### P < 0.001 as compared to lesion group.
projecting to the striatum. The depletion of DA and DAergic synapses in the striatum results in the loss of striatal neurons by excitotoxicity [42]. Furthermore, SCH 23390 and Haloperidol, as selective dopamine receptor antagonists, completely reduced the behavioral action of apelin13, suggesting a critical role of D1 receptors in apelin signaling processes [29]. Additionally, it has been reported that reducing the activity of DA2R receptors leads to a disinhibition of voltage-gated ion channels and increases Ca+2 influx which is a main factor in the destruction of the spines in MSNs [43]. It has been documented that metabotropic glutamate receptors (mGluRs) are promising new medication which are purposed for
changes in striatal synapses. It has been demonstrated that PSD-95 interacts with dopamine receptors in striatal synapses [8] and potentially regulates N-methyl-Daspartate (NMDA) and dopamine D1 receptors trafficking and function in the synapse [9,10]. Therefore, we decided to assess the levels of mGlu R1, DA1 and DA2 receptors in our study. The results demonstrated that apelin-13 could also improve the depletion of striatal D1 and DA2R expression induced by 6-OHDA. DA and DA receptors play an important role in the formation of synaptic plasticity. In fact, PD initially is a motor disorder described by a severe neurodegeneration of dopaminergic neurons of the SNc, 6
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 6. The effects of 6-OHDA and 6-OHDA plus apelin on striatal D1 receptor (A), and mGlu R1 (B). β-actin was used as an internal control. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control group. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion group. † p < 0.05 versus apelin-13 (1 μg/rat).
the striatum, whereas, apelin-13 (2 and 3 μg) restored the mGluR1 density close to the control levels. However, evaluation of other types of mGluRs need to be determined in further studies. In addition, some studies indicated that apelin/APJ system protects neurons against excitotoxicity mediated by glutamate [48–50]. It has been demonstrated that in primary culture of striatal neurons, signaling through mGluR1/5 and the DA1R class leads to increase the phosphorylation of the mitogen-activated extracellular signal-regulated kinase 2 (ERK2). Furthermore, co-activation of mGluR and DARs also enhanced phosphorylation of cAMP-response element binding protein (CREB) [51]. It had been reported that apelin-13 activates proliferative ERK2 signaling and induces protective effects [48]. Apelin attenuates glutamate-induced excitotoxicity via an APJ receptor by activating Akt and ERK1/2 [52], reducing cAMP production and thereby inhibiting adenyl cyclase formation [53], improving the intracellular Ca2+ concentration [50] and suppressing TNF-α expression [48]. Thus, apelin peptides are able to regulate numerous signaling pathways and such protective effects may be involved in the synapto protective effect of apelin which need to be clarified in further studies.
Fig. 5. The effects of 6-OHDA and 6-OHDA plus apelin on striatal PSD-95 (A), neurexin 1 (B) and neuroligin (C). β-actin was used as an internal control. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control group. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion group. † p < 0.05 versus apelin-13 (1 μg/rat).
reducing motor symptoms [44]. Group I mGluRs (mGluR1 and -5) receptors signal through Gq/11 to increase phosphoinositide hydrolysis and mobilize intracellular calcium stores [45]. Acute dopaminergic depletion changes the physiological effects of group I mGluR activation in striatum, substantia nigra and subthalamic nucleus [46,47]. The data showed that SNc lesion significantly increased the levels of mGluR1 in
7
Peptides 117 (2019) 170091
E. Haghparast, et al.
5. Conclusion [23]
Degeneration of DA projection to the striatum leads to motor disorders and changes in synaptic plasticity-related molecules and neuropeptide apelin-13 can significantly improve motor performance and preserve synaptic integrity in the striatum of 6-OHDA-lesioned rats.
[24]
[25]
Acknowledgments This work was supported by funds from Kerman Neuroscience Research Center (# 97-54), Kerman University of Medical Sciences and Iran National Science Foundation (INSF, project no. 93013457).
[26] [27]
[28]
References [29]
[1] J.L. Eriksen, Z. Wszolek, L. Petrucelli, Molecular pathogenesis of Parkinson disease, Arch. Neurol. 62 (2005) 353–357. [2] A.H. Schapira, P. Jenner, Etiology and pathogenesis of Parkinson’s disease, Mov. Disord. 26 (2011) 1049–1055. [3] L. Parnetti, A. Castrioto, D. Chiasserini, E. Persichetti, N. Tambasco, O. El-Agnaf, P. Calabresi, Cerebrospinal fluid biomarkers in Parkinson disease, Nat. Rev. Neurol. 9 (2013) 131. [4] B. Picconi, G. Piccoli, P. Calabresi, Synaptic dysfunction in Parkinson’s disease, synaptic plasticity, Springer (2012) 553–572. [5] A.C. Kreitzer, Physiology and pharmacology of striatal neurons, Ann. Rev. Neurosci. 32 (2009) 127–147. [6] P. Calabresi, B. Picconi, A. Tozzi, V. Ghiglieri, M. Di Filippo, Direct and indirect pathways of basal ganglia: a critical reappraisal, Nat. Neurosci. 17 (2014) 1022. [7] G. Porras, A. Berthet, B. Dehay, Q. Li, L. Ladepeche, E. Normand, S. Dovero, A. Martinez, E. Doudnikoff, M.-L. Martin-Négrier, PSD-95 expression controls LDOPA dyskinesia through dopamine D1 receptor trafficking, J. Clin. Invest. 122 (2012) 3977–3989. [8] C. Fiorentini, F. Gardoni, P. Spano, M. Di Luca, C. Missale, Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate Nmethyl-D-aspartate receptors, J. Biol. Chem. 278 (2003) 20196–20202. [9] Z. Ren, W.-L. Sun, H. Jiao, D. Zhang, H. Kong, X. Wang, M. Xu, Dopamine D1 and Nmethyl-D-aspartate receptors and extracellular signal-regulated kinase mediate neuronal morphological changes induced by repeated cocaine administration, Neuroscience 168 (2010) 48–60. [10] W.-D. Yao, R.D. Spealman, J. Zhang, Dopaminergic signaling in dendritic spines, Biochem. Pharmacol. 75 (2008) 2055–2069. [11] F. Gardoni, L. Schrama, A. Kamal, W. Gispen, F. Cattabeni, M. Di Luca, Hippocampal synaptic plasticity involves competition between Ca2+/calmodulindependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor, J. Neurosci. 21 (2001) 1501–1509. [12] A. Nezhadi, S. Esmaeili-Mahani, V. Sheibani, M. Shabani, F. Darvishzadeh, Neurosteroid allopregnanolone attenuates motor disability and prevents the changes of neurexin 1 and postsynaptic density protein 95 expression in the striatum of 6-OHDA-induced rats’ model of Parkinson’s disease, Biomed. Pharmacother. 88 (2017) 1188–1197. [13] N. Takagi, R. Logan, L. Teves, M.C. Wallace, J.W. Gurd, Altered interaction between PSD‐95 and the NMDA receptor following transient global ischemia, J. Neurochem. 74 (2000) 169–178. [14] C. Noelker, M. Schwake, M. Balzer-Geldsetzer, M. Bacher, J. Popp, J. Schlegel, K. Eggert, W.H. Oertel, T. Klockgether, R.C. Dodel, Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson’s disease, Neurosci. Lett. 507 (2012) 10–15. [15] A.G. Carter, B.L. Sabatini, State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons, Neuron 44 (2004) 483–493. [16] P.-C. Chen, M.R. Vargas, A.K. Pani, R.J. Smeyne, D.A. Johnson, Y.W. Kan, J.A. Johnson, Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson’s disease: critical role for the astrocyte, Proc. Natl. Acad. Sci. 106 (2009) 2933–2938. [17] N. Aytan, J.-K. Choi, I. Carreras, V. Brinkmann, N.W. Kowall, B.G. Jenkins, A. Dedeoglu, Fingolimod modulates multiple neuroinflammatory markers in a mouse model of Alzheimer’s disease, Sci. Rep. 6 (2016) 24939. [18] K. Tatemoto, M. Hosoya, Y. Habata, R. Fujii, T. Kakegawa, M.-X. Zou, Y. Kawamata, S. Fukusumi, S. Hinuma, C. Kitada, Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor, Biochem. Biophys. Res. Commun. 251 (1998) 471–476. [19] M. Wigglesworth, L. Wolfe, A. Wise, Orphan seven transmembrane receptor screening, GPCRs: from deorphanization to lead structure identification, Springer (2007) 105–144. [20] N. De Mota, Z. Lenkei, C. Llorens-Cortès, Cloning, pharmacological characterization and brain distribution of the rat apelin receptor, Neuroendocrinology 72 (2000) 400–407. [21] A.D. Medhurst, C.A. Jennings, M.J. Robbins, R.P. Davis, C. Ellis, K.Y. Winborn, K.W. Lawrie, G. Hervieu, G. Riley, J.E. Bolaky, Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin, J. Neurochem. 84 (2003) 1162–1172. [22] X. Zhang, X. Peng, M. Fang, C. Zhou, F. Zhao, Y. Zhang, Y. Xu, Q. Zhu, J. Luo,
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40] [41]
[42]
[43] [44]
[45]
[46]
[47]
[48]
[49]
8
G. Chen, Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model, Peptides 32 (2011) 1793–1799. J. Sauvant, J.-C. Delpech, K. Palin, N. De Mota, J. Dudit, A. Aubert, H. Orcel, P. Roux, S. Layé, F. Moos, Mechanisms involved in dual vasopressin/apelin neuron dysfunction during aging, PLoS One 9 (2014). E. Haghparast, S. Esmaeili-Mahani, M. Abbasnejad, V. Sheibani, Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats, Neuropeptides 68 (2018) 28–35. E. Pouresmaeili-Babaki, S. Esmaeili-Mahani, M. Abbasnejad, H. Ravan, Protective effect of neuropeptide apelin-13 on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y dopaminergic cells: involvement of its antioxidant and antiapoptotic properties, Rejuvenation Res. 20 (2) (2017) 116–123. K.A. Jellinger, Pathology of parkinsonism, Neurobiol. Aging 21 (2000) 135. H. Bao, X. Yang, Y. Huang, H. Qiu, G. Huang, H. Xiao, J. Kuai, The neuroprotective effect of apelin-13 in a mouse model of intracerebral hemorrhage, Neurosci. Lett. 628 (2016) 219–224. A. Kasai, T. Kinjo, R. Ishihara, I. Sakai, Y. Ishimaru, Y. Yoshioka, A. Yamamuro, K. Ishige, Y. Ito, S. Maeda, Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis, PLoS One 6 (2011). M. Jaszberenyi, E. Bujdoso, G. Telegdy, Behavioral, neuroendocrine and thermoregulatory actions of apelin-13, Neuroscience 129 (2004) 811–816. S.-Y. Lv, Y.-J. Qin, H.-T. Wang, N. Xu, Y.-J. Yang, Q. Chen, Centrally administered apelin-13 induces depression-like behavior in mice, Brain Res. Bull. 88 (2012) 574–580. J.L. Hudson, C.G. van Horne, I. Strömberg, S. Brock, J. Clayton, J. Masserano, B.J. Hoffer, G.A. Gerhardt, Correlation of apomorphine-and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats, Brain Res. 626 (1993) 167–174. E.R. Graf, X. Zhang, S.-X. Jin, M.W. Linhoff, A.M. Craig, Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins, Cell 119 (2004) 1013–1026. Y. Hata, S. Butz, T.C. Sudhof, CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins, J. Neurosci. 16 (1996) 2488–2494. C. Dean, F.G. Scholl, J. Choih, S. DeMaria, J. Berger, E. Isacoff, P. Scheiffele, Neurexin mediates the assembly of presynaptic terminals, Nat. Neurosci. 6 (2003) 708. J.N. Levinson, N. Chéry, K. Huang, T.P. Wong, K. Gerrow, R. Kang, O. Prange, Y.T. Wang, A. El-Husseini, Neuroligins mediate excitatory and inhibitory synapse formation involvement of PSD-95 and neurexin-1β in neuroligin-induced synaptic specificity, J. Biol. Chem. 280 (2005) 17312–17319. O. Prange, T.P. Wong, K. Gerrow, Y.T. Wang, A. El-Husseini, A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13915–13920. N. Masuko, K. Makino, H. Kuwahara, K. Fukunaga, T. Sudo, N. Araki, H. Yamamoto, Y. Yamada, E. Miyamoto, H. Saya, Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90 a possible regulatory role in molecular clustering at synaptic sites, J. Biol. Chem. 274 (1999) 5782–5790. M. Migaud, P. Charlesworth, M. Dempster, L.C. Webster, A.M. Watabe, M. Makhinson, Y. He, M.F. Ramsay, R.G. Morris, J.H. Morrison, Enhanced longterm potentiation and impaired learning in mice with mutant postsynaptic density95 protein, Nature 396 (1998) 433. R. Sattler, Z. Xiong, W.-Y. Lu, M. Hafner, J.F. MacDonald, M. Tymianski, Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein, Science 284 (1999) 1845–1848. J. Zhang, S.M. Lewis, B. Kuhlman, A.L. Lee, Supertertiary structure of the MAGUK core from PSD-95, Structure 21 (2013) 402–413. D.R. Cook, A.J. Gleichman, S.A. Cross, S. Doshi, W. Ho, K.L. Jordan‐Sciutto, D.R. Lynch, D.L. Kolson, NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury, J. Neurochem. 118 (2011) 1113–1123. D.J. Surmeier, J.N. Guzman, J. Sanchez-Padilla, J.A. Goldberg, What causes the death of dopaminergic neurons in Parkinson’s disease? Prog. Brain Res. Elsevier (2010) 59–77. B. Vincenza, G. Veronica, S. Carmelo, C. Paolo, P. Barbara, Synaptic dysfunction in Parkinson’s disease, Biochem. Soc. Trans. 38 (2010) 493–497. R. Galici, N.G. Echemendia, A.L. Rodriguez, P.J. Conn, A selective allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors has effects similar to an orthosteric mGlu2/3 receptor agonist in mouse models predictive of antipsychotic activity, J. Pharmacol. Exp. Ther. 315 (2005) 1181–1187. K.A. Johnson, C.K. Jones, M.N. Tantawy, M. Bubser, M. Marvanova, M.S. Ansari, R.M. Baldwin, P.J. Conn, C.M. Niswender, The metabotropic glutamate receptor 8 agonist (S)-3, 4-DCPG reverses motor deficits in prolonged but not acute models of Parkinson’s disease, Neuropharmacology 66 (2013) 187–195. O. Valenti, M.J. Marino, M. Wittmann, E. Lis, A.G. DiLella, G.G. Kinney, P.J. Conn, Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse, J. Neurosci. 23 (2003) 7218–7226. M. Wittmann, M.J. Marino, P.J. Conn, Dopamine modulates the function of group II and group III metabotropic glutamate receptors in the substantia nigra pars reticulata, J. Pharmacol. Exp. Ther. 302 (2002) 433–441. Y. Ishimaru, A. Sumino, D. Kajioka, F. Shibagaki, A. Yamamuro, Y. Yoshioka, S. Maeda, Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice, J. Pharmacol. Sci. 133 (2017) 34–41. L.A. O’Donnell, A. Agrawal, P. Sabnekar, M.A. Dichter, D.R. Lynch, D.L. Kolson, Apelin, an endogenous neuronal peptide, protects hippocampal neurons against
Peptides 117 (2019) 170091
E. Haghparast, et al.
Neurosci. 25 (2005) 3763–3773. [52] B. Cheng, J. Chen, B. Bai, Q. Xin, Neuroprotection of apelin and its signaling pathway, Peptides 37 (2012) 171–173. [53] N.A. Chapman, D.J. Dupré, J.K. Rainey, The apelin receptor: physiology, pathology, cell signalling, and. ligand modulation of a peptide-activated class A GPCR, Biochem. Cell Biol. 92 (2014) 431–440.
excitotoxic injury, J. Neurochem. 102 (2007) 1905–1917. [50] X.J. Zeng, S.P. Yu, L. Zhang, L. Wei, Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons, Exp. Cell Res. 316 (2010) 1773–1783. [51] P.J. Voulalas, L. Holtzclaw, J. Wolstenholme, J.T. Russell, S.E. Hyman, Metabotropic glutamate receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons, J.
9
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Apelin-13 attenuates motor impairments and prevents the changes in synaptic plasticity-related molecules in the striatum of Parkinsonism rats Elham Haghparasta,b, Vahid Sheibania, Mehdi Abbasnejadb, Saeed Esmaeili-Mahania,b, a b
T
⁎
Laboratory of Molecular Neuroscience, Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences. Kerman, Iran Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman. Kerman, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Apelin-13 Parkinson’s disease 6-OHDA Motor impairment Striatum Synaptic plasticity
The striatum plays a critical role in motor control and also learning and memory of motor skills. It has been reported that striatal synaptic components are significantly decreased in dopaminergic-denervated striatum. In this study the effects of apelin-13 were investigated on motor disorders and striatal synaptosomal expression of PSD-95, neurexin1, neuroligin, metabotropic glutamate receptor (mGlu R1) and dopaminergic receptors (DR1 and DR2) in rat parkinsonism experimental model. 6-hydroxydopamine (6-OHDA) was injected into the substantia nigra. Apelin-13 (1, 2 and 3 μg/rat) was administered into the substantia nigra one week after the 6-OHDA injection. Accelerating rotarod, beam-balance, beam-walking and bar tests were performed one month after the apelin injection. Immunohistochemistry staining of dopaminergic neurons was performed. The levels of synaptic proteins were determined by immunoblotting. 6-OHDA-treated animals showed a significant impairment in motor-skill tasks and a dramatically change in the expression levels of mentioned proteins. Apelin-13 (3 μg/rat) significantly attenuates the motor impairments and prevents the changes in striatal synaptic elements in 6-OHDA-treated animals. In addition, it could rescue the dopaminergic neurons of the substantia nigra. The data will potentially extend the possible benefic aspect of apelin in neurodegenerative disorders.
1. Introduction Parkinson’s disease (PD) is defined by the dopamine (DA) neurons degeneration originating in the substantia nigra pars compacta (SNc) that leads to profound depletion of dopamine in the striatum and consequently severe movement abnormalities such as tremors, catalepsy and bradykinesia [1,2]. PD pathology is associated not only with dopaminergic neurodegeneration, but also with abnormal patterns of firing of neural pathways. Dysfunctional synaptic plasticity at the onset of PD is associated with the development of the motor complications, such as dyskinesia [3,4]. Projection pathways of DA neurons from the nigrostriatal bundle to the dorsal striatum have a central role in control of fine motor functions. More than 95% of striatal neurons are medium spiny neurons (MSNs) that form asymmetric synapses with glutamatergic inputs and symmetric connections at the DA synapses [5]. Therefore, the
degeneration of the nigrostriatal dopaminergic pathway causes a significant change in the striatal neurons and then resulting in loss of striatal synaptic plasticity [6]. Postsynaptic density protein 95 (PSD-95) is a main scaffolding protein in the dendritic spines of MSNs in the striatum [7], which interacts with dopamine receptor [8] and potentially regulates N-methylD-aspartate (NMDA) and dopamine D1 receptors trafficking and function in the synapses [9,10]. It has been reported that striatal PSD-95 levels significantly decrease in dopaminergic-denervation striatum which support its role in PD [11–13]. The neuroligin and β-neurexin complex is a major component of synaptic connection and acts as balancer of pre- and post-synaptic proteins. The ability of neuroligin and β-neurexin to form and maintain excitatory and inhibitory synapses indicates that they have potential roles in the induction of neurological disorders such as PD [14]. Striatal MSNs exhibit numerous ionic conductance that shape their firing properties, and such conductances are sensitive to some
Abbreviations: DA, dopamine; SNc, substantia nigra pars compacta; PD, Parkinson's disease; 6-OHDA, 6-hydroxydopamine; CNS, central nervous system; APJ, apelin receptor; MSNs, medium spiny neurons; PSD-95, postsynaptic density protein 95; mGluR1, metabotropic glutamate receptor 1; DR1, dopamine receotor1; DR2, dopamine receotor2 ⁎ Corresponding author at: Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, P.O. Box 76135-133, Kerman, Iran. E-mail addresses: [email protected], [email protected] (S. Esmaeili-Mahani). https://doi.org/10.1016/j.peptides.2019.05.003 Received 7 February 2019; Received in revised form 24 April 2019; Accepted 17 May 2019 Available online 21 May 2019 0196-9781/ © 2019 Elsevier Inc. All rights reserved.
Peptides 117 (2019) 170091
E. Haghparast, et al.
allowed to recover from the anesthesia and then placed in individual cages. After one weeks, as a recovery period, apelin (phoenix pharmaceuticals INC, USA; cat # :057-18) was dissolved in ACSF and delivered into the right SNc over 2 min. The injection needles were left in place for an additional 1 min before they were slowly withdrawn [24].
neuromodulators such as DA. The expression of D1 receptors in striatonigral MSNs regulates sodium, potassium, and calcium channels which results in modulation of MSN excitability [15]. In PD, the loss of the DAergic neurons in the striatum leads to loss of DA-mediated inhibition, possibly results in excess excitatory transmission [16]. Neurotransmission, during PD-like neurodegeneration, can be regulated by dopamine (DA) and metabotropic glutamate (mGlu) receptors [17]. The apelin peptide is the endogenous ligand for G protein-coupled receptor, APJ. Apelin-13 is more potent than other type of apelin in competitive binding to the APJs [18,19]. The distribution of apelin and its receptors in the amygdala, hypothalamus, hippocampus, cerebellum, striatum, substantia nigra, and hypothalamus [20,21], suggests that apelin might have critical and broad roles in physiological and pathophysiological conditions. Recently, it has been shown that the apelinAPJ system might play an important role in the neuronal survival and signaling in the central nervous system and therefore, apelin should be further investigated as a potential protective neuropeptide in various neurological disorders that might cause brain injury [22]. It has been documented that the plasma concentrations of apelin are reduced with age, so that plasma apelin concentrations in aged rats are just half of those in adults [23]. We have recently reported that apelin significantly improves cognitive performance in 6-OHDA-induced rat model of PD [24] and also, has a protective effect on 6-OHDA-induced toxicity in SH-SY5Y dopaminergic cells [25]. The present study was design to find the possible neuromodulatory effects of apelin-13 on the movement disorders and some of the striatal synaptosomal proteins levels (mGlu, D1 and D2 receptors, neuroligin, neurexin1 and PSD-95 which play important roles in the maintaining and integrity of neural connection) in 6-OHDA-induced hemi parkinsonian male rats.
2.3. Accelerating rotarod The rotarod apparatus (TSE, SER-NO; 040910-02, Germany) was used for Motor-skill learning and motor performance at different progressively higher speeds. Briefly, at the first day, the rats were trained on the rotating rod (with persistent 10 rpm for 3 min) to adapt with the apparatus. Next day, the animals were located on the rotating rod with an accelerating protocol (10–60 rpm in 5 min). The latency to fall was measured in seconds. Each animal was given three trials with a 30 min inter-trial rest interval and the mean of trials was calculated (Bellum et al., 2013). 2.4. Balance beam and Beam traversal test The balance beam is a test for the evaluation of motor coordination in rodents. The beam consists of 80 cm long horizontal wooden rod (1.5 cm diameter) which was placed at a height of 80 cm above a thick foam. Each animal was placed on the center of the rod and the time that each animal remained on the beam was measured. Cut-off time was 60 s. Motor coordination and balance of the animals were assessed by measuring the ability of the rats to traverse of a narrow beam (2.5 × 100 cm) from bright light to reach a dark goal box placed on the opposite end of the beam. The wooden beam was fixed 80 cm above a thick foam. The animal was placed behind the starting line (facing dark goal box) and released. The latency to traverse the beam (beam Freeze time) and the time of the hind feet were placed entirely inside the goal box at the opposite end of the beam were recorded. If an animal was incapable to complete the beam run, the test was finished at 2 min (cutoff time) [12].
2. Materials and methods 2.1. Animals Adult male Wistar rats weighing 250–300 g were used in this study. Before and during the study, the animals were kept in polypropylene cages, four per cage, under controlled temperature (24 ± 1 °C) and 12h light-dark cycle and had free access to food (standard rodent diet) and water. All experimental protocols and treatments were approved by the Ethics Committee of Kerman Neuroscience Research Center (Ethics Code: EC/97-54/KNRC). The animals were randomly divided into several experimental groups, each containing 7 rats: (I) Control; (II) Neurotoxin sham (sham N); (III) Lesion; (IV) Lesion + Vehicle; (V) Lesion animals which received 1 μg/rat of apelin (Lesion + apelin 1); (VI) Lesion animals which had 2 μg/rat of apelin (Lesion + apelin 2); and (VII) lesion animals which were treated with 3 μg/rat of apelin (Lesion + apelin 3).
2.5. Bar test Anterior limbs of the rats were placed on a bar (9 mm in diameter) fixed at the height of 9 cm away from the surface. The duration of maintaining this position was determined as the intensity of catalepsy. The end point of catalepsy was considered when any exploratory head movements or displacement of one or both forearms was performed. The cut off time of the test was 120 sec and the test was done in three consecutive times with 10 min interval and the average latencies were calculated (Nayebi et al., 2010).
2.2. Experimental design
2.6. Immunohistochemistry (IHC)
Under ketamine and xylazine (80 and 10 mg/kg, i.p, respectively) anesthesia, the rats were fixed in a stereotaxic frame in the flat position with the incisor bar positioned 3.3 mm below the interaural line. A guide cannula (22-gauge stainless steel) was implanted and fixed just above (1 mm) the right SNc according the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986); coordinates with respect to bregma: 3.7 mm antero-posterior; 2.2 mm lateral to the midline; 7.7 mm dorsoventral from the dura surface. 6-OHDA (Sigma-Aldrich Co., LLC, USA) was dissolved in saline with 0.2 % ascorbic acid and gradually injected into the right SNc (8 μg/2 μl) at a rate of 1 μL/min. Neurotoxin sham animals were submitted to the same procedure but 2 μl of vehicle was infused into the SNc. Lesion + vehicle animals were also submitted to the same procedure and received 6-OHDA as a neurotoxin plus artificial cerebrospinal fluid (ACSF, consisting of (in mM): NaCl, 124; NaHCO3, 25; D glucose, 10; KCl, 4.4; MgCl2, 2; KH2PO4, H2O, 1.25 and CaCl2, 6H20, 2.) as vehicle of apelin. Animals were
Under deep anesthesia using intramuscular injection of ketamine and xylazine, the brains were fixed by cardiac perfusion with 0.9% NaCl followed by 4% paraformaldehyde, pH 7.4. The brains were removed and post fixed in the same fixative overnight. Then, the striatum and the SN were cut and dehydrated in alcohol series, cleared in xylene, infiltrated with paraffin and embedded in paraffin. The paraffin embedded tissue sections (5 mm sized) were de-paraffinized with xylene and endogenous peroxidase activity was quenched with 3% H2O2 in methanol for 30 min in the dark. Tissue sections were dehydrated through graded alcohols and subjected to antigen retrieval using 10 mM sodium citrate. The sections were washed with TBST (Tris Borate Saline Tween-20) and then blocked with 5% bovine serum albumin (BSA) for one hour. The SN sections were incubated with the respective mouse monoclonal primary antibody against tyrosine hydroxylase and striatal sections were incubated with anti-Dopamine D2 receptor (PA2234). The Slides were then washed for 5 min in TBST and incubated for 1 h 2
Peptides 117 (2019) 170091
E. Haghparast, et al.
with the respective HRP (Horseraddish Peroxidase) conjugated antimouse secondary antibody diluted with TBS in a ratio of 1:200. After washing, the slides were incubated with DAB (3,39-diaminobenzidine tetrahydrochloride) (Sigma) and immediately washed under tap water just after color development. The sections were then counter with hematoxylin. and mounted with DPX (dibutyl phthalate xylene) and then observed under a light microscope.
2.7. Western blot analysis for synaptic proteins The right striatum was homogenized in 10% (w/v) of 0.32 M sucrose-HEPES buffer (HEPES-buffer solution: 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 5 mM HEPES, pH 7.4) along with protease inhibitor. Further, homogenate was centrifuged at 4 °C for 10 min at 600 g. Then supernatant was diluted 1:1 with 1.3 M HEPES sucrose to produce a suspension at a final concentration of 0.8 M HEPES sucrose. This suspension was further centrifuged at two to three times at 12,000 g for 15 min at 4 °C. The supernatant was discarded each time. The pellet that consisting of synaptosomes was suspended in RIPA buffer (mixed with protease inhibitors: 1 mM phenyl methyl sulfonyl fluoride, 2.5 mg/ml leupeptin, and 10 mg/ml aprotinin) along with 0.2% Triton X-100 and centrifuged at 20,000 g for 30 min. This step is responsible for the disruption of synaptic membranes and enriches the synapse proteins. Further, the resulting pellet was re-suspended only in RIPA buffer (mixed with protease inhibitors). Proteins contents were quantified by the Bradford assay method. Bovine serum albumin (BSA; 0.01–0.1 mg/ml) was used as standard. Equal amounts of protein per sample (40 μg) were added to sample buffer, heated at 100 °C for 5 min and were resolved electrophoretically on 10% SDS–PAGE and then proteins were transferred to PVDF membranes (Hybond ECL, GE Healthcare Bio-Sciences Corp. NJ, USA). After blocking (2 h at room temperature) with 5% non-fat dried milk in Trisbuffered saline with Tween 20 (TBS-T: 20 mM Tris–HCl, 150 mMNaCl, pH 7.5, 0.1% Tween 20), the membranes were probed with anti-PSD95, anti-neurexin1, anti-neuroligin, anti-D1 or anti-mGlu1 receptors antibodies (Santa Cruz Biotechnology, USA, 1:1000) for overnight at 4 °C. After washing with TBS-T (three times for 5 min), the membranes were exposed to secondary anti-species antibody (1:10,000, Santa Cruz Biotechnology) for 1 h at room temperature with a secondary antibody attached to horseradish peroxidase (1:15,000, GE Healthcare BioSciences Corp. NJ, USA). All antibodies were diluted in blocking buffer. Pre-stained protein ladder (SM7012, Cinagen Co, Iran) was used for monitoring protein separation and estimating the molecular weight and integrated intensity of blotting bands. The antibody-antigen complexes were detected using the ECL system and exposed to Lumi-Film chemiluminescent detection film (Roche Germany). Lab Works analyzing software (UVP, UK) was used to evaluate the intensity of the blotting bands. β-actin (1:10,000) was used as loading control. The expression values were presented as tested proteins/β-actin ratio for each sample (Kamat et al., 2014).
Fig. 1. Effect of apelin on rotarod test (A) and bar test (B) performance. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control and sham N groups. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion and lesion + vehicle groups. † p < 0.05 versus apelin-13 (1 μg/rat).
3. Results 3.1. Effects of apelin on rotarod and bar test performance in 6-OHDAinjected rats There was no significant difference in rotarod performance between control and neurotoxin sham groups (P = 0.995; Fig. 1A). Injection of 6-OHDA caused a significant reduction in the latency to fall in lesion and drug sham groups [F (6,48) = 13.739; P = 0.000]. However, apelin-treated animals (3 μg/rat) significantly attenuated the decreasing effect of 6-OHDA on rotarod motor performance (P < 0.01). Apelin in doses of 1 and 2 μg/rat had no significant effect on 6-OHDAinduced motor impairment in rotarod test. Results of the bar test in different groups showed that control and surgical sham had no sign of catalepsy [F (6,48) = 53.880; P = 0.000; Fig. 1B]. 6-OHDA induced a significant catalepsy in the lesion and drug sham groups (P = 0.000). In contrast, all apelin-treated groups showed a significant and dose dependent (P < 0.0001) recovery in catalepsy time.
2.8. Statistical analysis 3.2. Effects of apelin on balance beam and beam traversal test in 6-OHDAinjected rats
All of the statistical analyses were performed using IBM SPSS Statistics 22 software (Chicago, IL, United States). All data were expressed as mean ± S.E.M. Analyses of variances were followed by the Tukey's post hoc multiple comparison test to assign points of significant difference. The blotting values were expressed as tested proteins/βactin ratio for each sample. The averages for different groups were compared by ANOVA, followed by the Tukey's post hoc. P < 0.05 was considered significant.
Control and neurotoxin sham animals were able to balance themselves on the beam for the maximum 60 s, while lesion and drug sham groups were able to stay on the beam for significantly (p < 0.0001) shorter times than control or surgical sham animals [F (6,48) = 124.968; P = 0.000]. All of the apelin-treated groups (1, 2 and 3 μg/rat) took significantly and dose dependently longer time on the balance beam as compared with 6-OHDA-treated animals (P < 0.0001, Fig. 2A). Results of the beam traversal task showed that 6-OHDA-treated and 3
Peptides 117 (2019) 170091
E. Haghparast, et al.
(P < 0.001) TH immunoreactive neurons in the SN region as compared to 6-OHDA treated group [F (2,11) = 608.149; P = 0.000; Fig. 3B]. 3.4. Apelin improve D2R proteins expression in the striatum IHC with anti-D2R antibody revealed a severe downregulation of striatal dopamine D2 receptor in 6-OHDA-treated animals (P < 0.001; Fig. 4A). Quantitative measurements revealed a 59.7% reduction in positive reaction to D2 receptor antibody in the striatum of the lesion group as compared to the control animals. Apelin-13 (3 μg/rat) treatment increased 91% immunoreactivity in rat striatum [F (2,11) = 41.112; P = 0.000; Fig. 4B]. 3.5. Effects of 6-OHDA and 6-OHDA plus apelin treatment on PSD-95, nurexin1, neuroligin, mGluR1 and DR1 proteins expression in the striatum The data showed that 6-OHDA-treated animals had a significant decrease in the level of PSD-95 (P = 0.001; Fig. 5A), neurexin 1 (P < 0.05; Fig. 5B) and neuroligin (P < 0.05; Fig. 5C). Apelin-treated animals (specially 3 μg/rat) showed a significant and higher level for PSD-95 (P < 0.001), neurexin (P < 0.01) and neuroligin (P < 0.05) than the lesion animals. Tukey's test following one-way ANOVA indicated that the animals in 6-OHDA group had a significant decrease in the level of D1 receptor (P = 0.001; Fig. 6A) and a significant increase in the level of mGluR1 (P = 0.000; Fig. 6B). Apelin-13 (2 and 3 μg/rat) significantly prevented the effect of 6-OHDA on D1 (P < 0.05) and mGlu1 (P < 0.05) receptors density. 4. Discussion It has been demonstrated that motor dysfunctions in PD are due to striatum and basal ganglia circuit dysfunction as well as morphological changes in the striatal synapses [26]. The data showed that intra-SNc injection of 6-OHDA resulted in motor impairments (increased catalepsy, freezing or traveling time and decreased motor-balance and motor coordination) in rats. Furthermore, the results showed that apelin-13 significantly improved motor impairments in 6-OHDA-treated rats. This result is also consistent with apelin-13 ability for alleviating motor function in mouse model of intracerebral hemorrhage [27]. In addition, it has been reported that spinal cord apelin/APJ system in amyotrophic lateral sclerosis has a neuroprotective effect against selective death of motor neurons [28]. Furthermore, Jaszberenyi and colleagues demonstrated that apelin-13 cause a markedly activation of locomotion in a dosedependent manner [29]. In contrast, it has been shown that apelin-13 has no influence in the rotarod test and wire hanging task in depressionlike behavior of mice [30]. However, apomorphine-induced rotational test showed that 6OHDA-induced substantia nigra damage can be attenuated by apelin-13 [24]. It has been demonstrated that there is a straight correlation between apomorphine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesion rats [31]. It seems that the nigrostriatal dopaminergic content was reserved or restored. Furthermore, IHC study showed the histological changes in lesion- and apelintreated rats. Overall, 6-OHDA administration caused a significant depletion of dopaminergic (TH positive) cells in the SNc of the lesion animals, but treatment with apelin-13 significantly preserved SN dopaminergic neurons. The destruction of the nigrostriatal dopaminergic pathway leads to main morphological and functional alterations in the striatal neuronal circuitry, and therefore, loss of striatal synaptic plasticity [6]. It has been demonstrated that the membrane-associated proteins, localized at synapses, are critical for synapse specification and integrity [32,33] and they act as balancer of inhibitory and excitatory synapses in CNS. A balance of excitation and inhibition is essential for almost all
Fig. 2. Effect of apelin on time to fall in balance test (A), the average of time to cross on the beam (B) and the average of the latency to begin (freeze time) the task (C). Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control and sham N groups. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion and lesion + vehicle groups. † p < 0.05 versus apelin-13 (1 μg/rat).
drug sham animals had a significant increase in the duration to cross beam (P < 0.001; Fig. 2B) and freezing time (P < 0.001; Fig. 2C) as compared to the control or surgical sham groups. There was a significant improvement in performance on the beam traversal test in the apelin-treated animals (2 and 3 μg/rat) [F (6,48) = 15.875; P = 0.000]. 3.3. Apelin attenuates dopaminergic neuron death in 6-OHDA-injected rats Immunohistochemistry was used to observe the changes in the number of tyrosine hydroxylase (TH)-positive neurons (Fig. 3). The TH immunoreactive neurons of the SN region of the rats in the control group displayed clear boundaries and morphology, strong immunoactivity, higher TH expression and clear neurites (Fig. 3A, upper pannel). 6-OHDA administration caused 85.44% reduction of the number of TH+ terminals and cell bodies in the SN, (P < 0.001), as well as reduced immunoactivity, lighter-stained cytoplasm and unclear cell boundaries (Fig. 3A, middle panel). Interestingly, treatment with apelin-13 (3 μg/rat) reduced the dopaminergic cell loss, and rescued 4
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 3. Comparative photomicrographs of tyrosine hydroxylase (TH) immunoreactivity which were taken from the substantia nigra (SN) of control, lesion and lesion plus apelin-treated group (A). Statistical comparison of the number of TH positive cells in the SN sections of the experimental groups (B). Data are shown as mean ± SEM (n = 4). *** p < 0.001 versus control group. ### P < 0.001 as compared to lesion group.
significantly decreased the levels of striatal PSD 95, neuroligin and neurexin and confirmed that the protective effects of apelin are accompanied with preserving the synaptic proteins in the striatum. It has been reported that striatal neurexin and PSD-95 levels significantly decrease in dopaminergic-denervation striatum which support its pathophysiological role in PD [11,12,4]. The data showed that apelin-13 treatment (3 μg/rats) significantly increased the striatal levels of PSD95, neurexin1 and neuroligin in SNc lesioned animals. It has also been reported that apelin can induce some mechanisms for the regulation of synaptic plasticity [41]. It seems apelin can also prevent molecular
physiological functions, including cognitive processes and motor control [34]. For instance, neurexins and PSD-95 can regulate excitatory and inhibitory synapses [35,36]. PSD-95 manages synaptic activity, arranges glutamate receptors and their related signaling proteins and intracellular signaling [37–39]. In addition to its role in the localization and anchoring of receptors and signaling proteins, PSD-95 plays an important role in stabilization, localization and trafficking of glutamate and DA receptors in the synapse [10,40]. Furthermore, presynaptic and postsynaptic differentiation are mediated by neurexins and neuroligins in the CNS excitatory synapses. The data indicated that SN lesion 5
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 4. The effects of 6-OHDA and 6-OHDA plus apelin-13 on striatal DA2 receptors (A), and percentage of positive reaction (B). Data are shown as mean ± SEM (n = 4). *p < 0.05 and *** p < 0.001 versus control group. ### P < 0.001 as compared to lesion group.
projecting to the striatum. The depletion of DA and DAergic synapses in the striatum results in the loss of striatal neurons by excitotoxicity [42]. Furthermore, SCH 23390 and Haloperidol, as selective dopamine receptor antagonists, completely reduced the behavioral action of apelin13, suggesting a critical role of D1 receptors in apelin signaling processes [29]. Additionally, it has been reported that reducing the activity of DA2R receptors leads to a disinhibition of voltage-gated ion channels and increases Ca+2 influx which is a main factor in the destruction of the spines in MSNs [43]. It has been documented that metabotropic glutamate receptors (mGluRs) are promising new medication which are purposed for
changes in striatal synapses. It has been demonstrated that PSD-95 interacts with dopamine receptors in striatal synapses [8] and potentially regulates N-methyl-Daspartate (NMDA) and dopamine D1 receptors trafficking and function in the synapse [9,10]. Therefore, we decided to assess the levels of mGlu R1, DA1 and DA2 receptors in our study. The results demonstrated that apelin-13 could also improve the depletion of striatal D1 and DA2R expression induced by 6-OHDA. DA and DA receptors play an important role in the formation of synaptic plasticity. In fact, PD initially is a motor disorder described by a severe neurodegeneration of dopaminergic neurons of the SNc, 6
Peptides 117 (2019) 170091
E. Haghparast, et al.
Fig. 6. The effects of 6-OHDA and 6-OHDA plus apelin on striatal D1 receptor (A), and mGlu R1 (B). β-actin was used as an internal control. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control group. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion group. † p < 0.05 versus apelin-13 (1 μg/rat).
the striatum, whereas, apelin-13 (2 and 3 μg) restored the mGluR1 density close to the control levels. However, evaluation of other types of mGluRs need to be determined in further studies. In addition, some studies indicated that apelin/APJ system protects neurons against excitotoxicity mediated by glutamate [48–50]. It has been demonstrated that in primary culture of striatal neurons, signaling through mGluR1/5 and the DA1R class leads to increase the phosphorylation of the mitogen-activated extracellular signal-regulated kinase 2 (ERK2). Furthermore, co-activation of mGluR and DARs also enhanced phosphorylation of cAMP-response element binding protein (CREB) [51]. It had been reported that apelin-13 activates proliferative ERK2 signaling and induces protective effects [48]. Apelin attenuates glutamate-induced excitotoxicity via an APJ receptor by activating Akt and ERK1/2 [52], reducing cAMP production and thereby inhibiting adenyl cyclase formation [53], improving the intracellular Ca2+ concentration [50] and suppressing TNF-α expression [48]. Thus, apelin peptides are able to regulate numerous signaling pathways and such protective effects may be involved in the synapto protective effect of apelin which need to be clarified in further studies.
Fig. 5. The effects of 6-OHDA and 6-OHDA plus apelin on striatal PSD-95 (A), neurexin 1 (B) and neuroligin (C). β-actin was used as an internal control. Data are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and *** p < 0.001 versus control group. # P < 0.05, ##P < 0.01 and ### P < 0.001 as compared to lesion group. † p < 0.05 versus apelin-13 (1 μg/rat).
reducing motor symptoms [44]. Group I mGluRs (mGluR1 and -5) receptors signal through Gq/11 to increase phosphoinositide hydrolysis and mobilize intracellular calcium stores [45]. Acute dopaminergic depletion changes the physiological effects of group I mGluR activation in striatum, substantia nigra and subthalamic nucleus [46,47]. The data showed that SNc lesion significantly increased the levels of mGluR1 in
7
Peptides 117 (2019) 170091
E. Haghparast, et al.
5. Conclusion [23]
Degeneration of DA projection to the striatum leads to motor disorders and changes in synaptic plasticity-related molecules and neuropeptide apelin-13 can significantly improve motor performance and preserve synaptic integrity in the striatum of 6-OHDA-lesioned rats.
[24]
[25]
Acknowledgments This work was supported by funds from Kerman Neuroscience Research Center (# 97-54), Kerman University of Medical Sciences and Iran National Science Foundation (INSF, project no. 93013457).
[26] [27]
[28]
References [29]
[1] J.L. Eriksen, Z. Wszolek, L. Petrucelli, Molecular pathogenesis of Parkinson disease, Arch. Neurol. 62 (2005) 353–357. [2] A.H. Schapira, P. Jenner, Etiology and pathogenesis of Parkinson’s disease, Mov. Disord. 26 (2011) 1049–1055. [3] L. Parnetti, A. Castrioto, D. Chiasserini, E. Persichetti, N. Tambasco, O. El-Agnaf, P. Calabresi, Cerebrospinal fluid biomarkers in Parkinson disease, Nat. Rev. Neurol. 9 (2013) 131. [4] B. Picconi, G. Piccoli, P. Calabresi, Synaptic dysfunction in Parkinson’s disease, synaptic plasticity, Springer (2012) 553–572. [5] A.C. Kreitzer, Physiology and pharmacology of striatal neurons, Ann. Rev. Neurosci. 32 (2009) 127–147. [6] P. Calabresi, B. Picconi, A. Tozzi, V. Ghiglieri, M. Di Filippo, Direct and indirect pathways of basal ganglia: a critical reappraisal, Nat. Neurosci. 17 (2014) 1022. [7] G. Porras, A. Berthet, B. Dehay, Q. Li, L. Ladepeche, E. Normand, S. Dovero, A. Martinez, E. Doudnikoff, M.-L. Martin-Négrier, PSD-95 expression controls LDOPA dyskinesia through dopamine D1 receptor trafficking, J. Clin. Invest. 122 (2012) 3977–3989. [8] C. Fiorentini, F. Gardoni, P. Spano, M. Di Luca, C. Missale, Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate Nmethyl-D-aspartate receptors, J. Biol. Chem. 278 (2003) 20196–20202. [9] Z. Ren, W.-L. Sun, H. Jiao, D. Zhang, H. Kong, X. Wang, M. Xu, Dopamine D1 and Nmethyl-D-aspartate receptors and extracellular signal-regulated kinase mediate neuronal morphological changes induced by repeated cocaine administration, Neuroscience 168 (2010) 48–60. [10] W.-D. Yao, R.D. Spealman, J. Zhang, Dopaminergic signaling in dendritic spines, Biochem. Pharmacol. 75 (2008) 2055–2069. [11] F. Gardoni, L. Schrama, A. Kamal, W. Gispen, F. Cattabeni, M. Di Luca, Hippocampal synaptic plasticity involves competition between Ca2+/calmodulindependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor, J. Neurosci. 21 (2001) 1501–1509. [12] A. Nezhadi, S. Esmaeili-Mahani, V. Sheibani, M. Shabani, F. Darvishzadeh, Neurosteroid allopregnanolone attenuates motor disability and prevents the changes of neurexin 1 and postsynaptic density protein 95 expression in the striatum of 6-OHDA-induced rats’ model of Parkinson’s disease, Biomed. Pharmacother. 88 (2017) 1188–1197. [13] N. Takagi, R. Logan, L. Teves, M.C. Wallace, J.W. Gurd, Altered interaction between PSD‐95 and the NMDA receptor following transient global ischemia, J. Neurochem. 74 (2000) 169–178. [14] C. Noelker, M. Schwake, M. Balzer-Geldsetzer, M. Bacher, J. Popp, J. Schlegel, K. Eggert, W.H. Oertel, T. Klockgether, R.C. Dodel, Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson’s disease, Neurosci. Lett. 507 (2012) 10–15. [15] A.G. Carter, B.L. Sabatini, State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons, Neuron 44 (2004) 483–493. [16] P.-C. Chen, M.R. Vargas, A.K. Pani, R.J. Smeyne, D.A. Johnson, Y.W. Kan, J.A. Johnson, Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson’s disease: critical role for the astrocyte, Proc. Natl. Acad. Sci. 106 (2009) 2933–2938. [17] N. Aytan, J.-K. Choi, I. Carreras, V. Brinkmann, N.W. Kowall, B.G. Jenkins, A. Dedeoglu, Fingolimod modulates multiple neuroinflammatory markers in a mouse model of Alzheimer’s disease, Sci. Rep. 6 (2016) 24939. [18] K. Tatemoto, M. Hosoya, Y. Habata, R. Fujii, T. Kakegawa, M.-X. Zou, Y. Kawamata, S. Fukusumi, S. Hinuma, C. Kitada, Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor, Biochem. Biophys. Res. Commun. 251 (1998) 471–476. [19] M. Wigglesworth, L. Wolfe, A. Wise, Orphan seven transmembrane receptor screening, GPCRs: from deorphanization to lead structure identification, Springer (2007) 105–144. [20] N. De Mota, Z. Lenkei, C. Llorens-Cortès, Cloning, pharmacological characterization and brain distribution of the rat apelin receptor, Neuroendocrinology 72 (2000) 400–407. [21] A.D. Medhurst, C.A. Jennings, M.J. Robbins, R.P. Davis, C. Ellis, K.Y. Winborn, K.W. Lawrie, G. Hervieu, G. Riley, J.E. Bolaky, Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin, J. Neurochem. 84 (2003) 1162–1172. [22] X. Zhang, X. Peng, M. Fang, C. Zhou, F. Zhao, Y. Zhang, Y. Xu, Q. Zhu, J. Luo,
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40] [41]
[42]
[43] [44]
[45]
[46]
[47]
[48]
[49]
8
G. Chen, Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model, Peptides 32 (2011) 1793–1799. J. Sauvant, J.-C. Delpech, K. Palin, N. De Mota, J. Dudit, A. Aubert, H. Orcel, P. Roux, S. Layé, F. Moos, Mechanisms involved in dual vasopressin/apelin neuron dysfunction during aging, PLoS One 9 (2014). E. Haghparast, S. Esmaeili-Mahani, M. Abbasnejad, V. Sheibani, Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats, Neuropeptides 68 (2018) 28–35. E. Pouresmaeili-Babaki, S. Esmaeili-Mahani, M. Abbasnejad, H. Ravan, Protective effect of neuropeptide apelin-13 on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y dopaminergic cells: involvement of its antioxidant and antiapoptotic properties, Rejuvenation Res. 20 (2) (2017) 116–123. K.A. Jellinger, Pathology of parkinsonism, Neurobiol. Aging 21 (2000) 135. H. Bao, X. Yang, Y. Huang, H. Qiu, G. Huang, H. Xiao, J. Kuai, The neuroprotective effect of apelin-13 in a mouse model of intracerebral hemorrhage, Neurosci. Lett. 628 (2016) 219–224. A. Kasai, T. Kinjo, R. Ishihara, I. Sakai, Y. Ishimaru, Y. Yoshioka, A. Yamamuro, K. Ishige, Y. Ito, S. Maeda, Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis, PLoS One 6 (2011). M. Jaszberenyi, E. Bujdoso, G. Telegdy, Behavioral, neuroendocrine and thermoregulatory actions of apelin-13, Neuroscience 129 (2004) 811–816. S.-Y. Lv, Y.-J. Qin, H.-T. Wang, N. Xu, Y.-J. Yang, Q. Chen, Centrally administered apelin-13 induces depression-like behavior in mice, Brain Res. Bull. 88 (2012) 574–580. J.L. Hudson, C.G. van Horne, I. Strömberg, S. Brock, J. Clayton, J. Masserano, B.J. Hoffer, G.A. Gerhardt, Correlation of apomorphine-and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats, Brain Res. 626 (1993) 167–174. E.R. Graf, X. Zhang, S.-X. Jin, M.W. Linhoff, A.M. Craig, Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins, Cell 119 (2004) 1013–1026. Y. Hata, S. Butz, T.C. Sudhof, CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins, J. Neurosci. 16 (1996) 2488–2494. C. Dean, F.G. Scholl, J. Choih, S. DeMaria, J. Berger, E. Isacoff, P. Scheiffele, Neurexin mediates the assembly of presynaptic terminals, Nat. Neurosci. 6 (2003) 708. J.N. Levinson, N. Chéry, K. Huang, T.P. Wong, K. Gerrow, R. Kang, O. Prange, Y.T. Wang, A. El-Husseini, Neuroligins mediate excitatory and inhibitory synapse formation involvement of PSD-95 and neurexin-1β in neuroligin-induced synaptic specificity, J. Biol. Chem. 280 (2005) 17312–17319. O. Prange, T.P. Wong, K. Gerrow, Y.T. Wang, A. El-Husseini, A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13915–13920. N. Masuko, K. Makino, H. Kuwahara, K. Fukunaga, T. Sudo, N. Araki, H. Yamamoto, Y. Yamada, E. Miyamoto, H. Saya, Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90 a possible regulatory role in molecular clustering at synaptic sites, J. Biol. Chem. 274 (1999) 5782–5790. M. Migaud, P. Charlesworth, M. Dempster, L.C. Webster, A.M. Watabe, M. Makhinson, Y. He, M.F. Ramsay, R.G. Morris, J.H. Morrison, Enhanced longterm potentiation and impaired learning in mice with mutant postsynaptic density95 protein, Nature 396 (1998) 433. R. Sattler, Z. Xiong, W.-Y. Lu, M. Hafner, J.F. MacDonald, M. Tymianski, Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein, Science 284 (1999) 1845–1848. J. Zhang, S.M. Lewis, B. Kuhlman, A.L. Lee, Supertertiary structure of the MAGUK core from PSD-95, Structure 21 (2013) 402–413. D.R. Cook, A.J. Gleichman, S.A. Cross, S. Doshi, W. Ho, K.L. Jordan‐Sciutto, D.R. Lynch, D.L. Kolson, NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury, J. Neurochem. 118 (2011) 1113–1123. D.J. Surmeier, J.N. Guzman, J. Sanchez-Padilla, J.A. Goldberg, What causes the death of dopaminergic neurons in Parkinson’s disease? Prog. Brain Res. Elsevier (2010) 59–77. B. Vincenza, G. Veronica, S. Carmelo, C. Paolo, P. Barbara, Synaptic dysfunction in Parkinson’s disease, Biochem. Soc. Trans. 38 (2010) 493–497. R. Galici, N.G. Echemendia, A.L. Rodriguez, P.J. Conn, A selective allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors has effects similar to an orthosteric mGlu2/3 receptor agonist in mouse models predictive of antipsychotic activity, J. Pharmacol. Exp. Ther. 315 (2005) 1181–1187. K.A. Johnson, C.K. Jones, M.N. Tantawy, M. Bubser, M. Marvanova, M.S. Ansari, R.M. Baldwin, P.J. Conn, C.M. Niswender, The metabotropic glutamate receptor 8 agonist (S)-3, 4-DCPG reverses motor deficits in prolonged but not acute models of Parkinson’s disease, Neuropharmacology 66 (2013) 187–195. O. Valenti, M.J. Marino, M. Wittmann, E. Lis, A.G. DiLella, G.G. Kinney, P.J. Conn, Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse, J. Neurosci. 23 (2003) 7218–7226. M. Wittmann, M.J. Marino, P.J. Conn, Dopamine modulates the function of group II and group III metabotropic glutamate receptors in the substantia nigra pars reticulata, J. Pharmacol. Exp. Ther. 302 (2002) 433–441. Y. Ishimaru, A. Sumino, D. Kajioka, F. Shibagaki, A. Yamamuro, Y. Yoshioka, S. Maeda, Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice, J. Pharmacol. Sci. 133 (2017) 34–41. L.A. O’Donnell, A. Agrawal, P. Sabnekar, M.A. Dichter, D.R. Lynch, D.L. Kolson, Apelin, an endogenous neuronal peptide, protects hippocampal neurons against
Peptides 117 (2019) 170091
E. Haghparast, et al.
Neurosci. 25 (2005) 3763–3773. [52] B. Cheng, J. Chen, B. Bai, Q. Xin, Neuroprotection of apelin and its signaling pathway, Peptides 37 (2012) 171–173. [53] N.A. Chapman, D.J. Dupré, J.K. Rainey, The apelin receptor: physiology, pathology, cell signalling, and. ligand modulation of a peptide-activated class A GPCR, Biochem. Cell Biol. 92 (2014) 431–440.
excitotoxic injury, J. Neurochem. 102 (2007) 1905–1917. [50] X.J. Zeng, S.P. Yu, L. Zhang, L. Wei, Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons, Exp. Cell Res. 316 (2010) 1773–1783. [51] P.J. Voulalas, L. Holtzclaw, J. Wolstenholme, J.T. Russell, S.E. Hyman, Metabotropic glutamate receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons, J.
9