Adrenomedullin Increases Camp Accumulation and BDNF
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Iranian Journal of Basic Medical Sciences ijbms.mums.ac.ir Adrenomedullin increases cAMP accumulation and BDNF expression in rat DRG and spinal motor neurons Mohsen Sisakht 1, Zahra Khoshdel 1, Amir Mahmoodazdeh 1 1, Mohammad Ali Takhshid 2, 3* 1 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, Sayed Mohammad Shafiee 2 Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran 3 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran A R T I C L E I N F O A B S T R A C T Article type: Objective(s): Adrenomedullin (AM) has high expression in the spinal cord. In this study, we investigated Original the expression of AM and its receptor components, including calcitonin receptor-like receptor (CLR) and receptor activity modifying proteins (RAMPs) in dorsal root ganglion (DRG) and spinal motor Article history: (SM) neurons. Furthermore, the effects of AM on cAMP/cAMP response element-binding protein Received: Jan 1, 2021 Accepted: Jun 23, 2021 derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were evaluated. Keywords: Materials(CREB), AKT/glycogen and Methods: synthase kinase-3 beta (GSK-3β) signaling pathways, and expressions of brain- Adrenomedullin Real-time PCR was used to assess expressions of AM, CLR, and RAMPs. cAMP levels, p-CREB, BDNF, Cyclic AMP Rat embryonic DRG and SM neurons were isolated, purified, and cultured. Nerve growth factors were determined by western blotting. Real-time PCR showed expressions of AM, CLR, RAMP2, and Signaling transduction andRAMP3 NT-3 in were both determined DRG and SM using neurons. an enzyme-linked immunosorbent assay. p-AKT and p-GSK-3β levels Spinal cord Results: AM increased cAMP accumulation and p-CREB levels in DRG and SM neurons. AM increased DRG and SM neurons. There was also an increase in NT-3 level in both DRG and SM neurons, which is p-AKT and p-GSK-3β in DRG, but not SM neurons. AM significantly increased BDNF expression in both Conclusion: These results showed both DRG and SM neurons are targets of AM actions in the spinal cord.statistically An increase significant in BDNF in SM expression neurons. by AM in both DRG and SM neurons suggests the possible ►Please cite this article as: beneficial role of AM in protecting, survival, and regeneration of sensory and motor neurons. Sisakht M, Khoshdel Z, Mahmoodazdeh A, Shafiee SM, Takhshid MA. Adrenomedullin increases cAMP accumulation and BDNF expression in rat IntroductionDRG and spinal motor neurons . Iran J Basic Med Sci 2021; 24:978-985. doi:(13), https://doi.org/ stimulatory10.22038/ijbms.2021.54796.12289 effects of AM on cAMP accumulation Adrenomedullin (AM) is a peptide with 52 amino and p-CREB were shown. AKT/GSK3 is another signaling acids, known initially as a vasodilator peptide (1). pathway with an important role in mediating AM effects. However, further investigation showed that AM had Ma et al. showed an increased level of phosphorylated high expression in various parts of the brain and spinal cord (2). In the spinal cord, AM expression occurs in layer of rat spinal dorsal horn, following intrathecal the dorsal root ganglion (DRG) and dorsal horn, both AKT(p-AKT)AM administration, and -GSK-3β suggesting (p-GSKβ3) the role in theof AKT/GSK3superficial neuronal and glial cells (2, 3). The bioactivity of AM pathway in AM-induced hyperalgesia (8). Furthermore, is mainly mediated through AM1 and AM2 receptors, in the cultured rat cardiomyocytes, AM overexpression heterodimeric receptors consist of calcitonin receptor- reduced ischemia/reperfusion-induced apoptosis like receptor (CLR) and one of the receptor-activity- through AKT/ signaling pathway (14). modifying proteins RAMP-2 or -3 (4). Both AM receptors, Brain-derived neurotrophic factor (BDNF) particularly AM1 (CLR/RAMP-2) are sensitive to the and NeurotrophinGSK-3β 3 (NT-3) are members of the neurotrophins family, essential for the growth, antagonistic effects of AM22–52 (5). Also, expression of both CLR and RAMPs was revealed in rat DRG and differentiation, and survival of neurons via activating spinal cord (6-8). Animal studies have shown increased tropomyosin receptor kinase receptors. BDNF is AM-like immunoreactivity in the DRG and spinal cord normally expressed in DRG neurons as well as sensory following several pathological conditions including and motor neurons of the spinal cord. BDNF expression spinal cord injuries (9), formalin- (10), capsaicin-, increases in the DRG and sensory neurons in response to various nociceptive stimuli, suggesting its role in pain pain (2), and repeated intrathecal (i.t.) injection of processing (15). completemorphine (11),Freund’s suggestive adjuvant-induced of the pronociceptive inflammatory role of of neuronal circuits that transmit signals from the CNS AM in both mechanical and chemical-induced pain (12). to muscles (16). Motor Animal neurons studies are revealed the final componentthe critical cAMP/protein kinase A (PKA)/phosphorylated cAMP role of BDNF in the functional properties and plasticity response element-binding protein (p-CREB) is the most of motor neurons (17). The importance of NT-3 has characterized AM signaling pathway (5). In many cell been highlighted in axonal regeneration, especially in lines and primary cells, including dissociated spinal cells the spinal cord (18, 19) and brain injuries (20). BDNF *Corresponding author: Mohammad Ali Takhshid. Shiraz University of Medical Sciences, Zand Blvd., Shiraz, Iran. Tel: +98-7112289113; Email: [email protected]. Adrenomedullin signaling pathway in spinal motor neurons Sisakht et al. and NT-3 are downstream targets of cAMP/PKA/CREB, of gestation (E15) were dislocated, and the DRGs were and their gene expression increases in response to the separated (Figure 1). The DRGs were gently digested increased cellular accumulation of cAMP (21, 22). using trypsin (0.125%, Sigma-Aldrich, Saint Louis, MO, To the best of our knowledge, no study has addressed USA) and mechanical trituration for 15 min at 37 °C. the expression of AM and its receptor components, The resulting cell suspension was seeded on poly-L- including CLR and RAMPs in the motor neurons of the lysine coated plates (48 well culture plate) and cultured spinal cord, yet. Herein, we evaluated and compared the at Neuro Basal-A medium supplemented with 2% B27 expression of AM, CLR, and RAMP2-3 in dissociated rat containing 2 mM GlutaMAX, 100 mg streptomycin, 100 ° DRG and motor neurons. We also investigated the effects units of penicillin, at 37 C with 5% CO2 and 80% relative of AM on the activation of cAMP/CREB and AKT/GSK- humidity. The cell density was adjusted to 1x 105 cells ml- pathways in both DRG and motor neurons and the 1. To remove non-neural cells, the day after incubation, expression of BDNF and NT-3. cells were treated with Materials3β and Methods Chemicals hr. 5-fluorodeoxyuridine (FUDR) Rat AM and AM 22-52 were obtained from Bachem and uridine at a final concentration of 20 mM for 72 Americas, Inc. (Torrance, CA, USA). All chemicals and Anti-β III-tubulin (neuronal marker) monoclonal western blot materials used in this study were obtained fluorescentthe cells were antibody placed andon aDAPI coverslip staining and were treated used with to from Sigma-Aldrich, Santa Cruz, and Abcam. ELISA kit determineice-cold 3.7% the formaldehyde purity of the inDRG PBS neurons for 10 min. (24). The Briefly, cells was obtained from Promega. were then washed with 0.5% bovine serum albumin (BSA) solution, blocked by 5% normal goat serum Animals supplemented with 0.5% BSA solution (1 hr at room Female Sprague-Dawley rats, weighing 200–250 g (n=120), were provided by the Laboratory Animal monoclonal antibody (Cell Signaling, diluted (1:1000) Center of Shiraz University of Medical Sciences, Shiraz, temperature),in a mixture of and 0.5% incubated BSA and with 0.01% β -III-tubulinTriton X-100) mouse for Iran. Rats were mated, and detection of the vaginal 1 hr at room temperature. Goat anti-mouse secondary antibody conjugated with Alexa 488 (Cell Signaling, were housed 4 to a cage in transparent cages (59×38×20 diluted (1:100) in the dilution buffer) along with 1 plaquecm) and was received considered water theand first food day ad libitumof pregnancy. under Ratsa 12 µg/ml DAPI (Sigma) was then added. Incubation was hr light/dark cycle. On day 14 rats were decapitated continued for 30 min in the dark at room temperature. Finally, the cells were washed and visualized under a after CO2 inhalation. All experimental protocols were performed in accordance with the National Institutes ope. of Health Guide for Care and Use of Laboratory Animals and were approved by the Medical and Research Ethics fluorescentaccording to microsc Wang et al spinal cords were Committee of the Shiraz University of Medical Sciences, digestedSM neurons in 0.025% were trypsinisolated, (Invitrogen), purified, and and cultured, spinal Shiraz, Iran. cells were dissociated. (25).using Briefly, trituration. Separating solution (12.4% of OptiPrep in DMEM) was added to the spinal cell suspension and centrifuged at 2200 rpm (10 Isolation and culture of embryonic rat DRG and SM min at 4 °C). Three distinct cellular layers (upper: cell neurons debris, interface: motor neurons, and lower: non-neural Embryonic rat DRG neurons culture was prepared cells) were separated (Figure 1). The motor neuron according to the method described by Burkey et al. layer was carefully separated, seeded on poly-L-lysine coated plates (48 well culture plate) and cultured in (23). Briefly, Sprague-Dawley rat embryos on day 15 Figure 1. Dorsal root ganglion (DRG) and spinal motor neuron cells isolated from rat embryo. Figure A shows the spinal cord and the associated DRGs.