Rapamycin suppresses PTZ-induced seizures at different developmental stages of zebrafish
Introduction
Epilepsy is a common neurological disorder, affecting around 65 million people worldwide. This disorder is characterized by the occurrence of recurrent and unpredictable seizures, which occur due to abnormal activity of neuronal cells (Moshé et al., 2014, Lin and Baines, 2014). The mechanist target of rapamycin (mTOR) protein is a 289-kDa serine-threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family and forms two distinct multi-protein complexes: mTORC1 and mTORC2. mTOR complexes integrate intracellular and extracellular signals and act as central regulators of cell activity, metabolism, growth, proliferation, and survival (Laplante and Sabatini, 2012, Laplante and Sabatini, 2013). Rapamycin is an anti-fungal macrolide compound that acts by inhibiting mTOR signaling and is produced by the soil bacterium Streptomyces hygroscopicus, which was isolated from a soil sample obtained in Rapa Nui (Vézina et al., 1975).
mTORC1 is the best characterized of the two mTOR complexes and integrates different inputs from intracellular and extracellular signals: growth factors, stress, energy status, hormones, and amino acids in the control of various cellular processes, including protein and lipid synthesis, differentiation, and autophagy (Laplante and Sabatini, 2013, Maiese et al., 2013). Regarding the central nervous system (CNS), the mTORC1 complex regulates a variety of neuronal functions: cell proliferation, survival, growth, and plasticity. Disruption of the mTORC1 pathway has been implicated in different neurological disorders, and previous studies have shown that the mTORC1 signaling overactivation is related to epilepsy occurrence (Wong, 2010). The mTORC1 overactivation has been verified in genetic and acquired epilepsies: tuberous sclerosis complex, focal cortical dysplasias, and animal models of epilepsy acquired after status epilepticus or trauma (Wong, 2010). Therefore, as a mTORC1 inhibitor, rapamycin could be an important alternative in epilepsy treatment (Ryther and Wong, 2012).
Several studies have shown that rapamycin has seizure suppressive and antiepileptogenic effects in animal models of epilepsy. Rapamycin treatment attenuated the development of posttraumatic epilepsy in a mouse model of traumatic brain injury, decreasing the frequency of seizure occurrence (Guo et al., 2013). Furthermore, rapamycin treatment showed protective effect following status epilepticus since rats treated with this compound presented less seizure frequency when compared with untreated animals (van Vliet et al., 2012). In addition, rapamycin treatment in epileptic rodents significantly reduced seizure frequency, suggesting an antiseizure effect (Huang et al., 2010). Therefore, different studies suggest a protective effect of rapamycin against epilepsy. However, whereas the antiepileptogenic effects of rapamycin have been shown to be a reality, the acute action of rapamycin in suppressing seizures needs to be further investigated in animal models of seizure. Moreover, the anticonvulsant effects of rapamycin appear to vary depending on the stage of development. In immature rats, rapamycin pretreatment had anticonvulsant effects against PTZ-induced seizures. However, the same treatment was ineffective against PTZ-induced seizures in adult animals (Chachua et al., 2012). Nevertheless, few studies have reported the effects of rapamycin throughout the development and more investigation is necessary to address this issue.
Zebrafish is a small freshwater teleost which has become widely used as a model organism to understand seizure modulation. PTZ-induced seizures both in larval and adult zebrafish caused behavioral, molecular, and electrographic alterations that would be expected from a seizure episode (Baraban et al., 2005, Pineda et al., 2011). In addition, both larval and adult zebrafish show response to classic antiepileptic drugs (AEDs), such as valproate (VPA), carbamazepine, and phenytoin (Berghmans et al., 2007, Lee et al., 2010, Siebel et al., 2013).
Despite the large number of clinically-used anticonvulsant drugs, approximately 30% of epileptic patients are refractory to current pharmacological treatments (Moshé et al., 2015). Therefore, novel therapeutic approaches that prevent or reverse the molecular and cellular mechanisms of epilepsy are necessary. Considering that the mTORC1 pathway is a prominent target in anticonvulsant therapies and zebrafish is an effective model widely used in seizure studies, the investigation of rapamycin effects on PTZ-induced seizures in zebrafish may improve our knowledge on the modulation of mTORC1 on seizure control.
Section snippets
Animals
Larval (7 days post fertilization, 7 dpf), juvenile (45 days post fertilization, 45 dpf), and adult (6–8 months) wild-type zebrafish (Danio rerio) used in this study were obtained from our breeding stock held at Pontifícia Universidade Católica do Rio Grande do Sul, Brazil. Zebrafish embryos were obtained from natural mating of adult zebrafish bred and maintained in an automated re-circulating tank system (Zebtec, Tecniplast Group, Buguggiate, VA, Italy). Fertilized eggs were collected, washed with
Locomotor activity response following classic AED treatment along different developmental stages
Results regarding the locomotion analysis have shown that the treatment with the classic AED VPA before PTZ-induced seizures did not alter the locomotor activity in larval, juvenile, and adult zebrafish (Fig. 1A).
Behavioral seizure response following classic AED treatment along different developmental stages
In order to investigate seizure development and zebrafish response to a classic AED along three different life stages (larval, juvenile, and adult), we analyzed the latencies to the first typical behavior characterizing each seizure stage (I, II, and III). Larvae, juveniles, and adults
Discussion
The mTOR signaling pathway acts as a molecular system integrator in response to nutrients, neurothrophic factors, and neurotransmitters, controlling protein synthesis and autophagy (Lipton and Sahin, 2014). This mechanism coordinates neural stem cell proliferation, synaptic plasticity, neuronal death, and neurogenesis (Lipton and Sahin, 2014). Concerning the cellular and molecular processes that may be regulated by mTORC1 in the CNS, pharmacological modulation of the mTOR signaling pathway
Acknowledgments
This work was supported by DECIT/SCTIE-MS through Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (Proc. 10/0036-5, conv. n. 700545/2008 — PRONEX). F.P.M. was the recipient of a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). B.D.P. was recipient of a fellowship from Programa Bolsa de Pesquisa para Alunos da Graduação (BPA/PUCRS). A.M.S. and C.D.B. were recipients
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