cAMP promotes differentiation of rodent neuronal progenitor cells

Numerous studies have described neuronal differentiation of neural progenitor cells derived from fetal tissue in vitro, but the biochemical mechanisms underlying this process remain largely unknown. In the present study, the role of cAMP in promoting functional maturation of neuronal progenitor cells (NPCs) from the subventricular zone (SVZ) of rodent fetal brain was investigated. NPCs were extracted from telencephalic vesicles of E14 rat embryos and then expanded in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). A mature neuronal fate was induced by: i) withdrawal of growth factors (basal condition); ii) addition of brain-derived neurotrophic factor (BDNF); or iii) addition of isobutylmethylxantine (IBMX). Whole cell patch clamping assessed electrophysiological properties. Immunocyto chemistry for MAP2 confirmed neuronal differentiation. Quantification of neuronal cells, and determination of their electrophysiological properties, was performed in the cited experimental groups. IBMX significantly enhanced the yield of MAP2-positive neurons in the culture system (3.7-fold increase). Application of a one-week differentiation protocol under IBMX induced functional maturity. MAP2-positive cells presented large Naand Kvoltagedependent currents, fired bursts of action potentials, and exhibited spontaneous synaptic activity. Further, IBMX proved more effective than BDNF for promoting neuronal maturation, leading to higher evoked peak currents and current densities, as well as greater yields of cells firing action potentials and presenting active synaptic contacts. Our data indicates the importance of cAMP-dependent mechanisms for maturation of neuronal progenitor cells in vitro. This knowledge can enable future manipulation of neurogenesis in vivo in order to promote the intrinsic regenerative capacity of the central nervous system. Introduction Various different stem cell types have been considered as potential matrices to generate new neurons for central nervous system (CNS) restoration. The most important of these types include neural stem cells (NSCs), extracted from adult and fetal neuronal tissue,1,2 embryonic stem cells (ESC), from the internal layer of blastocysts,3,4 mesenchymal stem cells (MSCs), from adult bone marrow,5,6 and even somatic cells, genetically induced to an immature state, the so-called induced pluripotent stem cells (iPSC).7 We have previously demonstrated that the ability to generate functional neurons varies significantly depending on the stem cell type used.5 Among the different stem cell types, NSCs from fetal tissue have been the most intensively investigated category to date. Studies have shown that neural progenitor cells (NPCs) and NSCs from fetal brains are able to survive in the host tissue, differentiate into mature neurons, and form active synaptic contacts.8-10 Additionally, NSCs are also able to release neurotransmitters and restore functional deficits in Parkinson’s disease,11-14 Huntington’s disease,15 spinal cord injuries,16 stroke17,18 and motor cortical lesions.19 Although much knowledge has been gained in recent years regarding stem cell biology and plasticity, few studies have demonstrated complete electrophysiological maturation of these cells in vitro,10,20 while the mechanisms underlying this process remain largely unknown. The key roles of cAMP and the PKA-pathway in neuronal differentiation have been described in previous studies.5,21-23 However, some controversy remains over these functions, with some authors suggesting that ATP acts preferentially on cell proliferation, serving as a negative regulator of terminal neuronal differentiation.24 cAMP on the other hand, has been shown to promote neurite elongation in NT2 embryonal carcinoma stem cells,21 human neuroblastoma cell lines23 and pheochromocytoma PC12 cells,22 during neuronal differentiation. This effect is thought to occur through phosphorylation of cAMP response-element binding protein (CREB) at the Ser133 position. CREB phosphorylation induces differentiation of hippocampal progenitor cells in vitro,25 and is also believed to regulate specific phases of adult neurogenesis in the subventricular zone/olfactory bulb system in vivo, since coexpression of pCREB and bromodeoxyuridine has been demonstrated in neuroblasts within the subventricular zone.26 In order to clarify the role of cAMP in neuronal differentiation of progenitor cells derived from the rodent subventricular zone, the immunocytochemical and electrophysiological properties, after induction of a neuronal phenotype, were explored under three experimental conditions: i) basal medium, after withdrawal of growth factors; ii) brain-derived neurotrophic factor (BDNF), and iii) isobutylmethylxantine (IBMX). The yield of MAP2-positive neurons was found to increase 1.6 fold under BDNF, and 3.7 fold under IBMX. Electrophysiological recordings with patch clamp revealed significantly higher amplitudes of peak Naand K-currents in cells differentiated under IBMX, as well as a higher number of cells firing action potentials and exhibiting spontaneous synaptic activity. These observations emphasize the importance of cAMP and the PKA-pathway in promoting functional maturation of neural progenitor cells. Materials and Methods Isolation and differentiation of neural progenitor cells from E14 rat embryos All experiments were approved by the Research Ethics Committee of the AlbertStem Cell Studies 2011; volume 1:e9 Correspondence: Guilherme Lepski, Department of Neurosurgery, Eberhard Karls University, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. Tel: +49.7071.29 80325 Fax: +49.7071.29.5245. E-mail: [email protected]