Dinitrophenol DNP is well known
2,4-Dinitrophenol (DNP) is well known to be cytotoxic at high concentrations. However, recent studies have demonstrated that at low micromolar, non-toxic concentrations, DNP has neuroprotective properties in a number of in vitro and in vivo models of neurodegeneration (reviewed in (De Felice and Ferreira, 2006)). Further, we have previously reported that DNP promotes neuritogenesis in neuronal primary cultures and induces neuronal differentiation of a neuroblastoma cell line (Wasilewska-Sampaio et al., 2005).
In an attempt to understand the early molecular changes underlying DNP-induced neurogenesis, we found that nestin and β-tubulin III levels were increased in DNP-treated EBs, suggesting that DNP triggers an initial commitment to a neuronal phenotype within the EBs. In addition, increased levels of glial fibrillary acidic protein (GFAP) suggest that astrocytes are also initially generated upon DNP treatment and may further contribute to in vitro neuronal development and survival, as previously suggested (Akanuma et al., 2012).
Although the mechanisms underlying DNP-induced neurogenesis and neuroprotection have not been fully elucidated, our finding that DNP increases ERK 1/2 phosphorylation suggests that ERK activation may be involved, as previously described for neuronal differentiation of stem cells (Li et al., 2006; Moon et al., 2009) and normal PF-3758309 cost development (Nuttall and Oteiza, 2011; Pucilowska et al., 2012). Previous studies from our group further support the notion that the neuroprotective actions of DNP involve stimulation of cyclic AMP (cAMP) signaling (Wasilewska-Sampaio et al., 2005; Sebollela et al., 2009). A crosstalk between PKA/cAMP and MEK/ERK appears to facilitate neural differentiation and neuritogenesis (De Felice et al., 2007) and thus might contribute to DNP-induced neurogenesis in ESCs. Indeed, ERK activation was found to be required for DNP-induced neuronal differentiation of a neuroblastoma cell line (Wasilewska-Sampaio et al., 2005).
We also report a protective action of DNP during neural differentiation of ESCs. As expected (Huang et al., 2013), RA-mediated differentiation involved an increase in the percentage of active caspase-3 cells within the EBs. On the other hand, DNP promoted neurogenesis while lowering caspase-3 activation, suggesting a cytoprotective role that might be relevant for cell survival and phenotype stability during and after neural differentation.
DNP is classically described as a mitochondrial uncoupling agent and we thus hypothesized that this property might underlie cytoprotection and neurogenesis in ESCs. Interestingly, however, we found that DNP increases ATP synthesis-linked O2 consumption. This supports the idea that DNP does not cause mitochondrial uncoupling at this concentration, despite evident neural differentiation status. Indeed, alterations in oxygen availability and mitochondrial bioenergetics have been linked to molecular signaling pathways that trigger differentiation across development (Birket et al., 2011; Mutoh et al., 2012). In this regard, future studies may help elucidate the mechanisms by which mitochondrial modulation is linked to the pro-neurogenic action of DNP.
Acknowledgments This work was supported by grants from Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (to STF and SKR) and National Institute of Translational Neuroscience (INNT) (to STF).
Introduction The ability to generate specific tissues from pluripotent stem cells (PSCs) is an important goal for developing human cell replacement therapies and to enable the generation of disease-specific models. While much attention has focused on the derivation of organs involved in metabolic diseases, such as the pancreas and liver, fewer studies have addressed organs derived from anterior foregut endoderm (AFE). Arising from a regional domain of definitive endoderm (DE), the AFE contributes to many organs including the salivary glands, esophagus, stomach, trachea, and lungs. It also gives rise to the pharyngeal endoderm, which in turn forms the thymus, parathyroid glands, thyroid gland and ultimobranchial bodies (Zorn & Wells, 2009).