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Neuronal maturation is a process that plays a key role in the development and regeneration of the central nervous system. Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal neuronal maturation are less understood. Among the mechanisms influencing such neuronal maturation process, apoptosis plays a key role. Several regulators have been described to modulate apoptosis, including post-transcriptional regulation by microRNAs. This study aimed to assess whether the strikingly induced miR-138-5p during neuronal maturation contributes to avoiding the neuronal death induced by apoptosis. Our results point out that the observed opposite expression of miR-138-5p and its target Caspase3 might modulate apoptosis favouring neuronal survival at distinct maturation stages. The unchanged expression of miR-138-5p in mature neurons contrasts with the significant downregulation in immature neurons upon apoptotic stimulation. Similarly, immunoblot and individual cellular assays confirmed that during maturation, not only the expression but processing of CASP-3 and caspase activity is reduced after apoptotic stimulation which resulted in a reduction of neuronal death. For all this data, we suggest that the upregulation of miR-138-5p during neuronal maturation is crucial in neuronal survival in pathological or traumatic conditions. Further studies would be needed to determine a more detailed role of miR-138-5p in apoptosis during neuronal maturation and the synergistic action of several microRNAs acting cooperatively on Caspase3 or other apoptotic targets.Graphical abstractHighligthsNeuronal maturation promotes cell survival.Neuronal maturation reduces the processing of CASP-3 and caspase activity after apoptotic stimulation.The expression changes of miR-138-5p and Caspase 3 are opposite during hippocampal neuron maturation.The increase of miR-138-5p expression throughout maturation is not influenced by apoptotic stimulation.MiR-138-5p is suggested as an essential factor concerning neuronal maturation, regulating Caspase 3 and favouring survival.

Nogo-A protein is a key myelin-associated inhibitor for axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in CNS such as neuronal differentiation, neuritogenesis, and plasticity. Our results showed that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3’UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.Graphical abstractHighlightsBioinformatics analyses show that miR-182-5p targets Nogo-A 3’UTR.MiR-182-5p downregulates Nogo-A protein expression in murine cell lines.MiR-182-5p promotes neurite outgrowth of rat primary hippocampal neurons in vitro.MiR-182-5p is suggested as a potential therapeutic tool for the promotion of axonal regeneration in different pathologies/diseases of the central nervous system.

Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti-inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231-2245, 2017. © 2016 Wiley Periodicals, Inc.

Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival, and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. J. Cell. Physiol. 232: 1501-1510, 2017. © 2016 Wiley Periodicals, Inc.

The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in various animal models of neuropathologies. We have previously shown the anti-inflammatory properties of TUDCA in an animal model of acute neuroinflammation. Here, we present a new anti-inflammatory mechanism of TUDCA through the regulation of transforming growth factor β (TGFβ) pathway. The bacterial lipopolysaccharide (LPS) was injected intravenously (iv) on TGFβ reporter mice (Smad-binding element (SBE)/Tk-Luc) to study in their brains the real-time activation profile of the TGFβ pathway in a non-invasive way. The activation of the TGFβ pathway in the brain of SBE/Tk-Luc mice increased 24 h after LPS injection, compared to control animals. This activation peak increased further in mice treated with both LPS and TUDCA than in mice treated with LPS only. The enhanced TGFβ activation in mice treated with LPS and TUDCA correlated with both an increase in TGFβ3 transcript in mouse brain and an increase in TGFβ3 immunoreactivity in microglia/macrophages, endothelial cells, and neurons. Inhibition of the TGFβ receptor with SB431542 drug reverted the effect of TUDCA on microglia/macrophages activation and on TGFβ3 immunoreactivity. Under inflammatory conditions, treatment with TUDCA enhanced further the activation of TGFβ pathway in mouse brain and increased the expression of TGFβ3. Therefore, the induction of TGFβ3 by TUDCA might act as a positive feedback, increasing the initial activation of the TGFβ pathway by the inflammatory stimulus. Our findings provide proof-of-concept that TGFβ contributes to the anti-inflammatory effect of TUDCA under neuroinflammatory conditions.

We designed and synthesized two anomeric oleyl glucosaminides as anti-cancer agents where the presence of a trifluoroacetyl group close to the anomeric center makes them resistant to hydrolysis by hexosaminidases. The oleyl glycosides share key structural features with synthetic and natural oleyl derivatives that have been reported to exhibit anti-cancer properties. While both glycosides showed antiproliferative activity on cancer cell lines, only the α-anomer caused endoplasmic reticulum (ER) stress and cell death on C6 glioma cells. Analysis of sphingolipids and glycosphingolipds in cells treated with the glycosides showed that the α-anomer caused a drastic accumulation of ceramide and glucosylceramide and reduction of lactosylceramide and GM3 ganglioside at concentrations above a threshold of 20 μM. In order to understand how ceramide levels increase in response to α-glycoside treatment, further investigations were done using specific inhibitors of sphingolipid metabolic pathways. The pretreatment with 3-O-methylsphingomyelin (a neutral sphingomyelinase inhibitor) restored sphingomyelin levels together with the lactosylceramide and GM3 ganglioside levels and prevented the ER stress and cell death caused by the α-glycoside. The results indicated that the activation of neutral sphingomyelinase is the main cause of the alterations in sphingolipids that eventually lead to cell death. The new oleyl glycoside targets a key enzyme in sphingolipid metabolism with potential applications in cancer therapy., We appreciate the help of Noelia Villarrubia Migallón (Flow cytometry and cell sorting unit, Instituto Cajal, CSIC) with flow cytometry determinations in cell death/apoptosis experiments. We acknowledge Dr. Ron Prywes, Columbia University for providing UPRE-pGL3 (previously called 5xATF6-pGL3) reporter plasmid. We greatly appreciate the financial support provided by the FISCAM-Servicio de Salud of Castilla-La Mancha (SESCAM) (PI2008/19 and PI2009/51), the Ministry of Economy and Competitiveness (PI11/00592, PI11/01436, SAF2009-11257 and SAF2012-40126), the Comunidad de Madrid (S2009/PPQ-1752), the CSIC (201280E091) and the Generalitat of Catalunya (Grant 2009SGR-1072) was greatly appreciated.

After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth.

Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti-inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231-2245, 2017. © 2016 Wiley Periodicals, Inc.

[Background] Bile acids are steroid acids found predominantly in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in different animal models of stroke and neurological diseases. However, the anti-inflammatory properties of TUDCA in the central nervous system (CNS) remain unknown. [Methods] The acute neuroinflammation model of intracerebroventricular (icv) injection with bacterial lipopolysaccharide (LPS) in C57BL/6 adult mice was used herein. Immunoreactivity against Iba-1, GFAP, and VCAM-1 was measured in coronal sections in the mice hippocampus. Primary cultures of microglial cells and astrocytes were obtained from neonatal Wistar rats. Glial cells were treated with proinflammatory stimuli to determine the effect of TUDCA on nitrite production and activation of inducible enzyme nitric oxide synthase (iNOS) and NFκB luciferase reporters. We studied the effect of TUDCA on transcriptional induction of iNOS and monocyte chemotactic protein-1 (MCP-1) mRNA as well as induction of protein expression and phosphorylation of different proteins from the NFκB pathway. [Results] TUDCA specifically reduces microglial reactivity in the hippocampus of mice treated by icv injection of LPS. TUDCA treatment reduced the production of nitrites by microglial cells and astrocytes induced by proinflammatory stimuli that led to transcriptional and translational diminution of the iNOS. This effect might be due to inhibition of the NFκB pathway, activated by proinflammatory stimuli. TUDCA decreased in vitro microglial migration induced by both IFN-γ and astrocytes treated with LPS plus IFN-γ. TUDCA inhibition of MCP-1 expression induced by proinflammatory stimuli could be in part responsible for this effect. VCAM-1 inmunoreactivity in the hippocampus of animals treated by icv LPS was reduced by TUDCA treatment, compared to animals treated with LPS alone. [Conclusions] We show a triple anti-inflammatory effect of TUDCA on glial cells: i) reduced glial cell activation, ii) reduced microglial cell migratory capacity, and iii) reduced expression of chemoattractants (e.g., MCP-1) and vascular adhesion proteins (e.g., VCAM-1) required for microglial migration and blood monocyte invasion to the CNS inflammation site. Our results present a novel TUDCA anti-inflammatory mechanism, with therapeutic implications for inflammatory CNS diseases., We greatly appreciate the help of Miriam Mecha and Carmen Guaza from the Neuroimmunology Group (Instituto Cajal, CSIC) with the microglial cell cultures. This work was supported by grants of the Spanish Ministry of Science and Innovation (SAF2009-11257), Spanish Ministry of Economics and Competitivity (SAF2012-40126) and grants from FISCAM Comunidad de CastillaLa Mancha (PI2008/19 and PI2009/51). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Central nervous system (CNS) injuries caused by cerebrovascular pathologies (e.g., stroke) or mechanical contusions (e.g., traumatic brain injury) disrupt the blood-brain barrier (BBB) that protects the CNS microenvironment from a direct contact with blood substances and cells. The initial neural damage caused by the trauma and the ischemic process is extended in time by a secondary neuronal loss due to the reactive microglial cells and blood leukocytes that migrate to the lesion site and produce inflammatory mediators (e.g., reactive oxygen species) that increase cell death. The severity of the neural damage in patients will determine the extension of the short- and long-term physical, cognitive and emotional impairments associated with these pathologies (McAllister, 2011). Glial cells (mainly astrocytes) and profibrotic mesenchymal cells (meningeal fibroblasts, perivascular fibroblasts and pericytes) react to the injury and migrate to the lesion site, secreting extracellular matrix proteins and inducing a new glia limitans called glial scar (Fawcett and Asher, 1999). This physical structure reduces the leakage of blood substances and the migration of blood cells to the lesion site, reducing cell death and facilitating the recovery of tissue homeostasis (Raposo and Schwartz, 2014). However, the glial scar is one of the main obstacles to axonal regeneration after injury (Fawcett and Asher, 1999). Secretory and transmembrane proteins are synthesized in the ribosomes coupled to the endoplasmic reticulum (ER). These proteins are folded by ER-resident chaperones that ensure a proper transport from ER to the Golgi apparatus. There, a quality control mechanism recognizes misfolded and/or unfolded proteins and induces their degradation in the proteasome, avoiding their accumulation in the lumen of the ER. Diverse pathological conditions (e.g., ischemia, trauma, viral and bacterial infections) may induce the accumulation of misfolded and/or unfolded proteins in the ER that trigger ER stress response. If this response cannot restore homeostasis, it may become chronic, resulting in cell death (Hetz and Mollereau, 2014). Salubrinal is a small molecule with cytoprotective effect on ER stress-induced cell death (Boyce et al., 2005). The neuroprotective effect of Salubrinal has been reported in an excitotoxic neuronal injury model in rat brain (Sokka et al., 2007), in a mouse model of sleep apnea (Zhu et al., 2008), in a cerebral ischemia/reperfusion injury model in rats (Nakka et al., 2010) and traumatic brain injury model in mice (Rubovitch et al., 2015). Moreover, Salubrinal had a cytoprotective effect on oligodendrocytes, reducing demyelination and improving functional recovery after spinal cord injury in mice (Ohri et al., 2013). Salubrinal treatment reduces cell death through the diminution of the ER stress response induced in CNS injury models (Sokka et al., 2007; Ohri et al., 2013), probably reducing ER protein overload in pathological conditions. The phosphorylation status of the translational initiator eIF2α regulates protein translation in the ER. eIF2α is phosphorylated by four different kinases: GCN2 (activated by amino acid starvation), HRI (activated by heme deprivation, as well as by osmotic and heat shocks), PKR (activated by viral infections, some cytokines and growth factors) and PERK (activated by ER stress and hypoxia). Increasing the phosphorylation status of eIF2α attenuates the translation of secretory proteins that are synthesized in the ER. Conversely, reducing the phosphorylation of eIF2α increases the translation of secretory proteins. PP1α phosphatase forms a complex with GADD34 or CReP protein that dephosphorylates eIF2α. Salubrinal is an inhibitor of the protein phosphatase PP1 that attenuates the translation of secretory proteins, maintaining eIF2α highly phosphorylated (Boyce et al., 2005). After CNS injury, reactive astrocytes express and secrete chondrotin sulfate proteoglycans (CSPGs), such as brevican, neurocan, versican and phosphacan, major axon growth inhibitory components of the glial scar (Fawcett and Asher, 1999). CSPGs consist of a large variety of core proteins, covalently linked to chondroitin sulfate glycosaminoglycans, synthesized in the ER and glycosylated in the Golgi apparatus. Both protein and glycosylated core of CSPGs have been described as axon growth inhibitors (Fawcett and Asher, 1999). Glial scar formation is regulated by cytokines and growth factors released from platelets, blood cells and CNS endogenous cells that initially respond to the lesion and then to the subsequent inflammation. Growth factors such as epidermal growth factor (EGF), transforming growth factor β (TGFβ) and connective tissue growth factor (CTGF), and cytokines such as interleukin-6 (IL-6), interferon gamma (IFNγ), and IL-1β, regulate the expression and secretion of CSPGs by astrocytes (Asher et al., 2000). Because astrocytes are the main producers of CSPGs and other profibrotic substances that form the glial scar, we studied in these cells the effect of Salubrinal on the expression and secretion of CSPGs (Barreda-Manso et al., 2015). Translational attenuation induced by Salubrinal (maintaining eIF2α phosphorylated) reduced the expression and secretion of CSPGs and other profibrotic proteins such as CTGF. Additionally, Salubrinal reduced the mRNAs for CSPGs and CTGF. These data suggests that Salubrinal might induce the degradation of non-translated ER-targeted protein mRNAs. This process will collaborate with the translational attenuation, to reduce protein overload in the ER. We used an in vitro model of glial scar to determine whether Salubrinal may have a beneficial effect on neurite outgrowth from cortical neurons. A coculture of astrocytes and fibroblasts was treated with the profibrotic growth factor TGFβ. Cortical neurons grown on top of astrocytes-fibroblasts cocultures, treated with TGFβ showed a reduced length of their neurites compared to control cocultures. However, pretreatment of the cocultures with Salubrinal, reverted the neurite outgrowth inhibition compared to cocultures treated with TGFβ only (Barreda-Manso et al., 2015). Although these data are preliminary and the effect of Salubrinal must be tested in an animal model of CNS injury before any conclusion, the data open the possibility of modulating extracellular matrix deposition and glial scar formation. As previously described, the glial scar is beneficial, because it reduces the leakage of blood substances and cells, helping to restore homeostasis in the injured tissue (Raposo and Schwartz, 2014). At this point, the question is, how much glial scar reduction is necessary to permit axonal regeneration and at the same time prevent leakage of blood content to the neural parenchyma? What dosage of Salubrinal and for how long the animals should be treated to have a beneficial effect? Most of the articles that studied the effect of Salubrinal on diverse animal models of CNS injury, treated the animals for the first three days after the injury with a concentration of Salubrinal ranging from 1 to 5 mg/kg (Sokka et al., 2007; Zhu et al., 2008; Ohri et al., 2013; Rubovitch et al., 2015). Only one study has followed-up the animals longer than 3 days after the injury (Ohri et al., 2013), with an open field BMS locomotor analysis, performed weekly. They found that animals treated with Salubrinal after a spinal cord injury showed significantly higher functional recovery than untreated injured animals (Ohri et al., 2013). The authors presented BMS scores until 7 weeks after spinal cord injury. The animals treated with Salubrinal for the first 3 days postlesion (acute treatment) had better outcome than injured untreated animals. These data suggest that acute Salubrinal treatment at the concentration tested may not increase leakage of blood content to the neural parenchyma and it may not impede glial scar formation. Obviously, more work is necessary before reaching further conclusions on the therapeutical effect of Salubrinal. However, the promising results of this drug deserve a careful trial. This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2009-11257), the Spanish Ministry of Economy and Competitivity (SAF2012-40126) and grants PI2008/19 and PI2009/51 from the FISCAM-Castilla-La Mancha Community.

The synthesis and biological activity of oleylN-acetyl-α- and β-d-glucosaminides (1 and 2, respectively) and their thioglycosyl analogues (3 and 4, respectively) are reported. The compounds exhibited antimitotic activity on rat glioma (C6) and human lung carcinoma (A549) cell cultures in the micromolar range. Analysis of cell extracts using ultra performance liquid chromatography–mass spectrometry showed that the synthetic glycosides produce alterations in glycosphingolipid metabolism, with variable effect on the level of glucosylceramide depending on the configuration of the antimitotic used. In vivo experiments in nude mice bearing an implanted C6 glioma showed that the α-thioglycoside 3 reduced tumor volume, while the O-glycosyl derivative was inactive, highlighting the importance of using enzyme resistant glycosides., The financial support provided by the Servicio de Salud de Castilla La Mancha Community (SESCAM, PI2008/19), the Ministry of of Science and Innovation (CTQ2007-67403/BQU and CTQ2010-15418), the Comunidad de Madrid (S2009/PPQ-1752), and Generalitat de Catalunya (Grant 2009SGR-1072) is greatly appreciated. We thank Dr. Leoncio Garrido for his help in obtaining MAS NMR spectra and Eva Dalmau for excellent technical assistance.