Lorenzo Romero-Ramírez

Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation

BackgroundBile 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.MethodsThe 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.ResultsTUDCA 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.ConclusionsWe 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.

Synthesis of Antimitotic Thioglycosides: In Vitro and in Vivo Evaluation of Their Anticancer Activity

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.

TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 with Anti-Inflammatory Effects in Microglial Cells.

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.

Salubrinal inhibits the expression of proteoglycans and favors neurite outgrowth from cortical neurons in vitro

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.

The Effect of Antitumor Glycosides on Glioma Cells and Tissues as Studied by Proton HR-MAS NMR Spectroscopy

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the 1H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death.

Tauroursodeoxycholic acid: more than just a neuroprotective bile conjugate

Neurological diseases and the neuroinflammatory response: Neurological diseases are usually accompanied by dramatic changes in the tissue homeostasis, inducing a neuroinflammatory environment that leads to the progressive activation of central nervous system (CNS) resident cells, and in certain cases, to the infiltration of leukocytes into the CNS. If this neuroinflammatory response persists, it is toxic for CNS resident cells, especially for neurons, and consequently is detrimental for the progression and outcome of neurological diseases. The blood-brain barrier (BBB) is a physical and functional barrier that facilitates the maintenance of homeostasis restricting the traffic of substances and cells from the blood to the CNS parenchyma. CNS resident cells (mainly glial cells) actively monitor and detect any alteration in tissue homeostasis. Any unbalance (such as infections, trauma, stroke, and neurodegeneration) leads to the activation of CNS glial cells (mainly astrocytes and microglia), to counterbalance the alteration and bring back homeostasis (Tian et al., 2012). Upon activation, microglial cells and astrocytes undergo severe morphological and structural changes, ranging from a resting to a reactive state. Reactive glial cells show enhanced release of proand anti-inflammatory mediators, phagocytic capacity and increased migration to the insult site. If this glial response cannot restore proper physiological condition, the inflammatory status is maintained by the secretion of pro-inflammatory mediators, resulting in chronic neuroinflammation and the loss of white and gray matter distinctive of CNS pathologies (Popovich et al., 2002). Moreover, pro-inflammatory cytokines and chemokines alter the BBB permeability, allowing the activation and recruitment of leukocytes to the CNS parenchyma (Weiss et al., 2009; Tian et al., 2012) that help to maintain the chronic neuroinflammation as a feedback mechanism with detrimental effects on the progression and outcome of neurological diseases.

All roads go to Salubrinal: endoplasmic reticulum stress, neuroprotection and glial scar formation.

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.

New oleyl glycoside as anti-cancer agent that targets on neutral sphingomyelinase.

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.

Specific Synthesis of Neurostatin and Gangliosides O -Acetylated in the Outer Sialic Acids Using a Sialate Transferase

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides’ outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.

Micellar Iron Oxide Nanoparticles Coated with Anti-Tumor Glycosides

The synthesis procedure of nanoparticles based on thermal degradation produces organic solvent dispersible iron oxide nanoparticles (OA-IONP) with oleic acid coating and unique physicochemical properties of the core. Some glycosides with hydrophilic sugar moieties bound to oleyl hydrophobic chains have antimitotic activity on cancer cells but reduced in vivo applications because of the intrinsic low solubility in physiological media, and are prone to enzymatic hydrolysis. In this manuscript, we have synthetized and characterized OA-IONP-based micelles encapsulated within amphiphilic bioactive glycosides. The glycoside-coated IONP micelles were tested as Magnetic Resonance Imaging (MRI) contrast agents as well as antimitotics on rat glioma (C6) and human lung carcinoma (A549) cell lines. Micelle antimitotic activity was compared with the activity of the corresponding free glycosides. In general, all OA-IONP-based micellar formulations of these glycosides maintained their anti-tumor effects, and, in one case, showed an unusual therapeutic improvement. Finally, the micelles presented optimal relaxometric properties for their use as T2-weighed MRI contrast agents. Our results suggest that these bioactive hydrophilic nano-formulations are theranostic agents with synergistic properties obtained from two entities, which separately are not ready for in vivo applications, and strengthen the possibility of using biomolecules as both a coating for OA-IONP micellar stabilization and as drugs for therapy.