Ana Perez-Castillo

Regulation of Inflammatory Response in Neural Cells in Vitro by Thiadiazolidinones Derivatives through Peroxisome Proliferator-activated Receptor γ Activation

In most neurodegenerative disorders, including multiple sclerosis, Parkinson disease, and Alzheimer disease, a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated astrocytes and microglia and their cytotoxic agents play a crucial pathological role. Current treatments for these diseases are not effective. In the present study we investigate the effect of thiadiazolidinone derivatives, which have been recently suggested to play a role in neurodegenerative disorders. We have found that thiadiazolidinones are potent neuroprotector compounds. Thiadiazolidinones inhibited inflammatory activation of cultured brain astrocytes and microglia by diminishing lipopolysaccharide-induced interleukin 6, tumor necrosis factor α, inducible nitric-oxide synthase, and inducible cyclooxygenase type 2 expression. In addition, thiadiazolidinones inhibited tumor necrosis factor-α and nitric oxide production and, concomitantly, protected cortical neurons from cell death induced by the cell-free supernatant from activated microglia. The neuroprotective effects of thiadiazolidinones are completely inhibited by the peroxisome proliferator-activated receptor γ antagonist GW9662. In contrast the glycogen synthase kinase 3β inhibitor LiCl did not show any effect. These findings suggest that thiadiazolidinones potently attenuate lipopolysaccharide-induced neuroinflammation and reduces neuronal death by a mechanism dependent of peroxisome proliferator-activated receptor γ activation.

NP031112, a Thiadiazolidinone Compound, Prevents Inflammation and Neurodegeneration under Excitotoxic Conditions: Potential Therapeutic Role in Brain Disorders

Inflammation and neurodegeneration coexist in many acute damage and chronic CNS disorders (e.g., stroke, Alzheimers disease, Parkinsons disease). A well characterized animal model of brain damage involves administration of kainic acid, which causes limbic seizure activity and subsequent neuronal death, especially in the CA1 and CA3 pyramidal cells and interneurons in the hilus of the hippocampus. Our previous work demonstrated a potent anti-inflammatory and neuroprotective effect of two thiadiazolidinones compounds, NP00111 (2,4-dibenzyl-[1,2,4]thiadiazolidine-3,5-dione) and NP01138 (2-ethyl-4-phenyl-[1,2,4]thiadiazolidine-3,5-dione), in primary cultures of cortical neurons, astrocytes, and microglia. Here, we show that injection of NP031112, a more potent thiadiazolidinone derivative, into the rat hippocampus dramatically reduces kainic acid-induced inflammation, as measured by edema formation using T2-weighted magnetic resonance imaging and glial activation and has a neuroprotective effect in the damaged areas of the hippocampus. Last, NP031112-induced neuroprotection, both in vitro and in vivo, was substantially attenuated by cotreatment with GW9662 (2-chloro-5-nitrobenzanilide), a known antagonist of the nuclear receptor peroxisome proliferator-activated receptor γ, suggesting that the effects of NP031112 can be mediated through activation of this receptor. As such, these findings identify NP031112 as a potential therapeutic agent for the treatment of neurodegenerative disorders.

CCAAT/Enhancer-binding Protein β Plays a Regulatory Role in Differentiation and Apoptosis of Neuroblastoma Cells

The C/EBPβ (CCAAT/enhancer-binding protein β) is a transcription factor that belongs to basic region-leucine zipper class DNA-binding proteins. There is a significant body of evidence that suggests that this protein plays a central role in adipocytic and eosinophilic differentiation. However, there is no information available regarding the role of this transcription factor in the development of mammalian neuronal tissues. In this study, we have examined the effect of C/EBPβ overexpression on the differentiation and survival of mouse Neuro2A cells. We found that C/EBPβ induces neuronal differentiation and that this process is inhibited by transfection with the C/EBP homologous protein 10 (CHOP), strongly suggesting that the extension of neurites is indeed due to the C/EBPβ transcriptional activity. As it has been suggested in adipocyte differentiation, here we show that C/EBPβ induces the expression of the endogenous C/EBPα gene and that this protein by itself is also able to induce a differentiated phenotype in Neuro2A cells. Neuronal differentiation induced by C/EBPβ requires activation of the phosphatidylinositol 3-kinase signaling pathway, whereas inhibition of the mitogen-activated protein kinase signaling does not have any effect. In addition, we show that C/EBPβ is expressed in the brain of neonatal rats, suggesting that this protein could play an important role in neuronal maturation. Finally, cell death was also induced by C/EBPβ through activation of the p53 protein and the cdk inhibitor p21.

Glycogen synthase kinase 3 inhibition promotes adult hippocampal neurogenesis in vitro and in vivo

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally identified as a regulator of glycogen metabolism but it also plays a pivotal role in numerous cellular functions, including differentiation, cell cycle regulation, and proliferation. The dentate gyrus of the hippocampus, together with the subventricular zone of the lateral ventricles, is one of the regions in which neurogenesis takes place in the adult brain. Here, using a chemical genetic approach that involves the use of several diverse inhibitors of GSK-3 as pharmacological tools, we show that inhibition of GSK-3 induces proliferation, migration, and differentiation of neural stem cells toward a neuronal phenotype in in vitro studies. Also, we demonstrate that inhibition of GSK-3 with the small molecule NP03112, called tideglusib, induces neurogenesis in the dentate gyrus of the hippocampus of adult rats. Taken together, our results suggest that GSK-3 should be considered as a new target molecule for modulating the production and integration of new neurons in the hippocampus as a treatment for neurodegenerative diseases or brain injury and, consequently, its inhibitors may represent new potential therapeutic drugs in neuroregenerative medicine.

The transcription factor early growth response factor-1 (EGR-1) promotes apoptosis of neuroblastoma cells.

Early growth response factor-1 (EGR-1) is an immediate early gene, which is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. EGR-1 has been involved in diverse biological functions such as cell growth, differentiation and apoptosis. Here we report that enforced expression of the EGR-1 gene induces apoptosis, as determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) analysis, in murine Neuro2A cells. In accordance with this role of EGR-1 in cell death, antisense oligonucleotides increase cell viability in cells cultured in the absence of serum. This apoptotic activity of the EGR-1 appears to be mediated by p73, a member of the p53 family of proteins, since an increase in the amount of p73 is observed in clones stably expressing the EGR-1 protein. We also observed an increase in the transcriptional activity of the mdm2 promoter in cells overexpressing EGR-1, which is paralleled by a marked decrease in the levels of p53 protein, therefore excluding a role of this protein in mediating EGR-1-induced apoptosis. Our results suggest that EGR-1 is an important factor involved in neuronal apoptosis.

Phosphodiesterase 7 Inhibition Preserves Dopaminergic Neurons in Cellular and Rodent Models of Parkinson Disease

BACKGROUND Phosphodiesterase 7 plays a major role in down-regulation of protein kinase A activity by hydrolyzing cAMP in many cell types. This cyclic nucleotide plays a key role in signal transduction in a wide variety of cellular responses. In the brain, cAMP has been implicated in learning, memory processes and other brain functions. METHODOLOGY/PRINCIPAL FINDINGS Here we show a novel function of phosphodiesterase 7 inhibition on nigrostriatal dopaminergic neuronal death. We found that S14, a heterocyclic small molecule inhibitor of phosphodiesterase 7, conferred significant neuronal protection against different insults both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. S14 treatment also reduced microglial activation, protected dopaminergic neurons and improved motor function in the lipopolysaccharide rat model of Parkinson disease. Finally, S14 neuroprotective effects were reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase A. CONCLUSIONS/SIGNIFICANCE Our findings demonstrate that phosphodiesterase 7 inhibition can protect dopaminergic neurons against different insults, and they provide support for the therapeutic potential of phosphodiesterase 7 inhibitors in the treatment of neurodegenerative disorders, particularly Parkinson disease.

THYROID HORMONE-REGULATED BRAIN MITOCHONDRIAL GENES REVEALED BY DIFFERENTIAL CDNA CLONING

Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain.

Peroxisome proliferator-activated receptor γ ligands regulate neural stem cell proliferation and differentiation in vitro and in vivo.

Peroxisome proliferator‐activated receptor gamma (PPARγ) belongs to a family of ligand‐activated nuclear receptors and its ligands are known to control many physiological and pathological situations. Its role in the central nervous system has been under intense analysis during the last years. Here we show a novel function for PPARγ in controlling stem cell expansion in the adult mammalian brain. Adult rats treated with pioglitazone, a specific ligand of PPARγ, had elevated numbers of proliferating progenitor cells in the subventricular zone and the rostral migratory stream. Electron microscopy analysis also showed important changes in the subventricular zone ultrastructure of pioglitazone‐treated animals including an increased number of migratory cell chains. These results were further confirmed in vitro. Neurosphere assays revealed significant increases in the number of neurosphere forming cells from pioglitazone‐ and rosiglitazone (two specific ligands of PPARγ receptor)‐treated cultures that exhibited enhanced capacity for cell migration and differentiation. The effects of pioglitazone were blocked by the PPARγ receptor antagonists GW9662 and T0070907, suggesting that its effects are mediated by a mechanism dependent on PPARγ activation. These results indicate for the first time that activation of PPARγ receptor directly regulates proliferation, differentiation, and migration of neural stem cells in vivo.

Enhancement of BRCA1 gene expression by the peroxisome proliferator-activated receptor γ in the MCF-7 breast cancer cell line

BRCA1 has been linked to the genetic susceptibility of a majority of familial breast and ovarian cancers. Several lines of evidence indicate that BRCA1 is a tumor suppressor and its expression is downregulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 gene expression might lead to new insights into the pathogenesis and treatment of these tumors. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor superfamily that has well-established roles in the regulation of adipocyte development and glucose homeostasis. More recently, it has been shown that ligands of PPARγ have a potent antitumorigenic activity in breast cancer cells. In the present study we have found that two distinct ligands of PPARγ; 15-deoxy-Δ-12,14-prostaglandin J2 (15dPG-J2) and rosiglitazone, increase the levels of BRCA1 protein in human MCF-7 breast cancer cells. Immunofluorescence microscopy analysis showed that, after treatment with 15dPG-J2, the BRCA1 protein is mainly localized in the nucleus. Functional analysis by transient transfection of different 5′-flanking region fragments, as well as gel mobility shift assays and mutagenic analysis, suggests that the effects of 15dPG-J2 and rosiglitazone are mediated through a functional DR1 located between the nucleotides −241 and −229, which is a canonical PPARγ type response element. Our data suggest that PPARγ is a crucial gene regulating BRCA1 gene expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

New Melatonin–N,N-Dibenzyl(N-methyl)amine Hybrids: Potent Neurogenic Agents with Antioxidant, Cholinergic, and Neuroprotective Properties as Innovative Drugs for Alzheimer’s Disease

Here, we describe a new family of melatonin-N,N-dibenzyl(N-methyl)amine hybrids that show a balanced multifunctional profile covering neurogenic, antioxidant, cholinergic, and neuroprotective properties at low-micromolar concentrations. They promote maturation of neural stem cells into a neuronal phenotype and thus they could contribute to CNS repair. They also protect neural cells against mitochondrial oxidative stress, show antioxidant properties, and inhibit human acetylcholinesterase (AChE). Moreover, they displace propidium from the peripheral anionic site of AChE, preventing the β-amyloid aggregation promoted by AChE. In addition, they show low cell toxicity and can penetrate into the CNS. This multifunctional profile highlights these melatonin-N,N-dibenzyl(N-methyl)amine hybrids as useful prototypes in the research of innovative drugs for Alzheimers disease.