Multitarget neuroprotection by quercetin: Changes in gene expression in two perinatal asphyxia models.

Hypoxic-ischemic encephalopathy (HIE) causes mortality and long-term neurologic morbidities in newborns, affecting pathways related to energy failure, excitotoxicity and oxidative stress that often lead to cell death. The whole process of HIE injury is coupled to changes in the expression of a great array of proteins. A nanoliposomal preparation of the flavonoid quercetin has been shown to exert neuroprotective effects in perinatal asphyxia models. This study aimed to identify neonatal HIE markers and explore the effect of quercetin administration in two perinatal asphyxia models: newborn rats and piglets. In the rat model, nanoliposomal quercetin administration reduced mortality after asphyxia. In the piglet model, quercetin partially overrode the reduction of HIF-1α mRNA levels in the cortex induced by asphyxia. Quercetin administration also reduced increased level of HO-1 mRNA in asphyctic piglets. These results suggest that quercetin neuroprotection might be involved in the regulation of HIF-1α, HO-1 and their targets. A proteomic approach revealed that the glycolytic pathway is strongly regulated by quercetin in both species. We also identified a set of proteins differentially expressed that could be further considered as markers. In piglets, this set includes Acidic Leucine-rich nuclear phosphoprotein 32 (ANP32A), associated with nervous system differentiation, proteins related with death pathways and alpha-enolase which can be converted to neuron-specific enolase, a glycolytic enzyme that may promote neuroprotection. In newborn rats, other promising proteins associated with neurogenesis and neuroprotection emerged, such as dihydropyrimidinase-related proteins, catalytic and regulatory subunits of phosphatases and heterogeneous nuclear ribonucleoprotein K (hnRNPK). Our results show that a nanoliposomal preparation of quercetin, with protective effect in two HIE mammal models, modulates the expression of proteins involved in energy metabolism and other putative neuroprotective signals in the cortex. Identification of these signals could reveal potential molecular pathways involved in disease onset and the novel quercetin neuroprotective strategy.

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis.

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8% of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCl depolarization reaching an inhibition of 49% with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8 percent of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCI depolarization reaching an inhibition of 49 percent with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.

Expression of the growth hormone gene and the pituitary-specific transcription factor GHF-1 in diabetic rats.

Diabetes in the rat is associated with poor growth and decreased GH in the pituitary. In this study we have examined whether this reduction reflects an impairment of GH gene expression. Diabetes was induced by the administration of streptozotocin (7 mg/100 g BW), and 18 days later, GH content, GH mRNA, and GH transcription rate were determined. GH mRNA levels were reduced by more than 80% in the pituitaries of diabetic rats, which had a similarly reduced GH content. The differences observed in transcription fully account for the changes in mRNA concentration, since the transcription rate of the gene was also reduced by a factor of 10 in the diabetic pituitaries. Insulin therapy (3 U/15 days) partially restored these parameters. The expression of the specific transcription factor GHF-1/Pit-1 in diabetic rats was also analyzed. Both GHF-1 mRNA levels and the binding of nuclear proteins to an oligodeoxynucleotide conforming to the GHF-1 proximal binding site in the promoter of the GH gene were normal in the diabetic pituitaries, thus excluding the possibility that decreased availability of this factor could be responsible for the decreased GH transcription. Since diabetes produced an approximately 3-fold reduction of circulating T3, the potential role of thyroid hormones on GH gene expression was also evaluated in thyroidectomized and thyroidectomized diabetic rats. Thyroidectomy decreased GH and GH mRNA to less than 5% of the values found in intact animals, and a single saturating injection of T3 (250 micrograms/100 g BW) resulted in a 8- to 10-fold induction of GH mRNA after 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of thyroid hormone receptor and c-erbA mRNA levels by butyrate in neuroblastoma (N2A) and glioma (C6) cells.

Butyrate produced a biphasic modulation of the thyroid hormone receptor in neuroblastoma N2A cells increasing receptor number by 20-35% at concentrations 0.25-0.75 mM and decreasing receptor levels by 30-55% at 2-4 mM. The half-life of the receptor, as assessed by its disappearance after incubation with 18 microM cycloheximide was 8.4 hr in control cells and 10.3 hr and 5.0 hr in cells incubated with 0.25 and 4 mM butyrate, respectively. This compound increased the abundance of multyacetylated forms of histone H4 from 30% in control cells to almost 70% with butyrate 4 mM. In glioma C6 cells, the fatty acid produced a dose-dependent increase of receptor levels (up to 3-4-fold with 2-5 mM butyrate) and had little effect in increasing multiacetylation (from 30% in controls to 42-46% with 2-5 mM butyrate). Recent studies have shown that the c-erbA proto-oncogen codes for the thyroid hormone receptor. In N2A and C6 cells, 2 c-erbA-related mRNAs, one measuring 2.6 kb and the other 6 kb, were detected. Both forms were differently regulated by butyrate. This compound decreased the abundance of the 2.6 kb forms in both cell types, even at the concentrations at which there was an elevation of receptor levels. Only the largest mRNA correlated with receptor concentration increasing by 2-3-fold after treatment of C6 cells with butyrate, and undergoing a smaller but biphasic change in N2A cells. Our data suggest that modification of chromatin structure probably secondary to acetylation induces changes in thyroid hormone receptor levels in neuroblastoma and glioma cells by affecting both receptor stability and receptor mRNA levels.

Retinoic acid regulates growth hormone gene expression.

Vitamin A is required for normal growth and development, and retinoic acid (RA) may be the active metabolite in this process. Recent evidence indicates that RA acts through binding to a nuclear receptor which belongs to the steroid/thyroid hormone receptor superfamily. The receptors seem to associate with hormone-response elements in the target genes resulting in the activation (or inhibition) of transcription. Although no interaction of RA-receptor complex with specific DNA sequences has yet been reported, the homology of the different receptors suggests their mechanisms of action are similar. We therefore examined whether the effects of RA on growth could be related to changes in the expression of the growth hormone gene which is known to be transcriptionally regulated by both thyroid and glucocorticoid hormones. Our results show that RA controls growth hormone production in pituitary GH1 cells and that its effect is synergistic with that caused by these hormones.