Potent activation of FGF-2 IRES-dependent mechanism of translation during brain development.

Fibroblast growth factor-2 (FGF-2) plays a fundamental role in brain functions. This role may be partly achieved through the control of its expression at the translational level via an internal ribosome entry site (IRES)-dependent mechanism. Transgenic mice expressing a bicistronic mRNA allowed us to study in vivo and ex vivo where this translational mechanism operates. Along brain development, we identified a stringent spatiotemporal regulation of FGF-2 IRES activity showing a peak at post-natal day 7 in most brain regions, which is concomitant with neuronal maturation. At adult age, this activity remained relatively high in forebrain regions. By the enrichment of this activity in forebrain synaptoneurosomes and by the use of primary cultures of cortical neurons or cocultures with astrocytes, we showed that this activity is indeed localized in neurons, is dependent on their maturation, and correlates with endogenous FGF-2 protein expression. In addition, this activity was regulated by astrocyte factors, including FGF-2, and spontaneous electrical activity. Thus, neuronal IRES-driven translation of the FGF-2 mRNA may be involved in synapse formation and maturation.

Protective effect of tert-butylhydroquinone on the quinolinic-acid-induced toxicity in rat striatal slices: role of the Nrf2-antioxidant response element pathway.

Tert-butylhydroquinone (tBHQ) is a xenobiotic with reported antioxidant properties. tBHQ has been shown to induce nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2) to further activate the antioxidant response element (ARE). In turn, the Nrf2/ARE pathway is responsible for the induction of phase 2 antioxidant enzymes that detoxify oxidant promoters from different toxic insults. In this work, the antioxidant and protective actions of tBHQ were explored for the first time on different biomarkers of the neurotoxic model produced by the excitotoxic and pro-oxidant molecule quinolinic acid (QUIN) in rat striatal slices. For comparison purposes, 3-nitropropionic acid was used as reference model. Our results show that tBHQ (25 µM) prevented the QUIN-induced lipid peroxidation and mitochondrial dysfunction. In addition, tBHQ enhanced glutathione-S-transferase activity, partially recovering its depletion induced by QUIN treatment. Our results also demonstrated that tBHQ was able to induce nuclear accumulation of Nrf2 and further antioxidant protection: while QUIN alone decreased the nuclear Nrf2, a treatment with tBHQ preserved the nuclear levels Nrf2 in the presence of QUIN. Therefore, the tBHQ-mediated Nrf2/ARE induction constitutes a signaling-mediated antioxidant strategy and therapeutic tool to be tested in different neurotoxic models.

A two-step synthetic strategy to obtain a water-soluble derivative of curcumin with improved antioxidant capacity and in vitro cytotoxicity in C6 glioma cells.

A novel water-soluble derivative of curcumin (Cur-[G-2]-OH) was designed and synthesized from accessible raw materials in only two steps with an overall yield of 80%. The modification of curcumin phenol groups with second-generation polyester dendrons (dendronization) as a strategy to achieve an optimal hydrophilic/hydrophobic balance allows the complete water solubilization of the new curcumin derivative (5mg/ml) at room temperature. The therapeutic potential of Cur-[G-2]-OH was investigated in terms of antioxidant capacity, intracellular uptake and cytotoxicity in both rat glioblastoma cells and normal human dermal fibroblasts. Although the phenolic groups of curcumin were locked by dendronization, Cur-[G-2]-OH exhibited antioxidant capacity in water that was even higher than curcumin in dimethylsulfoxide (DMSO). This compound showed a steady cellular uptake contrasted with curcumin, which has a saturation capture at high concentrations. Combined with improved stability, this property seems to allow the intracellular accumulation of Cur-[G-2]-OH. Furthermore, the new compound exhibited increased cytotoxicity in rat C6 glioma cells in a time- and concentration-dependent manner, whereas in normal human fibroblasts, its IC50 value was >600µM versus the IC50 of curcumin found between 100 and 200µM. Surprisingly, Cur-[G-2]-OH drives cell death of C6 cells by a different mechanism of apoptosis triggered by curcumin. Together, these results suggest that curcumin dendronization could promote molecular and cellular mechanisms that are different from those induced by curcumin, presumably due to structural factors and not only for improved water solubility.

The MLN4924 inhibitor exerts a neuroprotective effect against oxidative stress injury via Nrf2 protein accumulation.

It was explored the cytoprotective and antioxidant effect of MLN4924, a specific inhibitor of Nedd8-activating enzyme (NAE), against hydrogen peroxide (H2O2)-induced damage in cerebellar granule neurons (CGNs). Primary cultures of CGNs were exposed to H2O2 after preincubation with MLN4924. The compounds were removed, and CGNs were incubated in culture medium for 24h in order to determine cell viability by 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyl-tetrazolium bromide (MTT) and fluorescein diacetate (FDA) assays. It was demonstrated that MLN4924 remarkably attenuated H2O2-induced cell damage. Meanwhile reactive oxygen species (ROS) production was evaluated with the fluorescent probe dihydroethidium (DHE). Interestingly H2O2-induced ROS production was inhibited by pretreatment with MLN4924. MLN4924 treatment in CGNs resulted in nuclear factor E2-related factor 2 (Nrf2) protein accumulation. Intriguingly this effect was observed in the cytosolic and nuclear compartments of the CGNs. The cytoprotective effect of MLN4924 was associated with its ability to diminish ROS production induced by H2O2 and the accumulation of Nrf2 protein levels in the cytoplasm and nucleus of the CGNs.

Hyperglycemia upregulates translation of the fibroblast growth factor 2 mRNA in mouse aorta via internal ribosome entry site.

Fibroblast growth factor 2 (FGF-2) is normally synthesized at low levels but is elevated in various pathophysiological conditions including diabetes-associated vascular diseases. FGF-2 expression is regulated translationally through an internal ribosome entry site (IRES) located in its mRNA, which allows a nonclassical cap-independent translation. We addressed the pathophysiological regulation of the IRES in vivo by using a streptozotocin-induced hyperglycemic model known to suppress markedly overall translation. Evaluation of FGF-2 IRES-dependent translation was performed with transgenic mice expressing dual luciferase bicistronic mRNA containing the FGF-2 IRES. FGF-2 IRES-dependent reporter activity increased 240% of control in the diabetic aorta although the reporter mRNA levels significantly decreased. Expression of endogenous FGF-2 protein in the aorta closely correlated with the IRES activity but not with FGF-2 mRNA levels. Moreover, the biosynthesis of endogenous FGF-2 protein was stimulated in an IRES-dependent manner by high glucose that significantly suppressed global protein synthesis in aortic smooth muscle cells from the transgenic mice. These results suggest that IRES-dependent translational regulation could play a pathological role in FGF-2 expression in vivo, especially in the cardiovascular consequences of diabetes.

Cullin 3 as a novel target in diverse pathologies.

Recent evidence suggests that the malfunctioning disposal system of cell protein called ubiquitin-proteasome system (UPS) plays an important role in the development of disorders, including cancer and neurodegenerative diseases. Accumulating evidence suggests that the abnormal regulation of the E3 ubiquitin ligases, essential components of the UPS, contributes to uncontrolled proliferation, genomic instability and cancer, since these ligases and their substrates are involved in the regulation of cell cycle progression, gene transcription, signal transduction, DNA replication and others. Through selective degradation of specific substrates, E3 ligases regulate different biological processes. Cullins are a family of proteins that confer substrate specificity to multimeric complex of E3 ligases acting as scaffold proteins. So far, seven members of the cullin family of proteins have been identified. Interestingly, the data generated by several groups indicate that cullin 3 (Cul3) has begun to emerge as a protein involved in the etiopathology of multiple diseases. In this paper we examine the latest advances in basic research on the biology of Cul3 and how it could help to direct drug discovery efforts on this target.

Protective effect of L-kynurenine and probenecid on 6-hydroxydopamine-induced striatal toxicity in rats: implications of modulating kynurenate as a protective strategy.

The neuroactive metabolite at the kynunerine pathway, kynurenic acid (KYNA), is a well-known competitive antagonist at the co-agonist glycine site of the N-methyl-D-aspartate receptor (NMDAr), and also decreases the extracellular levels of glutamate by blocking α7-nicotinic acetylcholine receptor (α7-nAchr) located on glutamatergic terminals. KYNA has been often reported to be neuroprotective in different neurotoxic models. The systemic administration of L-kynurenine (L-KYN)--the precursor of KYNA--together with probenecid (PROB)--an inhibitor of organic acids transport--to rodents increases KYNA levels in the brain in a dose-dependent manner. The striatal infusion of the toxin 6-hydroxydopamine (6-OHDA) to rodents is one of the common models used to simulate Parkinson's disease (PD). Different studies have linked PD alterations with excessive glutamatergic transmission in the striatum since NMDAr antagonists exert beneficial effects in PD models. In this work we investigated the effect that a systemic administration of L-KYN+PROB exerted on the toxic model induced by 6-OHDA in rats. PROB (50 mg/kg, i.p.) + L-KYN (75 mg/kg, i.p.) were given to rats for seven consecutive days. On day two of treatment, the animals were infused with a single injection of 6-OHDA (20 µg/2 µl) into the right striatum. Fourteen days post-lesion, rotation behavior was assessed as a marker of motor impairment. The total levels of dopamine (DA) were also estimated in striatal tissue samples of 6-OHDA-treated animals as a neurochemical marker of damage. In addition, twenty eight days post-lesion, the striatal damage was assessed by hematoxylin/eosin staining and immunohistochemistry against glial fibrillary acidic protein (GFAP) in the same animals. Neurodegeneration was also assessed by Fluoro Jade staining. 6-OHDA infusion increased rotation behavior, striatal reactive gliosis and neurodegeneration, while DA levels were decreased. For all markers evaluated, we observed protective effects of L-KYN+PROB on the dopaminergic damage induced by 6-OHDA. Our results suggest that this strategy was useful to mitigate dopaminergic toxicity in the hemiparkinsonian model. The combined use of L-KYN and PROB is a valuable tool to modulate glutamatergic and cholinergic activities, presumably by means of increased levels of endogenous KYNA.