No abstract

The Fragile X syndrome (FXS) is the most frequent form of inherited mental disability and is considered a monogenic cause of autism spectrum disorder. FXS is caused by a triplet expansion that inhibits the expression of the FMR1 gene. The gene product, the Fragile X Mental Retardation Protein (FMRP), regulates mRNA metabolism in brain and nonneuronal cells. During brain development, FMRP controls the expression of key molecules involved in receptor signaling, cytoskeleton remodeling, protein synthesis and, ultimately, spine morphology. Symptoms associated with FXS include neurodevelopmental delay, cognitive impairment, anxiety, hyperactivity, and autistic-like behavior. Twenty years ago the first Fmr1 KO mouse to study FXS was generated, and several years later other key models including the mutant Drosophila melanogaster, dFmr1, have further helped the understanding of the cellular and molecular causes behind this complex syndrome. Here, we review to which extent these biological models are affected by the absence of FMRP, pointing out the similarities with the observed human dysfunction. Additionally, we discuss several potential treatments under study in animal models that are able to partially revert some of the FXS abnormalities.

The stability of expression of an internal control is required for accurate and reliable normalization in quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) experiments. However, expression of commonly used reference genes can be regulated under specific experimental conditions, particularly in response to stimuli that exert multiple effects on gene expression. The neurotrophin brain-derived neurotrophic factor (BDNF) regulates gene expression through activation of multiple signaling cascades, and we have conducted an expression study for the proper validation of internal control genes in BDNF-stimulated cultured hippocampal neurons. geNorm and NormFinder were applied to eight potential genes to identify the most stable genes to be used in the relative quantification of the effects of BDNF on gene expression. Our data show that Tbp (TATA box binding protein), Ppia (peptidylprolyl isomerase A), Pgk1 (phosphoglycerate kinase 1), and Hprt1 (hypoxanthine guanine phosphoribosyl transferase I) are the most stable genes under the experimental conditions used, contrasting with Tuba1 (tubulin alpha1-A chain) and Gapdh (glyceraldehydes-3-phosphate dehydrogenase), two genes widely used as control genes, which showed an unstable expression in hippocampal neurons stimulated with BDNF. Analysis of the BDNF-induced changes in expression of Sars, Tufm, and Egr3 by using different sets of control genes showed distinct results, with a combination of three to four of the genes Tbp/Ppia/Pgk1/Hprt1 providing the most consistent results. Our data reinforce the need for proper validation of the internal control genes for an accurate quantification of qRT-PCR results, particularly when analyzing cellular responses to agents (e.g., neurotrophins) that cause multiple changes in gene expression.

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin-proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.