Leonor Pérez-Martínez

Tissue inhibitor of metalloproteinase-2 promotes neuronal differentiation by acting as an anti-mitogenic signal.

Although traditionally recognized for maintaining extracellular matrix integrity during morphogenesis, the function of matrix metallo-proteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in the mature nervous system is essentially unknown. Here, we report that TIMP-2 induces pheochromocytoma PC12 cell-cycle arrest via regulation of cell-cycle regulatory proteins, resulting in differentiation and neurite outgrowth. TIMP-2 decreases cyclins B and D expression and increases p21Cip expression. Furthermore, TIMP-2 promotes cell differentiation via activation of the cAMP/Rap1/ERK (extracellular signal-regulated kinase) pathway. Expression of dominant-negative Rap1 blocks TIMP-2-mediated neurite outgrowth. Both the cell-cycle arrest and neurite outgrowth induced by TIMP-2 was independent of MMP inhibitory activity. Consistent with the PC12 cell data, primary cultures of TIMP-2 knock-out cerebral cortical neurons exhibit significantly reduced neurite length, which is rescued by TIMP-2. These in vitro results were corroborated in vivo. TIMP-2 deletion causes a delay in neuronal differentiation, as demonstrated by the persistence of nestin-positive progenitors in the neocortical ventricular zone. The interaction of TIMP-2 with α3β1 integrin in the cerebral cortex suggests that TIMP-2 promotes neuronal differentiation and maintains mitotic quiescence in an MMP-independent manner through integrin activation. The identification of molecules responsible for neuronal quiescence has significant implications for the ability of the adult brain to generate new neurons in response to injury and neurological disorders, such as Alzheimers and Parkinsons diseases.

Role of microRNAs in central nervous system development and pathology

Gene expression regulation is essential for correct functioning of the cell. Complex processes such as development, apoptosis, cell differentiation, and cell cycling require a fine tuning of gene expression. MicroRNAs (miRNAs) are small RNAs that have been recognized as key components of the gene expression regulatory machinery. By sequence complementarity, miRNAs recognize target mRNAs and inhibit their function through degradation or by repressing their translation. The development of the central nervous system (CNS) requires precise and exquisitely regulated gene expression patterns. It is now widely recognized that miRNAs have the capacity to provide such fine regulation both in time and in space. High‐throughput analyses as well as classical molecular biology approaches have allowed the identification of essential miRNAs for CNS development and function. Moreover, recent studies in several model organisms are beginning to show intricate regulatory networks involving miRNAs, transcription factors, and epigenetic regulators during CNS development. Here we review recent findings on the role that miRNAs play in the development of the CNS as well as in neuropathologies such as schizophrenia, Parkinson disease, and Alzheimers disease, among others.

Shaping synaptic plasticity: the role of activity-mediated epigenetic regulation on gene transcription.

Learning and memory are basic functions of the brain that allowed human evolution. It is well accepted that during learning and memory formation the dynamic establishment of new active synaptic connections is crucial. Persistent synaptic activation leads to molecular events that include increased release of neurotransmitters, increased expression of receptors on the postsynaptic neuron, thus creating a positive feedback that results in the activation of distinct signaling pathways that temporally and permanently alter specific patterns of gene expression. However, the epigenetic changes that allow the establishment of long term genetic programs that control learning and memory are not completely understood. Even less is known regarding the signaling events triggered by synaptic activity that regulate these epigenetic marks. Here we review the current understanding of the molecular mechanisms controlling activity‐dependent gene transcription leading synaptic plasticity and memory formation. We describe how Ca2+ entry through N‐methyl‐d‐aspartate‐type glutamate neurotransmitter receptors result in the activation of specific signaling pathways leading to changes in gene expression, giving special emphasis to the recent data pointing out different epigenetic mechanisms (histone acetylation, methylation and phosphorylation as well as DNA methylation and hydroxymethylation) underlying learning and memory.

microRNAs: key triggers of neuronal cell fate

Development of the central nervous system (CNS) requires a precisely coordinated series of events. During embryonic development, different intra- and extracellular signals stimulate neural stem cells to become neural progenitors, which eventually irreversibly exit from the cell cycle to begin the first stage of neurogenesis. However, before this event occurs, the self-renewal and proliferative capacities of neural stem cells and neural progenitors must be tightly regulated. Accordingly, the participation of various evolutionary conserved microRNAs is key in distinct central nervous system (CNS) developmental processes of many organisms including human, mouse, chicken, frog, and zebrafish. microRNAs specifically recognize and regulate the expression of target mRNAs by sequence complementarity within the mRNAs 3′ untranslated region and importantly, a single microRNA can have several target mRNAs to regulate a process; likewise, a unique mRNA can be targeted by more than one microRNA. Thus, by regulating different target genes, microRNAs let-7, microRNA-124, and microRNA-9 have been shown to promote the differentiation of neural stem cells and neural progenitors into specific neural cell types while microRNA-134, microRNA-25 and microRNA-137 have been characterized as microRNAs that induce the proliferation of neural stem cells and neural progenitors. Here we review the mechanisms of action of these two sets of microRNAs and their functional implications during the transition from neural stem cells and neural progenitors to fully differentiated neurons. The genetic and epigenetic mechanisms that regulate the expression of these microRNAs as well as the role of the recently described natural RNA circles which act as natural microRNA sponges regulating post-transcriptional microRNA expression and function during the early stages of neurogenesis is also discussed.

Dexamethasone Rapidly Regulates TRH mRNA Levels in Hypothalamic Cell Cultures: Interaction with the cAMP Pathway

The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1–3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10–10–10–4 M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10–10M, an increase above control at 10–8–10–6M and a reduction at higher concentrations (10–5– 10–4M). The stimulatory effect of 10–8M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10–7M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10–3M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system.

The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl‐CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2’s function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post‐transcriptional and post‐translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals.

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1-24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.

Multifactorial Modulation of TRH Metabolism

Abstract1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through medianeminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary.2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell.3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure.4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times.TRH mRNA are thus controlled by transsynaptic and hormonal influences.5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglu-tamyl peptidase II (PPII).6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity.7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.

MiR-7 Promotes Epithelial Cell Transformation by Targeting the Tumor Suppressor KLF4

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression and their misregulation is common in different types of cancer. Although it has been shown that miR-7 plays an oncogenic role in different cellular contexts, the molecular mechanisms by which miR-7 promotes cell transformation are not well understood. Here we show that the transcription factor KLF4 is a direct target of miR-7 and present experimental evidence indicating that the regulation of KLF4 by miR-7 has functional implications in epithelial cell transformation. Stable overexpression of miR-7 into lung and skin epithelial cells enhanced cell proliferation, cell migration and tumor formation. Alteration of these cellular functions by miR-7 resulted from misregulation of KLF4 target genes involved in cell cycle control. miR-7-induced tumors showed decreased p21 and increased Cyclin D levels. Taken together, these findings indicate that miR-7 acts as an oncomiR in epithelial cells in part by directly regulating KLF4 expression. Thus, we conclude that miR-7 acts as an oncomiR in the epithelial cellular context, where through the negative regulation of KLF4-dependent signaling pathways, miR-7 promotes cellular transformation and tumor growth.