Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons. 相似文献
The introduction of induced pluripotent stem (iPS) cells has been a milestone in the field of regenerative medicine and drug discovery. iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy. Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease, Alzheimer’s disease, Huntington disease, ischemic heart disease, diabetes and so on. Moreover, iPS cells derived from patients suffering from unique incurable diseases can be developed into patient- and disease-specific cell lines. These cells can be used as an effective approach to study the mechanisms of diseases, providing useful tools for drug discovery, development and evaluation. The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening. Furthermore, iPS cells can be applied in the field of toxicological and pharmacokinetics tests. This review focuses on the applications of iPS cells in the field of pharmaceutical industry. 相似文献
The excitatory neurotransmitter, glutamate, generates a characteristic burst-pause type of firing in midbrain dopamine neurons in association with the reward behavior, but the cellular mechanism by which glutamate generates these bursts is unknown. Here, we show that the bursts in spontaneously firing dopamine neurons can be generated by the combinative actions of the brief stimulatory and the subsequent Ca(2+)-dependent inhibitory signals in response to glutamate stimulation. The two Ca(2+)-dependent firing-extinction signals are activated by different glutamate receptors. Although the activation of metabotropic glutamate receptors rapidly stopped the enhanced firing through the Ca(2+) release from intracellular stores, the activation of NMDA and AMPA/kainate receptors abolished the firing immediately after termination of the stimulation due to the Ca(2+) accumulation in the cell. These two Ca(2+)-dependent inhibitory mechanisms appear to participate in the generation of characteristic bursts in dopamine neurons by controlling the maximum firing number of single bursts and the duration of post-firing pauses. 相似文献
Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming. 相似文献
Patient‐specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long‐QT syndromes (LQTS). To study the LQT2‐associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC‐derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC‐derived CMs. Further characterization of N996I‐HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype. 相似文献
Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants. 相似文献
Fragile X (FX) is the most common genetic cause of intellectual disability and autism. Previous studies have shown that partial inhibition of metabotropic glutamate receptor signaling is sufficient to correct behavioral phenotypes in a mouse model of FX, including audiogenic seizures, open‐field hyperactivity and social behavior. These phenotypes model well the epilepsy (15%), hyperactivity (20%) and autism (30%) that are comorbid with FX in human patients. Identifying reliable and robust mouse phenotypes to model cognitive impairments is critical considering the 90% comorbidity of FX and intellectual disability. Recent work characterized a five‐choice visuospatial discrimination assay testing cognitive flexibility, in which FX model mice show impairments associated with decreases in synaptic proteins in prefrontal cortex (PFC). In this study, we sought to determine whether instrumental extinction, another process requiring PFC, is altered in FX model mice, and whether downregulation of metabotropic glutamate receptor signaling pathways is sufficient to correct both visuospatial discrimination and extinction phenotypes. We report that instrumental extinction is consistently exaggerated in FX model mice. However, neither the extinction phenotype nor the visuospatial discrimination phenotype is corrected by approaches targeting metabotropic glutamate receptor signaling. This work describes a novel behavioral extinction assay to model impaired cognition in mouse models of neurodevelopmental disorders, provides evidence that extinction is exaggerated in the FX mouse model and suggests possible limitations of metabotropic glutamate receptor‐based pharmacotherapy. 相似文献
The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) have been reported to functionally interact in the striatum. The aim of the present work was to verify the hypothesis that the state of activation of A2A Rs could influence mGlu5R-mediated effects in the striatum. In electrophysiological experiments (extracellular recording in rat corticostriatal slices), the ability of the selective mGlu5R agonist CHPG to potentiate the reduction of the field potential amplitude induced by NMDA was prevented not only by the selective mGlu5R antagonist MPEP, but also by the selective A2AR antagonist ZM 241385. Analogously, the application of CHPG potentiated NMDA-induced toxicity (measured by LDH release) in cultured striatal neurons, an effect that was abolished by both MPEP and ZM 241385. Finally, the A2AR agonist CGS 21680 potentiated CHGP effects, an action that was reproduced and abolished, respectively, by forskolin (an activator of the cAMP/protein kinase A, PKA, pathway) and KT 5720 (a PKA inhibitor). The results indicate that A2ARs exert a permissive role on mGlu5R-induced effects in the striatum. Such an interaction may represent an additional target for the development of therapeutic strategies towards striatal disorders. 相似文献
The glutamate metabotropic receptor 5 (mGluR5) and the adenosine A2A receptor (A2AR) represent major non‐dopaminergic therapeutic targets in Parkinson's disease (PD) to improve motor symptoms and slow down/revert disease progression. The 6‐hydroxydopamine rat model of PD was used to determine/compare the neuroprotective and behavioral impacts of single and combined administration of one mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), and two A2AR antagonists, (E)‐phosphoric acid mono‐[3‐[8‐[2‐(3‐methoxyphenyl)vinyl]‐7‐methyl‐2,6‐dioxo‐1‐prop‐2‐ynyl‐1,2,6,7‐tetrahydropurin‐3‐yl]propyl] (MSX‐3) and 8‐ethoxy‐9‐ethyladenine (ANR 94). Chronic treatment with MPEP or MSX‐3 alone, but not with ANR 94, reduced the toxin‐induced loss of dopaminergic neurons in the substantia nigra pars compacta. Combining MSX‐3 and MPEP further improved the neuroprotective effect of either antagonists. At the behavioral level, ANR 94 and MSX‐3 given alone significantly potentiated l ‐DOPA‐induced turning behavior. Combination of either A2AR antagonists with MPEP synergistically increased L‐DOPA‐induced turning. This effect was dose‐dependent and required subthreshold drug concentration, which per se had no motor stimulating effect. Our findings suggest that co‐treatment with A2AR and mGluR5 antagonists provides better therapeutic benefits than those produced by either drug alone. Our study sheds some light on the efficacy and advantages of combined non‐dopaminergic PD treatment using low drug concentration and establishes the basis for in‐depth studies to identify optimal doses at which these drugs reach highest efficacy.
