Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS

The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y4 subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y4 subtype with the aid of specific anti-P2Y4 antiserum, and heterologously transfected P2Y4-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y4 receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y4 homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y4 receptors and antitag antibodies. The structural propensity of the P2Y4 protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in <it>Drosophila </it>different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in <it>Drosophila </it>Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response.</p> <p>Results</p> <p>Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3^rdinstar larvae and we show that 20E-induced levels of <it>EcR </it>isoforms <it>EcR-RA, ER-RC</it>, and <it>EcR-RD/E </it>differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types.</p> <p>Conclusions</p> <p>We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.</p

Regulation of type 1 diabetes development and B-cell activation in nonobese diabetic mice by early life exposure to a diabetogenic environment

Microbes, including viruses, influence type 1 diabetes (T1D) development, but many such influences remain undefined. Previous work on underlying immune mechanisms has focussed on cytokines and T cells. Here, we compared two nonobese diabetic (NOD) mouse colonies, NODlow and NODhigh, differing markedly in their cumulative T1D incidence (22% vs. 90% by 30 weeks in females). NODhigh mice harbored more complex intestinal microbiota, including several pathobionts; both colonies harbored segmented filamentous bacteria (SFB), thought to suppress T1D. Young NODhigh females had increased B-cell activation in their mesenteric lymph nodes. These phenotypes were transmissible. Co-housing of NODlow with NODhigh mice after weaning did not change T1D development, but T1D incidence was increased in female offspring of co-housed NODlow mice, which were exposed to the NODhigh environment both before and after weaning. These offspring also acquired microbiota and B-cell activation approaching those of NODhigh mice. In NODlow females, the low rate of T1D was unaffected by cyclophosphamide but increased by PD-L1 blockade. Thus, environmental exposures that are innocuous later in life may promote T1D progression if acquired early during immune development, possibly by altering B-cell activation and/or PD-L1 function. Moreover, T1D suppression in NOD mice by SFB may depend on the presence of other microbial influences. The complexity of microbial immune regulation revealed in this murine model may also be relevant to the environmental regulation of human T1D

Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies

INTRODUCTION: Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called “cerebral reperfusion injury”. Multiple pathological processes are involved in this injury, including leukocyte infiltration, platelet and complement activation, postischemic hyperperfusion, and breakdown of the blood–brain barrier. METHODS/RESULTS AND CONCLUSIONS: Magnetic resonance imaging (MRI) can provide extensive information on this process of injury, and may have a role in the future in stratifying patients’ risk for reperfusion injury following recanalization. Moreover, different MRI modalities can be used to investigate the various mechanisms of reperfusion injury. Antileukocyte antibodies, brain cooling and conditioned blood reperfusion are potential therapeutic strategies for lessening or eliminating reperfusion injury, and interventionalists may play a role in the future in using some of these therapies in combination with thrombolysis or embolectomy. The present review summarizes the mechanisms of reperfusion injury and focuses on the way each of those mechanisms can be evaluated by different MRI modalities. The potential therapeutic strategies are also discussed

The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease.

The miR-17/92 cluster is among the best-studied microRNA clusters. Interest in the cluster and its members has been increasing steadily and the number of publications has grown exponentially since its discovery with more than 1000 articles published in 2012 alone. Originally found to be involved in tumorigenesis, research work in recent years has uncovered unexpected roles for its members in a wide variety of settings that include normal development, immune diseases, cardiovascular diseases, neurodegenerative diseases and aging. In light of its ever-increasing importance and ever-widening regulatory roles, we review here the latest body of knowledge on the cluster\u27s involvement in health and disease as well as provide a novel perspective on the full spectrum of protein-coding and non-coding transcripts that are likely regulated by its members