Prion protein attenuates excitotoxicity by inhibiting NMDA receptors

It is well established that misfolded forms of cellular prion protein (PrP [PrPC]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrPC remains incompletely understood. To determine the physiological role of PrPC, we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)–evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrPC. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPC mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits

Characterization of extracellular proteases of psychrotrophic pseudomonads

For this study 8 cultures of P. fluorescens were selected. A protease was isolated from bacterial culture number T20 and was partially purified by a combination of ammonium sulfate precipitation, gel filtration on Sephadex G-200 and affinity column chromatography. The molecular weight of this protease was estimated to be 43,000 by gel filtration on Sephadex G-200. The proteases investigated in this study were active over a wide range of temperatures (5-45°C) with maximum activity at the temperature of 35°C. The pH optimum for T20 protease was 7.2; other proteases were also maximally active at neutral pH. All of the proteases investigated retained considerable activity after a heat exposure of ten minutes at 100°C. The T20 protease was stable over a wide range of pH (5.5-9.0) when stored at 10°C for four days. All of the proteases were found to be metalloproteases requiring divalent cations for activity and were inactivated slightly by sulfhydryl reagents. The synthesis of all proteases by the bacteria was stimulated by addition of milk to the growth medium. The protease activity of all isolates began to increase in early logarithmic phase and continued to increase till late stationary phase, and then began to decline. The proteases from various isolates were found to possess similar antigenic determinants. Each bacterial culture produced only one extracellular protease

Mechanism of production of multiple mRNAs for the major sialoglycoprotein of human erythrocytes, glycophorin A

Glycophorins are a family of human erythrocyte membrane glycoproteins. At least four different sialoglycoproteins glycophorins, A (or α), B (or δ), C (or β) and D (or γ) have been detected in humans. Glycophorin A is the major sialic acid-containing protein of human erythrocyte membranes. It consists of 131 amino acids distributed in three structural domains. Glycophorin A is encoded by a single gene which gives rise to three different mRNAs, large (2.8 kb ), medium (1.7 kb) and small (1.0 kb) in reticulocytes and in K562, a human erythroleukaemia cell line expressing glycophorin A on its surface. -- Six clones were isolated from a cDNA library constructed with K562 cell mRNA in λgt10 phage using as a probe a synthetic oligonucleotide (GPA-N2) encoding amino acid numbers 30 to 40 of glycophorin A. Nucleotide sequencing of the six clones revealed that all contain an identical protein coding region except for the well known glycophorin AM-AN polymorphism and essentially identical 5" untranslated regions. In contrast, clones differ substantially in the length of their 3" untranslated regions. Examination of the 3 ’ untranslated region of the largest clone revealed seven poly(A) addition signals (AATAAA). To study how the single gene encoding glycophorin A generates three different mRNAs, primer extension analysis and Northern blotting experiments were performed. These experiments supported the findings of the cDNA sequencing and revealed that the three glycophorin A mRNAs differ in the length of their 3 ' untranslated region. The primary structure of the three glycophorin A mRNAs is deduced based upon the nucleotide sequence of various cDNAs, primer extension analysis and Northern blotting experiments. A mechanism is proposed for the generation of the three glycophorin A mRNAs from a single glycophorin A gene that involves differential processing of the 3 ' end of glycophorin A pre-mRNA utilizing multiple poly(A) addition signals

Block of T-type calcium channels by protoxins I and II

Article deposited according to publisher policies: http://www.biomedcentral.com/about/copyright [July 9, 2014].YesFunding provided by the Open Access Authors Fund

Modulation of NMDA receptors by prion proteins

Background: The precise physiological function of endogenous cellular prion protein (PrPC) remains unclear. It has been shown that PrP-null mice exhibit reduced LTP and greater susceptability to seizure mortality in several in vivo (e.g. kainic acid) models of epilepsy. In addition, PrP-null mice exhibit greater exctitotoxic cell death in response to kainic acid exposure. Methods: In our study we investigated the synaptic properties of WT and PrP-null mice. Results: Recordings in the CA1 layer of adult hippocampal slices showed that PrP-null mice exhibit a reduced threshold to evoked responses and no difference in paired-pulse facilitation relative to WT animals. In addition, greater excitability was observed in PrP-null slices in response to zero-Mg2+ induced seizure-like events. Recordings from mature hippocampal cultures showed slightly altered AMPA and GABAA miniature synaptic currents. NMDA mEPSCs were observed to be increased in amplitude and significantly prolonged in decay time. NMDA-evokved currents also exhibited markedly prolonged deactivation kinetics. This effect on evoked NMDA currents was reproduced in WT neurons by PrP-RNAi transfection, and eliminated by PrPC transfection into PrP-null neurons. Conclusions: These data suggest enhanced NMDA activity in PrP-null neurons. Consistent with this finding, in vitro and in vivo excitotoxicity assays demonstrated increased neuronal cell death in PrP-null cultures and animals upon transient exposure to NMDA. The prolonged deactivation kinetics were most consistent with functional activity/augmentation of NR2D NMDA receptor subunits, and PrP coimmunoprecipiated with NR2D NMDA receptor subunits. This enhanced NMDA receptor function was paralleleld by increased excitotoxicy in Prp-null mice. Our findings demonstrate a novel functional role for PrP as a modulator of synaptic NMDA currents and attributes a neuroprotective function to PrP

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation

D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC