Findings In Oocytes Research Articles

Effect of a GRIN2A de novo mutation associated with epilepsy and intellectual disability on NMDA receptor currents and Mg2+ block in cultured primary cortical neurons

Background GRIN2A encodes the GluN2A subunit of the NMDA receptor (NMDAR), an ionotropic glutamate receptor that has important roles in synaptogenesis and synaptic plasticity. Some individuals with early onset epilepsies and intellectual disability carry heterozygous missense mutations in this gene, including a de-novo mutation in the receptor pore region (GluN2AN615K). We hypothesised that this mutation underlies the carrier's brain disorder and sought to explore its functional consequences. MethodsWe made two-electrode voltage clamp recordings from Xenopus laevis oocytes expressing GluN1/GluN2AN615K (N615K) NMDARs and compared them with wild-type (WT) NMDARs to assess the mutation's effect on potency of inhibition by Mg2+ and other channel blockers. We then used whole-cell patch-clamping to evaluate NMDAR-mediated currents in mouse primary cortical pyramidal neurons transfected with either GluN2AWT or GluN2AN651K subunits. Means were compared by use of independent two-tailed t tests. FindingsIn oocytes, Mg2+ (1 mM) block at −60 mV was significantly decreased (N615K [n=13], mean 5% [SE 8] vs WT [n=15], 89 [4]; p<0·0001). Furthermore, in N615K (n=17) and WT (n=17) oocytes, block by 10 μM memantine was also reduced (mean 26% [6] vs 75 [7], p<0·0001) as was block by 100 μM amantadine (18% [4] vs 44 [12], p<0·0001). Block by ketamine (N615K, n=14; WT, n=14) was not significantly affected, whereas block by dextromethorphan was increased (N615K [n=9], 56% [8] vs WT [n=8], 44 [6]; p=0·003). In N615K (n=10) and WT (n=10) neurons we observed a significant decrease in Mg2+ sensitivity (49% [18] vs 95 [5], p<0·0001) and a significant decrease in current density (42 pA/pF [19] vs 61 [20], p=0·044). InterpretationThis study suggests that the disease-associated mutation GluN2AN615K has substantial effects on NMDAR inhibition by both endogenous and exogenous channel blockers, and on NMDA current density. It is plausible that these changes underlie the carrier's phenotype. FundingWellcome Trust via an Edinburgh Clinical Academic Training PhD Fellowship.

Function and regulation of the mammalian Musashi mRNA translational regulator

The evolutionarily conserved RNA-binding protein, Musashi, regulates neural stem cell self-renewal. Musashi expression is also indicative of stem cell populations in breast and intestinal tissues and is linked to cell overproliferation in cancers of these tissues. Musashi has been primarily implicated as a repressor of target mRNAs in stem cell populations. However, little is known about the mechanism by which Musashi exerts mRNA translational control or how Musashi function is regulated. Recent findings in oocytes of the frog, Xenopus, indicate an unexpected role for Musashi as an activator of a number of maternal mRNAs during meiotic cell cycle progression. Given the importance of Musashi function in stem cell biology and the implications of aberrant Musashi expression in cancer, it is critical that we understand the molecular processes that regulate Musashi function.

Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions.

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.