Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Providing Home Treatment for Acute Mental Illness During the COVID-19 Pandemic.

The Intensive Home Treatment Team in Edinburgh provides care at home for those with acute and severe mental health problems. During the first COVID-19 lockdown, the team conducted and evaluated video and telephone calls but also continued seeing most patients face to face to ensure adequate care. The in-person care was achieved safely, without an increase in staff sickness events. During the lockdown, the team observed more cases of psychosis, particularly acute and transient psychosis and first-episode psychosis, particularly among women.

Dialysis and plasmapheresis for schizophrenia: a systematic review.

Increasing evidence suggests that circulating factors and immune dysfunction may contribute to the pathogenesis of schizophrenia. In particular, proinflammatory cytokines, complement and autoantibodies against CNS epitopes have recently been associated with psychosis. Related concepts in previous decades led to several clinical trials of dialysis and plasmapheresis as treatments for schizophrenia. These trials may have relevance for the current understanding of schizophrenia. We aimed to identify whether dialysis or plasmapheresis are beneficial interventions in schizophrenia. We conducted a systematic search in major electronic databases for high-quality studies (double-blinded randomised trials with sham controls) applying either haemodialysis or plasmapheresis as an intervention in patients with schizophrenia, published in English from the start of records until September 2018. We found nine studies meeting inclusion criteria, reporting on 105 patients in total who received either sham or active intervention. One out of eight studies reported a beneficial effect of haemodialysis on schizophrenia, one a detrimental effect and six no effect. The sole trial of plasmapheresis found it to be ineffective. Adverse events were reported in 23% of patients. Studies were at unclear or high risk of bias. It is unlikely that haemodialysis is a beneficial treatment in schizophrenia, although the studies were of small size and could not consider potential subgroups. Plasmapheresis was only addressed by one study and warrants further exploration as a treatment modality in schizophrenia.

The human NMDA receptor GluN2AN615K variant influences channel blocker potency.

N-methyl-D-aspartate (NMDA) receptors are glutamate receptors with key roles in synaptic plasticity, due in part to their Mg2+ mediated voltage-dependence. A large number of genetic variants affecting NMDA receptor subunits have been found in people with a range of neurodevelopmental disorders, including GluN2AN615K (GRIN2AC1845A) in two unrelated individuals with severe epileptic encephalopathy. This missense variant substitutes a lysine in place of an asparagine known to be important for blockade by Mg2+ and other small molecule channel blockers. We therefore measured the impact of GluN2AN615K on a range of NMDA receptor channel blockers using two-electrode voltage clamp recordings made in Xenopus oocytes. We found that GluN2AN615K resulted in block by Mg2+ 1 mmol/L being greatly reduced (89% vs 8%), block by memantine 10 µmol/L (76% vs 27%) and amantadine 100 µmol/L (45% vs 17%) being substantially reduced, block by ketamine 10 µmol/L being modestly reduced (79% vs 73%) and block by dextromethorphan 10 µmol/L being enhanced (45% vs 55%). Coapplying Mg2+ with memantine or amantadine did not reduce the GluN2AN615K block seen with either small molecule. In addition, we measured single-channel conductance of GluN2AN615K-containing NMDA receptors in outside-out patches pulled from Xenopus oocytes, finding a 4-fold reduction in conductance (58 vs 15 pS). In conclusion, the GluN2AN615K variant is associated with substantial changes to important physiological and pharmacological properties of the NMDA receptor. Our findings are consistent with GluN2AN615K having a disease-causing role, and inform potential therapeutic strategies.

Functional assessment of triheteromeric NMDA receptors containing a human variant associated with epilepsy.

KEY POINTS: NMDA receptors are neurotransmitter-gated ion channels that are critically involved in brain cell communication Variations in genes encoding NMDA receptor subunits have been found in a range of neurodevelopmental disorders. We investigated a de novo genetic variant found in patients with epileptic encephalopathy that changes a residue located in the ion channel pore of the GluN2A NMDA receptor subunit. We found that this variant (GluN2AN615K ) impairs physiologically important receptor properties: it markedly reduces Mg2+ blockade and channel conductance, even for receptors in which one GluN2AN615K is co-assembled with one wild-type GluN2A subunit. Our findings are consistent with the GluN2AN615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers. ABSTRACT: NMDA receptors are ionotropic calcium-permeable glutamate receptors with a voltage-dependence mediated by blockade by Mg2+ . Their activation is important in signal transduction, as well as synapse formation and maintenance. Two unrelated individuals with epileptic encephalopathy carry a de novo variant in the gene encoding the GluN2A NMDA receptor subunit: a N615K missense variant in the M2 pore helix (GRIN2AC1845A ). We hypothesized that this variant underlies the neurodevelopmental disorders in carriers and explored its functional consequences by electrophysiological analysis in heterologous systems. We focused on GluN2AN615K co-expressed with wild-type GluN2 subunits in physiologically relevant triheteromeric NMDA receptors containing two GluN1 and two distinct GluN2 subunits, whereas previous studies have investigated the impact of the variant in diheteromeric NMDA receptors with two GluN1 and two identical GluN2 subunits. We found that GluN2AN615K -containing triheteromers showed markedly reduced Mg2+ blockade, with a value intermediate between GluN2AN615K diheteromers and wild-type NMDA receptors. Single-channel conductance was reduced by four-fold in GluN2AN615K diheteromers, again with an intermediate value in GluN2AN615K -containing triheteromers. Glutamate deactivation rates were unaffected. Furthermore, we expressed GluN2AN615K in cultured primary mouse cortical neurons, observing a decrease in Mg2+ blockade and reduction in current density, confirming that the variant continues to have significant functional impact in neuronal systems. Our results demonstrate that the GluN2AN615K variant has substantial effects on NMDA receptor properties fundamental to the roles of the receptor in synaptic plasticity, even when expressed alongside wild-type subunits. This work strengthens the evidence indicating that the GluN2AN615K variant underlies the disabling neurodevelopmental phenotype in carriers.

GRIN2A-related disorders: genotype and functional consequence predict phenotype.

Alterations of the N-methyl-d-aspartate receptor (NMDAR) subunit GluN2A, encoded by GRIN2A, have been associated with a spectrum of neurodevelopmental disorders with prominent speech-related features, and epilepsy. We performed a comprehensive assessment of phenotypes with a standardized questionnaire in 92 previously unreported individuals with GRIN2A-related disorders. Applying the criteria of the American College of Medical Genetics and Genomics to all published variants yielded 156 additional cases with pathogenic or likely pathogenic variants in GRIN2A, resulting in a total of 248 individuals. The phenotypic spectrum ranged from normal or near-normal development with mild epilepsy and speech delay/apraxia to severe developmental and epileptic encephalopathy, often within the epilepsy-aphasia spectrum. We found that pathogenic missense variants in transmembrane and linker domains (misTMD+Linker) were associated with severe developmental phenotypes, whereas missense variants within amino terminal or ligand-binding domains (misATD+LBD) and null variants led to less severe developmental phenotypes, which we confirmed in a discovery (P = 10-6) as well as validation cohort (P = 0.0003). Other phenotypes such as MRI abnormalities and epilepsy types were also significantly different between the two groups. Notably, this was paralleled by electrophysiology data, where misTMD+Linker predominantly led to NMDAR gain-of-function, while misATD+LBD exclusively caused NMDAR loss-of-function. With respect to null variants, we show that Grin2a+/- cortical rat neurons also had reduced NMDAR function and there was no evidence of previously postulated compensatory overexpression of GluN2B. We demonstrate that null variants and misATD+LBD of GRIN2A do not only share the same clinical spectrum (i.e. milder phenotypes), but also result in similar electrophysiological consequences (loss-of-function) opposing those of misTMD+Linker (severe phenotypes; predominantly gain-of-function). This new pathomechanistic model may ultimately help in predicting phenotype severity as well as eligibility for potential precision medicine approaches in GRIN2A-related disorders.

The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences.

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This "switch" is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.

Application of n-of-1 treatment trials in schizophrenia: systematic review.

BACKGROUND: Single patient or 'n-of-1' trials are a pragmatic method to achieve optimal, evidence-based treatments for individual patients. Such trials could be particularly valuable in chronic, heterogeneous, difficult to treat illnesses such as schizophrenia.AimsTo identify how often, and in what way, n-of-1 trials have been used in schizophrenia. METHOD: We performed a systematic search in the major electronic databases for studies adopting n-of-1 methodology in schizophrenia, published in English from the start of records until the end of January 2017. RESULTS: We identified six studies meeting inclusion criteria. There was wide variability in study methodology and analysis. Each trial reported positive outcomes for their respective intervention, but all studies were at high risk of bias. CONCLUSIONS: In conclusion, n-of-1 trials are currently underutilised in schizophrenia. Existing trials suggest the method is well tolerated and potentially effective in achieving optimal treatments for patients, but more standardised methods of design, execution and analysis are required in future trials.Declaration of interestS.M.L. has received grants and personal fees from Janssen, and personal fees from Otsuka and Sunovion, in the past 3 years, outside the submitted work.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Functional assessment of the NMDA receptor variant GluN2A R586K.

Background: The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that has important roles in synaptogenesis, synaptic transmission, and synaptic plasticity. Recently, a large number of rare genetic variants have been found in NMDAR subunits in people with neurodevelopmental disorders, and also in healthy individuals. One such is the GluN2AR586K variant, found in a person with intellectual disability. Identifying the functional consequences, if any, of such variants allows their potential contribution to pathogenesis to be assessed. Here, we assessed the effect of the GluN2AR586K variant on NMDAR pore properties. Methods: We expressed recombinant NMDARs with and without the GluN2AR586K variant in Xenopus laevis oocytes and in primary cultured mouse neurons, and made electrophysiological recordings assessing Mg2+ block, single-channel conductance, mean open time and current density. Results: The GluN2AR586K variant was not found to influence any of the properties assessed. Conclusions: Our findings suggest it is unlikely that the GluN2AR586K variant contributes to the pathogenesis of neurodevelopmental disorder.

Synthesis, radio-synthesis and in vitro evaluation of terminally fluorinated derivatives of HU-210 and HU-211 as novel candidate PET tracers.

We report the synthesis of terminally fluorinated HU-210 and HU-211 analogues (HU-210F and HU-211F, respectively) and their biological evaluation as ligands of cannabinoid receptors (CB1 and CB2) and N-methyl d-aspartate receptor (NMDAR). [18F]-labelled HU-210F was radiosynthesised from the bromo-substituted precursor. In vitro assays showed that both HU-210F and HU-211F retain the potent pharmacological profile of HU-210 and HU-211, suggesting that [18F]-radiolabelled HU-210F and HU-211F could have potential as PET tracers for in vivo imaging.

Antipsychotics and abnormal liver function tests: systematic review.

OBJECTIVE: Systematic assessment of the prevalence and pattern of liver function test (LFT) abnormalities associated with regular antipsychotics in adult humans and consideration of management of such abnormalities. DATA SOURCES: Systematic search identifying cohort, cross-sectional or case studies/series, reporting LFT abnormalities in patients receiving regular antipsychotics. EMBASE, PsychINFO, and MEDLINE were searched for studies in English from record onset. STUDY SELECTION: Abstracts were independently screened for eligibility by 2 researchers. Ineligible studies included those that did not report LFT reference ranges, those that studied fewer than 10 patients on a given antipsychotic, and those studying children. DATA EXTRACTION: Key variables in group studies were extracted. Case studies/series were examined for patient outcome. DATA SYNTHESIS: Ten group studies and 91 case studies/series were eligible, although quality was poor. All groups receiving regular antipsychotics had a prevalence of LFT abnormalities greater than chance. The median percentage of patients with any abnormal LFT on any antipsychotic was 32%, with a range of 5% to 78%. The median percentage of patients with clinically significant elevations was 4%, with a range of 0% to 15%. Transaminases were most commonly elevated. Abnormalities were generally asymptomatic, arose within 6 weeks, and were either stably persistent or resolved with continued treatment. Case reports suggested that antipsychotics can be associated with severe hepatitis, fatal in a small minority of cases. Chlorpromazine is most commonly associated with acute liver injury. CONCLUSIONS: The LFT abnormalities in patients receiving regular antipsychotics are common but generally mild and transient. Very rarely, a severe or fatal hepatic injury can emerge.