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Vascular endothelial growth factor A (VEGF-A) has therapeutic cardiovascular effects but delivery challenges have impeded clinical development. We report the first clinical study of naked mRNA encoding VEGF-A (AZD8601) injected into the human heart. EPICCURE (NCT03370887) was a randomized, double-blind study of AZD8601 in patients with left ventricular ejection fraction (LVEF) of 30-50% undergoing elective coronary artery bypass surgery. Thirty epicardial injections of AZD8601 (total 3 mg) or placebo in citrate-buffered saline were targeted to ischemic but viable myocardial regions mapped using quantitative [

We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in MAP3K15 were 30% less likely to develop diabetes ( P = 5.7 × 10 −10 ) and had lower glycosylated hemoglobin (β = −0.14 SD units, P = 1.1 × 10 −24 ). These associations were independent of body mass index, suggesting protection against insulin resistance even in the setting of obesity. We replicated these findings in 96,811 Admixed Americans in the Mexico City Prospective Study ( P < 0.05)Moreover, the protective effect of MAP3K15 variants was stronger in individuals who did not carry the Latino-enriched SLC16A11 risk haplotype ( P = 6.0 × 10 −4 ). Separately, we identified a Finnish-enriched MAP3K15 protein-truncating variant associated with decreased odds of both type 1 and type 2 diabetes ( P < 0.05) in FinnGen. No adverse phenotypes were associated with protein-truncating MAP3K15 variants in the UKB, supporting this gene as a therapeutic target for diabetes.

We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in

Background: Early intramuscular administration of SARS-CoV-2-neutralising monoclonal antibody combination, tixagevimab–cilgavimab, to non-hospitalised adults with mild to moderate COVID-19 has potential to prevent disease progression. We aimed to evaluate the safety and efficacy of tixagevimab–cilgavimab in preventing progression to severe COVID-19 or death. Methods: TACKLE is an ongoing, phase 3, randomised, double-blind, placebo-controlled study conducted at 95 sites in the USA, Latin America, Europe, and Japan. Eligible participants were non-hospitalised adults aged 18 years or older with a laboratory-confirmed SARS-CoV-2 infection (determined by RT-PCR or an antigen test) from any respiratory tract specimen collected 3 days or less before enrolment and who had not received a COVID-19 vaccination. A WHO Clinical Progression Scale score from more than 1 to less than 4 was required for inclusion and participants had to receive the study drug 7 days or less from self-reported onset of mild to moderate COVID-19 symptoms or measured fever. Participants were randomly assigned (1:1) to receive either a single tixagevimab–cilgavimab 600 mg dose (two consecutive 3 mL intramuscular injections, one each of 300 mg tixagevimab and 300 mg cilgavimab) or placebo. Randomisation was stratified (using central blocked randomisation with randomly varying block sizes) by time from symptom onset, and high-risk versus low-risk of progression to severe COVID-19. Participants, investigators, and sponsor staff involved in the treatment or clinical evaluation and monitoring of the participants were masked to treatment-group assignments. The primary endpoints were severe COVID-19 or death from any cause through to day 29, and safety. This study is registered with ClinicalTrials.gov, NCT04723394. Findings: Between Jan 28, 2021, and July 22, 2021, 1014 participants were enrolled, of whom 910 were randomly assigned to a treatment group (456 to receive tixagevimab–cilgavimab and 454 to receive placebo). The mean age of participants was 46·1 years (SD 15·2). Severe COVID-19 or death occurred in 18 (4%) of 407 participants in the tixagevimab–cilgavimab group versus 37 (9%) of 415 participants in the placebo group (relative risk reduction 50·5% [95% CI 14·6–71·3]; p=0·0096). The absolute risk reduction was 4·5% (95% CI 1·1–8·0; p Interpretation: A single intramuscular tixagevimab–cilgavimab dose provided statistically and clinically significant protection against progression to severe COVID-19 or death versus placebo in unvaccinated individuals and safety was favourable. Treating mild to moderate COVID-19 earlier in the disease course with tixagevimab–cilgavimab might lead to more favourable outcomes.

We report primary results of a phase 3 trial of AZD7442 (tixagevimab/cilgavimab) for post-exposure prophylaxis to prevent symptomatic coronavirus disease 2019 (COVID-19).Adults without prior SARS-CoV-2 infection or COVID-19 vaccination were enrolled within 8 days of exposure to a SARS-CoV-2-infected individual and randomized 2:1 to a single 300-mg AZD7442 dose (one 1.5-mL intramuscular injection each of tixagevimab and cilgavimab consecutively) or placebo. Primary endpoints were safety and first post-dose SARS-CoV-2 reverse-transcription-polymerase-chain-reaction (RT-PCR)-positive symptomatic COVID-19 event before day 183.1121 participants were randomized and dosed (mean age 46 years; 49% females; AZD7442, n=749; placebo, n=372). Median (range) follow-up was 49 (5-115) and 48 (20-113) days for AZD7442 and placebo, respectively. Adverse events occurred in 162/749 (21.6%) and 111/372 (29.8%) participants with AZD7442 and placebo, respectively, mostly mild/moderate. RT-PCR-positive symptomatic COVID-19 occurred in 23/749 (3.1%) and 17/372 (4.6%) AZD7442- and placebo-treated participants, respectively (relative risk reduction 33.3%; 95% confidence interval [CI] -25.9 to 64.7; P=.21). In predefined subgroup analyses of 1073 (96%) participants who were SARS-CoV-2 RT-PCR-negative (n=974 [87%]) or missing an RT-PCR result (n=99 [9%]) at baseline, AZD7442 reduced RT-PCR-positive symptomatic COVID-19 by 73.2% (95% CI 27.1 to 90.1) versus placebo.This study did not meet the primary efficacy endpoint of post-exposure prevention of symptomatic COVID-19 with AZD7442 versus placebo. However, predefined analysis of participants who were SARS-CoV-2 RT-PCR-negative or missing an RT-PCR result at baseline support a role for AZD7442 in preventing symptomatic COVID-19.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABABreceptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+. Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENTDysregulation of GABABreceptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+. We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

The monoclonal-antibody combination AZD7442 is composed of tixagevimab and cilgavimab, two neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that have an extended half-life and have been shown to have prophylactic and therapeutic effects in animal models. Pharmacokinetic data in humans indicate that AZD7442 has an extended half-life of approximately 90 days.In an ongoing phase 3 trial, we enrolled adults (≥18 years of age) who had an increased risk of an inadequate response to vaccination against coronavirus disease 2019 (Covid-19), an increased risk of exposure to SARS-CoV-2, or both. Participants were randomly assigned in a 2:1 ratio to receive a single dose (two consecutive intramuscular injections, one containing tixagevimab and the other containing cilgavimab) of either 300 mg of AZD7442 or saline placebo, and they were followed for up to 183 days in the primary analysis. The primary safety end point was the incidence of adverse events after a single dose of AZD7442. The primary efficacy end point was symptomatic Covid-19 (SARS-CoV-2 infection confirmed by means of reverse-transcriptase-polymerase-chain-reaction assay) occurring after administration of AZD7442 or placebo and on or before day 183.A total of 5197 participants underwent randomization and received one dose of AZD7442 or placebo (3460 in the AZD7442 group and 1737 in the placebo group). The primary analysis was conducted after 30% of the participants had become aware of their randomized assignment. In total, 1221 of 3461 participants (35.3%) in the AZD7442 group and 593 of 1736 participants (34.2%) in the placebo group reported having at least one adverse event, most of which were mild or moderate in severity. Symptomatic Covid-19 occurred in 8 of 3441 participants (0.2%) in the AZD7442 group and in 17 of 1731 participants (1.0%) in the placebo group (relative risk reduction, 76.7%; 95% confidence interval [CI], 46.0 to 90.0; P0.001); extended follow-up at a median of 6 months showed a relative risk reduction of 82.8% (95% CI, 65.8 to 91.4). Five cases of severe or critical Covid-19 and two Covid-19-related deaths occurred, all in the placebo group.A single dose of AZD7442 had efficacy for the prevention of Covid-19, without evident safety concerns. (Funded by AstraZeneca and the U.S. government; PROVENT ClinicalTrials.gov number, NCT04625725.).

Diabetes mellitus is a chronic health condition that can result in significant end-organ complications and is estimated to impact at least 8.5% of the global adult population. Here, we performed gene-level collapsing analysis on exome sequences from 454,796 multi-ancestry UK Biobank participants to detect genetic associations with diabetes. Rare non-synonymous variants in GCK, GIGYF1, HNF1A, and HNF4A were significantly associated (P-8) with increased risk of diabetes, whereas rare non-synonymous variants in MAP3K15 were significantly associated with reduced risk of diabetes. Recessive carriers of rare non-synonymous variants in the X chromosome gene MAP3K15 had a 30% reduced risk of diabetes (OR=0.70, 95% CI: [0.62,0.79], P=5.7×10-10), along with reduced blood glucose (beta=-0.13, 95% CI: [-0.15,-0.10], P=5.5×10-18) and reduced glycosylated haemoglobin levels (beta=-0.14, 95% CI: [-0.16,-0.11], P=1.1×10-24). Hemizygous males carrying protein-truncating variants (PTVs) in MAP3K15 demonstrated a 40% reduced risk of diabetes (OR=0.60, 95% CI: [0.45,0.81], P=0.0007). These findings were independently replicated in FinnGen, with a MAP3K15 PTV associating with decreased risk of both type 1 diabetes (T1DM) and type 2 diabetes (T2DM) (pMAP3K15 loss on diabetes was independent of body mass index, suggesting its protective effect is unlikely to be mediated via the insulin resistance pathway. Tissue expression profile of MAP3K15 indicates a possible involvement of pancreatic islet cell or stress response pathways. No safety concerns were identified among heterozygous or recessive MAP3K15 PTV carriers across over 15,719 studied endpoints in the UK Biobank. Human population genetic evidence supports MAP3K15 inhibition as a novel therapeutic target for diabetes.

Background The safety and efficacy of the AZD1222 (ChAdOx1 nCoV-19) vaccine in a large, diverse population at increased risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United States, Chile, and Peru has not been known. Methods In this ongoing, double-blind, randomized, placebo-controlled, phase 3 clinical trial, we investigated the safety, vaccine efficacy, and immunogenicity of two doses of AZD1222 as compared with placebo in preventing the onset of symptomatic and severe coronavirus disease 2019 (Covid-19) 15 days or more after the second dose in adults, including older adults, in the United States, Chile, and Peru. Results A total of 32,451 participants underwent randomization, in a 2:1 ratio, to receive AZD1222 (21,635 participants) or placebo (10,816 participants). AZD1222 was safe, with low incidences of serious and medically attended adverse events and adverse events of special interest; the incidences were similar to those observed in the placebo group. Solicited local and systemic reactions were generally mild or moderate in both groups. Overall estimated vaccine efficacy was 74.0% (95% confidence interval [CI], 65.3 to 80.5; P

Despite the success of SARS-CoV-2 vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of COVID-19. Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19, and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct non-overlapping epitopes on the spike protein receptor binding domain to potently neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and abrogate effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry. Together, these two mAbs create a higher barrier to viral escape and a wider breadth of coverage, neutralizing all known SARS-CoV-2 variants of concern. In a non-human primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, while therapeutic administration accelerated virus clearance from lung. In an ongoing Phase I study in healthy participants (NCT04507256), 300 mg intramuscular AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers >10-fold above those of convalescent sera for ≥3 months, which remained 3-fold above those of convalescent sera 9 months post-AZD7442 administration. Approximately 1–2% of serum AZD7442 levels were detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentrations suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABA(B)R2 subunit, promotes internalization of the GABA(B) receptor (GABA(B)R) and leads to smaller GABA(B)R-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABA(B)R1 subunit, and that GABA(B)Rs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABA(B)R interaction can be regulated by intracellular Ca(2+). Finally, a peptide that potentially reduces recruitment of PP2A to GABA(B)Rs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABA(B)Rs may be a useful strategy to increase receptor signaling for treating diseases. SIGNIFICANCE STATEMENT Dysregulation of GABA(B) receptors (GABA(B)Rs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABA(B)Rs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABA(B)R1 subunit, and that this interaction is sensitive to intracellular Ca(2+). We demonstrate that a short peptide corresponding to the PP2A interaction site on GABA(B)R1 competes for PP2A binding, enhances phosphorylation GABA(B)R2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABA(B)Rs may provide a specific strategy to modulate GABA(B)R signaling in disease conditions.

A new model for translational research and drug repositioning has recently been established based on three-way partnerships between public funders, the pharmaceutical industry and academic investigators. Through two pioneering initiatives - one involving the Medical Research Council in the United Kingdom and one involving the National Center for Advancing Translational Sciences of the National Institutes of Health in the United States - new investigations of highly characterized investigational compounds have been funded and are leading to the exploration of known mechanisms in new disease areas. This model has been extended beyond these first two initiatives. Here, we discuss the progress to date and the unique requirements and challenges for this model.

Our knowledge of how estrogen signaling can influence inhibitory synaptic transmission is rudimentary and is addressed here. Collectively, our data suggest that estrogen modulates the dynamics of surface GABAA receptors and hence efficacy of synaptic inhibition, via a postsynaptic mechanism that relies on disrupting the postsynaptic scaffold. This regulatory mechanism may have profound effects on the efficacy of neuronal inhibition and therefore synaptic plasticity and thus play a role in the pathophysiology of estrogen-related seizure and cognitive disorders.

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

The identification of highly potent and orally active phenylpyrazines for the inhibition of PDE10A is reported. The new analogues exhibit subnanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired druglike properties. Employing structure-based drug design approaches, we methodically explored two key regions of the binding pocket of the PDE10A enzyme to alter the planarity of the parent compound 1 and optimize its affinity for PDE10A. Bulky substituents at the C9 position led to elimination of the mutagenicity of 1, while a crucial hydrogen bond interaction with Glu716 markedly enhanced its potency and selectivity. A systematic assessment of the ADME and PK properties of the new analogues led to druglike development candidates. One of the more potent compounds, 96, displayed an IC(50) for PDE10A of 0.7 nM and was active in predictive antipsychotic animal models.

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer’s disease. SAR studies of the S2′ region of the BACE1 ligand binding pocket with pyrazolyl and thienyl P2′ side chains are reported. These analogs exhibit low nanomolar potency for BACE1, and demonstrate >50- to 100-fold selectivity for the structurally related aspartyl proteases BACE2 and cathepsin D. Small groups attached at the nitrogen of the P2′ pyrazolyl moiety, together with the P3 pyrimidine nucleus projecting into the S3 region of the binding pocket, are critical components to ligand’s potency and selectivity. P2′ thiophene side chain analogs are highly potent BACE1 inhibitors with excellent selectivity against cathepsin D, but only modest selectivity against BACE2. The cell-based activity of these new analogs tracked well with their increased molecular binding with EC50 values of 0.07–0.2 μM in the ELISA assay for the most potent analogs.

The 5-hydroxytryptamine 2C (5-HT 2C ) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT 2C receptor-selective agonist vabicaserin [(−)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT 2B receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced 125 I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT 2C receptor sites in Chinese hamster ovary cell membranes with a K i value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT 2B receptor subtype using [ 3 H]5HT was 14 nM. Vabicaserin was a potent and full agonist (EC 50 , 8 nM; E max , 100%) in stimulating 5-HT 2C receptor-coupled calcium mobilization and exhibited 5-HT 2A receptor antagonism and 5-HT 2B antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT 2B receptors, vabicaserin failed to induce a 5-HT 2B receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT 2B competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT 2B receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT 2B receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT 2B receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT 2C receptor agonist.

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Novel imidazo[1,5-a]pyrido[3,2-e]pyrazines have been synthesized and characterized as both potent and selective phosphodiesterase 10A (PDE10A) inhibitors. For in vitro characterization, inhibition of PDE10A mediated cAMP hydrolysis was used and a QSAR model was established to analyze substitution effects. The outcome of this analysis was complemented by the crystal structure of PDE10A in complex with compound 49. Qualitatively new interactions between inhibitor and binding site were found, contrasting with previously published crystal structures of papaverine-like inhibitors. In accordance with the known antipsychotic potential of PDE10A inhibitors, MK-801 induced stereotypy and hyperactivity in rats were reversed by selected compounds. Thus, a promising compound class has been identified for the treatment of schizophrenia that could circumvent side effects connected with current therapies.

J. Neurochem. (2010) 113, 601–614. Abstract The critical sequence of molecular, neurotransmission and synaptic disruptions that underpin the emergence of psychiatric disorders like schizophrenia remain to be established with progress only likely using animal models that capture key features of such disorders. We have related the emergence of behavioural, neurochemical and synapse ultrastructure deficits to transcriptional dysregulation in the medial prefrontal cortex of Wistar rats reared in isolation. Isolation reared animals developed sensorimotor deficits at postnatal day 60 which persisted into adulthood. Analysis of gene expression prior to the emergence of the sensorimotor deficits revealed a significant disruption in transcriptional control, notably of immediate early and interferon-associated genes. At postnatal day 60 many gene transcripts relating particularly to GABA transmission and synapse structure, for example Gabra4, Nsf, Syn2 and Dlgh1, transiently increased expression. A subsequent decrease in genes such as Gria2 and Dlgh2 at postnatal day 80 suggested deficits in glutamatergic transmission and synapse integrity, respectively. Microdialysis studies revealed decreased extracellular glutamate suggesting a state of hypofrontality while ultrastructural analysis showed total and perforated synapse complement in layer III to be significantly reduced in the prefrontal cortex of postnatal day 80 isolated animals. These studies provide a molecular framework to understand the developmental emergence of the structural and behavioural characteristics that may in part define psychiatric illness.

Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.

Estrogens are hormones that modulate a diverse array of effects during development and adulthood. The effects of estrogen are mediated by two estrogen receptor (ER) isotypes, ERalpha and ERbeta, which classically function as transcription factors to modulate specific target gene expression and in addition regulate a growing list of intracellular signaling cascades. These receptors share protein sequence homology and protein-motif organization but have distinct differences in their tissue distribution and binding affinities for their ligands. In the nervous system estrogen has been implicated to play a role in a number of processes which regulate synaptic plasticity including synaptogenesis and neurogenesis. The role for estrogen in a range of neurological and neuropsychiatric diseases is also becoming very apparent. Estrogen is able to regulate processes and behaviours relevant for both Alzheimer's disease and schizophrenia and to modulate neuroendocrine and inflammatory processes important in neuroinflammation, anxiety and depressive disorders as well as chronic pain. We will consider the rationale for estrogen-based therapies for diseases of the nervous system. In particular we will highlight the molecular mechanisms and signal transduction pathways most likely underlying the effects of estrogen in the CNS.

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of γ-aminobutyric acid type B (GABAB) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABAB receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABAB receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABAB receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABAB receptors in central neurons, consistent with their enrichment at glutamatergic synapses.

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH2) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERalpha) and beta (ERbeta) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERbeta. Selective ERbeta agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERbeta knockout mice. In hippocampal slices, ERbeta activation enhanced long-term potentiation, an effect that was absent in slices from ERbeta knockout mice. ERbeta activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERbeta agonist, but not an ERalpha agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERbeta can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca 2+ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca 2+ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca 2+ -induced cell death by modulating Ca 2+ channels and promote neurite outgrowth via FKBP52 binding.

A beam-walking apparatus has been evaluated for its ability to detect motor impairments in mice acutely treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg, s.c., single or double administration). Mice subjected to MPTP lesioning showed deficits in motor performance on the beam-walking task, for up to 6 days post-MPTP administration, as compared to saline-treated controls. In addition, MPTP-treated mice were detected to have a marked depletion in striatal dopamine levels and a concomitant reduction in substantia nigra (SN) tyrosine hydroxylase (TH) immunoreactivity, at 7 days post-MPTP administration, indicative of dopaminergic neuronal loss. Pre-administration of the potent MAO-B inhibitor R-(-)-deprenyl at 3 or 10 mg/kg, 30 min, s.c, significantly inhibited the MPTP-induced reduction in SN TH-immunoreactivity, striatal dopamine depletions and impairments in mouse motor function. The data described in the present study provides further evidence that functional deficits following an acute MPTP dosing schedule in mice can be quantified and are related to nigro-striatal dopamine function.

Disorders of the central nervous system (CNS) are some of the most prevalent, devastating and yet poorly treated illnesses. The development of new therapies for CNS disorders such as Alzheimer's disease has the potential to provide patients with significant improvements in quality of life, as well as reduce the future economic burden on health-care systems. However, few truly innovative CNS drugs have been approved in recent years, suggesting that there is a considerable need for strategies to enhance the productivity of research and development in this field. In this article, using illustrative examples from neurological and psychiatric disorders, we describe various approaches that are being taken to discover CNS drugs, discuss their relative merits and consider how risk can be balanced and attrition reduced.

GABAA receptors mediate the majority of fast synaptic inhibition in the brain. The accumulation of these ligand-gated ion channels at synaptic sites is a prerequisite for neuronal inhibition, but the molecular mechanisms underlying this phenomenon remain obscure. To further understand these processes, we have examined the cellular origins of synaptic GABAA receptors. To do so, we have created fluorescent GABAA receptors that are capable of binding α-bungarotoxin (Bgt), facilitating the visualization of receptor endocytosis, exocytosis and delivery to synaptic sites. Imaging with Bgt in hippocampal neurons revealed that GABAA receptor endocytosis occurred exclusively at extrasynaptic sites, consistent with the preferential colocalization of extrasynaptic receptors with the AP2 adaptin. Receptor insertion into the plasma membrane was also predominantly extrasynaptic, and pulse-chase analysis revealed that these newly inserted receptors were then able to access directly synaptic sites. Therefore, our results demonstrate that synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. Moreover, they illustrate a dynamic mechanism for neurons to modulate GABAA receptor number at inhibitory synapses by controlling the stability of extrasynaptic receptors.

Many neurodegenerative diseases are caused by intracellular, aggregate-prone proteins, including polyglutamine-expanded huntingtin in Huntington's disease (HD) and mutant tau in fronto-temporal dementia/tauopathy. Previously, we showed that rapamycin, an autophagy inducer, enhances mutant huntingtin fragment clearance and attenuated toxicity. Here we show much wider applications for this approach. Rapamycin enhances the autophagic clearance of different proteins with long polyglutamines and a polyalanine-expanded protein, and reduces their toxicity. Rapamycin also reduces toxicity in Drosophila expressing wild-type or mutant forms of tau and these effects can be accounted for by reductions in insoluble tau. Thus, our studies suggest that the scope for rapamycin as a potential therapeutic in aggregate diseases may be much broader than HD or even polyglutamine diseases.

Parkinson's disease (PD) is a chronic neurodegenerative disease, predominantly affecting the elderly, for which only symptomatic treatments are currently available. In the clinic, PD is viewed primarily as a disorder of the nigrostriatal dopaminergic pathway because presenting symptoms typically involve motor disturbances that can be modulated with dopamine agonists. However, PD is increasingly being viewed as a spectrum of complex impairments that emerge from the abnormal function of multiple neurochemical systems. Recent research integrates many of these findings and suggests potentially useful targets for disease modifying therapies. Here, we will compare and contrast the various cellular mechanisms thought to be important in the pathophysiology and etiology of this debilitating disease.

Summary: Alzheimer’s disease is a progressive neurodegenerative disorder and the leading cause of dementia in the Western world. Postmortem, it is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles, and a profound gray matter loss. Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability. The extracellular amyloid plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid-β (Aβ) peptide and have led to the foundation of the amyloid hypothesis. This hypothesis postulates that Aβ is one of the principal causative factors of neuronal death in the brains of Alzheimer’s patients. With multiple drugs now moving through clinical development for the treatment of Alzheimer’s disease, we will review current and future treatment strategies aimed at improving both the cognitive deficits associated with the disease, as well as more novel approaches that may potentially slow or halt the deadly neurodegenerative progression of the disease.

Transgenic mice (Tg2576) overexpressing the Swedish mutation of the human amyloid precursor protein display biochemical, pathological, and behavioral markers consistent with many aspects of Alzheimer's disease, including impaired hippocampal function. Impaired, hippocampal-dependent, contextual fear conditioning (CFC) is observed in mice as young as 20 weeks of age. This impairment can be attenuated after treatment before training with the phosphodiesterase-4 inhibitor rolipram (0.1 mg/kg, i.p.). A rolipram-associated improvement is also observed in the littermate controls, suggesting that the effect of rolipram is independent of β-amyloid. Acute treatment before training (but not after training or before testing) with the γ-secretase inhibitor (GSI)N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butylester (DAPT), at a dose that reduces brain concentrations of β-amyloid (100 mg/kg), attenuates the impairment in 20- to 65-week-old Tg2576 mice. Importantly, DAPT had no effect on performance of control littermates. These data are supportive of a role of β-amyloid in the impairment of CFC in Tg2576 mice. Furthermore, they suggest that acute treatment with GSI may provide improved cognitive functioning as well as disease-modifying effects in Alzheimer's disease.

The efficacy of synaptic inhibition depends on the number of γ-aminobutyric acid type A receptors (GABA A Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA A R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA A R β subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the β3 subunit) incorporates the major sites of serine phosphorylation within receptor β subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepβ3) corresponding to the AP2 binding motif in the GABA A R β3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepβ3 peptide, but not its phosphorylated equivalent (pepβ3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA A R current. These effects of pepβ3 on GABA A R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepβ3 on GABA A R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA A Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

AD (Alzheimer's disease) is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles and profound grey matter loss. The ‘amyloid’ hypothesis postulates that the toxic Aβ (amyloid β) peptide, enzymatically derived from the proteolytic processing of a larger protein called APP (amyloid precursor protein), is one of the principal causative factors of neuronal cell death in the brains of AD patients. As such, methods for lowering Aβ levels in the brain are of significant interest with regard to identifying novel disease modifying therapies for the treatment of AD. In this review, we will review a variety of approaches and mechanisms capable of modulating levels of Aβ.

gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.

The mammalian homologues of the Drosophila transient receptor potential (TRP) channel are plasma membrane proteins involved in the regulation of cellular Ca(2+) influx. These ion channels can be activated subsequent to either depletion of Ca(2+) from internal stores or through receptor-mediated processes. The mRNA expression patterns of several individual mammalian short transient receptor potential channels (TRPCs) have been described. Cross-comparisons between these data, however, are at best difficult predominantly due to the non-quantitative methods used. Furthermore there is limited data on the expression of TRPC family members in human tissues. In the present study we used a single technique, namely TaqMan real-time quantitative RT-PCR, to investigate the mRNA distribution of human TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (hTRPCs) in discrete human brain areas, peripheral tissues as well as a panel of cell-lines. All hTRPCs studied were widely expressed within CNS and significant peripheral expression was often observed. Despite this, each channel exhibited a distinctive hallmark distribution profile. hTRPC1 was widely expressed in CNS and peripheral tissues, whereas hTRPC3 and hTRPC5 were predominantly expressed in tissues of CNS. hTRPC4 mRNA was detected in CNS and certain peripheral tissues such as bone, heart and prostate. hTRPC6 was homogeneously expressed throughout the CNS and peripheral tissues with the highest levels in placenta and lung. hTRPC7 mRNA was also broadly expressed in CNS as well as some peripheral tissues. The pattern of expression of the TRPCs was quite different in the various cell lines examined. TRPC3 and TRPC6 were selectively present in HEK-293 cells whilst TRPC1 was broadly distributed in the cell lines analyzed. In contrast TRPC4 and TRPC5 mRNAs were predominantly expressed in HK-2 and HEK-293 cell lines respectively. TRPC7 was selectively expressed in COS-1, COS-7 and HK-2 cell lines. These results show tissue- and cell-specific co-expression of multiple TRPC forms indicating widespread potential for formation of heteromeric channels. These data will be useful in the complex task of relating channel subunit composition to function in native cells.