A Neurofunctional Domains Approach to Evaluate D1/D5 Dopamine Receptor Partial Agonism on Cognition and Motivation in Healthy Volunteers With Low Working Memory Capacity.

BACKGROUND: Dopamine D1 receptor signaling plays key roles in core domains of neural function, including cognition and reward processing; however, many questions remain about the functions of circuits modulated by dopamine D1 receptor, largely because clinically viable, selective agonists have yet to be tested in humans. METHODS: Using a novel, exploratory neurofunctional domains study design, we assessed the safety, tolerability, pharmacodynamics, and pharmacokinetics of PF-06412562, a selective D1/D5R partial agonist, in healthy male volunteers who met prespecified criteria for low working memory capacity. Functional magnetic resonance imaging, electrophysiologic endpoints, and behavioral paradigms were used to assess working memory, executive function, and motivation/reward processing following multiple-dose administration of PF-06412562. A total of 77 patients were assigned PF-06412562 (3 mg twice daily and 15 mg twice daily) or placebo administered for 5 to 7 days. Due to the exploratory nature of the study, it was neither powered for any specific treatment effect nor corrected for multiple comparisons. RESULTS: Nominally significant improvements from baseline in cognitive endpoints were observed in all 3 groups; however, improvements in PF-06412562-treated patients were less than in placebo-treated participants. Motivation/reward processing endpoints were variable. PF-06412562 was safe and well tolerated, with no serious adverse events, severe adverse events, or adverse events leading to dose reduction or temporary discontinuation except for 1 permanent discontinuation due to increased orthostatic heart rate. CONCLUSIONS: PF-06412562, in the dose range and patient population explored in this study, did not improve cognitive function or motivation/reward processing more than placebo over the 5- to 7-day treatment period. CLINICALTRIALS.GOV IDENTIFIER: NCT02306876.

Learning from Principles of Evidence-Based Medicine to Optimize Nonclinical Research Practices.

Thousands of pharmacology experiments are performed each day, generating hundreds of drug discovery programs, scientific publications, grant submissions, and other efforts. Discussions of the low reproducibility and robustness of some of this research have led to myriad efforts to increase data quality and thus reliability. Across the scientific ecosystem, regardless of the extent of concerns, debate about solutions, and differences among goals and practices, scientists strive to provide reliable data to advance frontiers of knowledge. Here we share our experience of current practices in nonclinical neuroscience research across biopharma and academia, examining context-related factors and behaviors that influence ways of working and decision-making. Drawing parallels with the principles of evidence-based medicine, we discuss ways of improving transparency and consider how to better implement best research practices. We anticipate that a shared framework of scientific rigor, facilitated by training, enabling tools, and enhanced data sharing, will draw the conversation away from data unreliability or lack of reproducibility toward the more important discussion of how to generate data that advances knowledge and propels innovation.

Cellular plasticity induced by anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis antibodies.

OBJECTIVE: Autoimmune-mediated anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood. METHODS: The effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses. RESULTS: We show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase. INTERPRETATION: These results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder.

Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice.

Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients' antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients' antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients' or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients' cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13-18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.

Acute mechanisms underlying antibody effects in anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: A severe but treatable form of immune-mediated encephalitis is associated with antibodies in serum and cerebrospinal fluid (CSF) against the GluN1 subunit of the N-methyl-D-aspartate receptor (NMDAR). Prolonged exposure of hippocampal neurons to antibodies from patients with anti-NMDAR encephalitis caused a reversible decrease in the synaptic localization and function of NMDARs. However, acute effects of the antibodies, fate of the internalized receptors, type of neurons affected, and whether neurons develop compensatory homeostatic mechanisms were unknown and are the focus of this study. METHODS: Dissociated hippocampal neuron cultures and rodent brain sections were used for immunocytochemical, physiological, and molecular studies. RESULTS: Patient antibodies bind to NMDARs throughout the rodent brain, and decrease NMDAR cluster density in both excitatory and inhibitory hippocampal neurons. They rapidly increase the internalization rate of surface NMDAR clusters, independent of receptor activity. This internalization likely accounts for the observed decrease in NMDAR-mediated currents, as no evidence of direct blockade was detected. Once internalized, antibody-bound NMDARs traffic through both recycling endosomes and lysosomes, similar to pharmacologically induced NMDAR endocytosis. The antibodies are responsible for receptor internalization, as their depletion from CSF abrogates these effects in hippocampal neurons. We find that although anti-NMDAR antibodies do not induce compensatory changes in glutamate receptor gene expression, they cause a decrease in inhibitory synapse density onto excitatory hippocampal neurons. INTERPRETATION: Our data support an antibody-mediated mechanism of disease pathogenesis driven by immunoglobulin-induced receptor internalization. Antibody-mediated downregulation of surface NMDARs engages homeostatic synaptic plasticity mechanisms, which may inadvertently contribute to disease progression.

Encephalitis and antibodies to dipeptidyl-peptidase-like protein-6, a subunit of Kv4.2 potassium channels.

OBJECTIVE: To report a novel cell surface autoantigen of encephalitis that is a critical regulatory subunit of the Kv4.2 potassium channels. METHODS: Four patients with encephalitis of unclear etiology and antibodies with a similar pattern of neuropil brain immunostaining were selected for autoantigen characterization. Techniques included immunoprecipitation, mass spectrometry, cell-base experiments with Kv4.2 and several dipeptidyl-peptidase-like protein-6 (DPPX) plasmid constructs, and comparative brain immunostaining of wild-type and DPPX-null mice. RESULTS: Immunoprecipitation studies identified DPPX as the target autoantigen. A cell-based assay confirmed that all 4 patients, but not 210 controls, had DPPX antibodies. Symptoms included agitation, confusion, myoclonus, tremor, and seizures (1 case with prominent startle response). All patients had pleocytosis, and 3 had severe prodromal diarrhea of unknown etiology. Given that DPPX tunes up the Kv4.2 potassium channels (involved in somatodendritic signal integration and attenuation of dendritic back-propagation of action potentials), we determined the epitope distribution in DPPX, DPP10 (a protein homologous to DPPX), and Kv4.2. Patients' antibodies were found to be specific for DPPX, without reacting with DPP10 or Kv4.2. The unexplained diarrhea led to a demonstration of a robust expression of DPPX in the myenteric plexus, which strongly reacted with patients' antibodies. The course of neuropsychiatric symptoms was prolonged and often associated with relapses during decreasing immunotherapy. Long-term follow-up showed substantial improvement in 3 patients (1 was lost to follow-up). INTERPRETATION: Antibodies to DPPX are associated with a protracted encephalitis characterized by central nervous system hyperexcitability (agitation, myoclonus, tremor, seizures), pleocytosis, and frequent diarrhea at symptom onset. The disorder is potentially treatable with immunotherapy.

The in vivo contribution of motor neuron TrkB receptors to mutant SOD1 motor neuron disease.

Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) are widely expressed in the vertebrate nervous system and play a central role in mature neuronal function. In vitro BDNF/TrkB signaling promotes neuronal survival and can help neurons resist toxic insults. Paradoxically, BDNF/TrkB signaling has also been shown, under certain in vitro circumstances, to render neurons vulnerable to insults. We show here that in vivo conditional deletion of TrkB from mature motor neurons attenuates mutant superoxide dismutase 1 (SOD1) toxicity. Mutant SOD1 mice lacking motor neuron TrkB live a month longer than controls and retain motor function for a longer period, particularly in the early phase of the disease. These effects are subserved by slowed motor neuron loss, persistence of neuromuscular junction integrity and reduced astrocytic and microglial reactivity within the spinal cord. These results suggest that manipulation of BDNF/TrkB signaling might have therapeutic efficacy in motor neuron diseases.

Genetic deletion of trkB.T1 increases neuromuscular function.

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB (trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1(-/-) animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.

Determinants of synaptic strength vary across an axon arbor.

We used synaptophysin-pHluorin expressed in hippocampal neurons to address how functional properties of terminals, namely, evoked release, total vesicle pool size, and release fraction, vary spatially across individual axon arbors. Consistent with previous reports, over short arbor distances (≈ 100 µm), evoked release was spatially heterogeneous when terminals contacted different postsynaptic dendrites or neurons. Regardless of the postsynaptic configuration, the evoked release and total vesicle pool size spatially covaried, suggesting that the fraction of synaptic vesicles available for release (release fraction) was similar over short distances. Evoked release and total vesicle pool size were highly correlated with the amount of NMDA receptors and PSD-95 in postsynaptic specialization. However, when individual axons were followed over longer distances (several hundred micrometers), a significant increase in evoked release was observed distally that was associated with an increased release fraction in distal terminals. The increase in distal release fraction can be accounted for by changes in individual vesicle release probability as well as readily releasable pool size. Our results suggest that for a single axon arbor, presynaptic strength indicated by evoked release over short distances is correlated with heterogeneity in total vesicle pool size, whereas over longer distances presynaptic strength is correlated with the spatial modulation of release fraction. Thus the mechanisms that determine synaptic strength differ depending on spatial scale.

Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis.

We recently described a severe, potentially lethal, but treatment-responsive encephalitis that associates with autoantibodies to the NMDA receptor (NMDAR) and results in behavioral symptoms similar to those obtained with models of genetic or pharmacologic attenuation of NMDAR function. Here, we demonstrate that patients' NMDAR antibodies cause a selective and reversible decrease in NMDAR surface density and synaptic localization that correlates with patients' antibody titers. The mechanism of this decrease is selective antibody-mediated capping and internalization of surface NMDARs, as Fab fragments prepared from patients' antibodies did not decrease surface receptor density, but subsequent cross-linking with anti-Fab antibodies recapitulated the decrease caused by intact patient NMDAR antibodies. Moreover, whole-cell patch-clamp recordings of miniature EPSCs in cultured rat hippocampal neurons showed that patients' antibodies specifically decreased synaptic NMDAR-mediated currents, without affecting AMPA receptor-mediated currents. In contrast to these profound effects on NMDARs, patients' antibodies did not alter the localization or expression of other glutamate receptors or synaptic proteins, number of synapses, dendritic spines, dendritic complexity, or cell survival. In addition, NMDAR density was dramatically reduced in the hippocampus of female Lewis rats infused with patients' antibodies, similar to the decrease observed in the hippocampus of autopsied patients. These studies establish the cellular mechanisms through which antibodies of patients with anti-NMDAR encephalitis cause a specific, titer-dependent, and reversible loss of NMDARs. The loss of this subtype of glutamate receptors eliminates NMDAR-mediated synaptic function, resulting in the learning, memory, and other behavioral deficits observed in patients with anti-NMDAR encephalitis.

mll ortholog containing functional domains of human MLL is expressed throughout the zebrafish lifespan and in haematopoietic tissues.

Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

Astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis.

Astrocytes modulate the formation and function of glutamatergic synapses in the CNS, but whether astrocytes modulate GABAergic synaptogenesis is unknown. We demonstrate that media conditioned by astrocytes, but not other cells, enhanced GABAergic but not glutamatergic axon length and branching, and increased the number and density of presynaptically active GABAergic synapses in dissociated hippocampal cultures. Candidate mechanisms and factors, such as activity, neurotrophins, and cholesterol were excluded as mediating these effects. While thrombospondins secreted by astrocytes are necessary and sufficient to increase hippocampal glutamatergic synaptogenesis, they do not mediate astrocyte effects on GABAergic synaptogenesis. We show that the factors in astrocyte conditioned media that selectively affect GABAergic neurons are proteins. Taken together, our results show that astrocytes increase glutamatergic and GABAergic synaptogenesis via different mechanisms and release one or more proteins with the novel functions of increasing GABAergic axon length, branching and synaptogenesis.

The Effects of a Novel Non-catechol Dopamine Partial Agonist on Working Memory in the Aged Rhesus Monkey.

Aged-related declines in cognition, especially working memory and executive function, begin in middle-age and these abilities are known to be mediated by the prefrontal cortex (PFC) and more specifically the dopamine (DA) system within the PFC. In both humans and monkeys, there is significant evidence that the PFC is the first cortical region to change with age and the PFC appears to be particularly vulnerable to age-related loss of dopamine (DA). Therefore, the DA system is a strong candidate for therapeutic intervention to slow or reverse age related declines in cognition. In the present study, we administered a novel selective, potent, non-catechol DA D1 R agonist PF-6294 (Pfizer, Inc.) to aged female rhesus monkeys and assessed their performance on two benchmark tasks of working memory - the Delayed Non-match to Sample Task (DNMS) and Delayed Recognition Span Task (DRST). The DNMS task was administered first with the standard 10 s delay and then with 5 min delays, with and without distractors. The DRST was administered each day with four trials with unique sequences and one trial of a repeated sequence to assess evidence learning and retention. Overall, there was no significant effect of drug on performance on any aspect of the DNMS task. In contrast, we demonstrated that a middle range dose of PF-6294 significantly increased memory span on the DRST on the first and last days of testing and by the last day of testing the increased memory span was driven by the performance on the repeated trials.

Mechanisms underlying autoimmune synaptic encephalitis leading to disorders of memory, behavior and cognition: insights from molecular, cellular and synaptic studies.

Recently, several novel, potentially lethal and treatment-responsive syndromes that affect hippocampal and cortical function have been shown to be associated with auto-antibodies against synaptic antigens, notably glutamate or GABA-B receptors. Patients with these auto-antibodies, sometimes associated with teratomas and other neoplasms, present with psychiatric symptoms, seizures, memory deficits and decreased levels of consciousness. These symptoms often improve dramatically after immunotherapy or tumor resection. Here we review studies of the cellular and synaptic effects of these antibodies in hippocampal neurons in vitro and preliminary work in rodent models. Our work suggests that patient antibodies lead to rapid and reversible removal of neurotransmitter receptors from synaptic sites, leading to changes in synaptic and circuit function that in turn are likely to lead to behavioral deficits. We also discuss several of the many questions raised by these and related disorders. Determining the mechanisms underlying these novel anti-neurotransmitter receptor encephalopathies will provide insights into the cellular and synaptic bases of the memory and cognitive deficits that are hallmarks of these disorders, and potentially suggest avenues for therapeutic intervention.

AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location.

OBJECTIVE: To report the clinical and immunological features of a novel autoantigen related to limbic encephalitis (LE) and the effect of patients' antibodies on neuronal cultures. METHODS: We conducted clinical analyses of 10 patients with LE. Immunoprecipitation and mass spectrometry were used to identify the antigens. Human embryonic kidney 293 cells expressing the antigens were used in immunocytochemistry and enzyme-linked immunoabsorption assay. The effect of patients' antibodies on cultures of live rat hippocampal neurons was determined with confocal microscopy. RESULTS: Median age was 60 (38-87) years; 9 were women. Seven had tumors of the lung, breast, or thymus. Nine patients responded to immunotherapy or oncological therapy, but neurological relapses, without tumor recurrence, were frequent and influenced the long-term outcome. One untreated patient died of LE. All patients had antibodies against neuronal cell surface antigens that by immunoprecipitation were found to be the glutamate receptor 1 (GluR1) and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Human embryonic kidney 293 cells expressing GluR1/2 reacted with all patients' sera or cerebrospinal fluid, providing a diagnostic test for the disorder. Application of antibodies to cultures of neurons significantly decreased the number of GluR2-containing AMPAR clusters at synapses with a smaller decrease in overall AMPAR cluster density; these effects were reversed after antibody removal. INTERPRETATION: Antibodies to GluR1/2 associate with LE that is often paraneoplastic, treatment responsive, and has a tendency to relapse. Our findings support an antibody-mediated pathogenesis in which patients' antibodies alter the synaptic localization and number of AMPARs.

Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome.

The roles of astrocytes in the CNS have been expanding beyond the long held view of providing passive, supportive functions. Recent evidence has identified roles in neuronal development, extracellular matrix maintenance, and response to inflammatory challenges. Therefore, insights into astrocyte secretion are critically important for understanding physiological responses and pathological mechanisms in CNS diseases. Primary astrocyte cultures were treated with inflammatory cytokines for either a short (1 day) or sustained (7 days) exposure. Increased interleukin-6 secretion, nitric oxide production, cyclooxygenase-2 activation, and nerve growth factor (NGF) secretion confirmed the astrocytic response to cytokine treatment. MS/MS analysis, computational prediction algorithms, and functional classification were used to compare the astrocyte protein secretome from control and cytokine-exposed cultures. In total, 169 secreted proteins were identified, including both classically and nonconventionally secreted proteins that comprised components of the extracellular matrix and enzymes involved in processing of glycoproteins and glycosaminoglycans. Twelve proteins were detected exclusively in the secretome from cytokine-treated astrocytes, including matrix metalloproteinase-3 (MMP-3) and members of the chemokine ligand family. This compilation of secreted proteins provides a framework for identifying factors that influence the biochemical environment of the nervous system, regulate development, construct extracellular matrices, and coordinate the nervous system response to inflammation.

Heterogeneity in synaptic vesicle release at neuromuscular synapses of mice expressing synaptopHluorin.

Mammalian neuromuscular junctions are useful model synapses to study the relationship between synaptic structure and function, although these have rarely been studied together at the same synapses. To do this, we generated transgenic lines of mice in which the thy1.2 promoter drives expression of synaptopHluorin (spH) as a means of optically measuring synaptic vesicle distribution and release. SpH is colocalized with other synaptic vesicle proteins in presynaptic terminals and does not alter normal synaptic function. Nerve stimulation leads to readily detectable and reproducible fluorescence changes in motor axon terminals that vary with stimulus frequency and, when compared with electrophysiological recordings, are reliable indicators of neurotransmitter release. Measurements of fluorescence intensity changes reveal a surprising amount of heterogeneity in synaptic vesicle release throughout individual presynaptic motor axon terminals. Some discrete terminal regions consistently displayed a greater rate and extent of release than others, regardless of stimulation frequency. The amount of release at a particular site is highly correlated to the relative abundance of synaptic vesicles there, indicating that a relatively constant fraction of the total vesicular pool, approximately 30%, is released in response to activity. These studies reveal previously unknown relationships between synaptic structure and function at mammalian neuromuscular junctions and demonstrate the usefulness of spH expressing mice as a tool for studying neuromuscular synapses in adults, as well as during development and diseases that affect neuromuscular synaptic function.

A novel approach to evaluate the pharmacodynamics of a selective dopamine D1/D5 receptor partial agonist (PF-06412562) in patients with stable schizophrenia.

BACKGROUND: PF-06412562 is an orally bioavailable, selective dopamine D1/D5 receptor partial agonist with a non-catechol structure under evaluation for treatment of cognitive impairment in schizophrenia. AIMS: This randomized, double-blind, placebo-controlled, parallel-group, Phase 1b study examined the pharmacokinetics and pharmacodynamics of three doses of PF-06412562 (3 mg, 9 mg, and 45 mg twice daily) over 15 days in patients with schizophrenia receiving antipsychotics. METHODS: Primary endpoints included adjunctive safety/tolerability and effects on MATRICS Consensus Cognitive Battery Working Memory domain and reward processing (Monetary Incentive Delay) tasks. Exploratory endpoints included other behavioral/neurophysiological tasks, including the N-back task. RESULTS: Among 95 subjects (78% male; mean age 34.8 years), baseline characteristics were similar across groups. The MATRICS Consensus Cognitive Battery Working Memory composite change from baseline on Day 13 improved in all groups, the smallest improvement was observed in the 45 mg group and was significantly smaller than that in the placebo group (two-sided p=0.038). For the Monetary Incentive Delay task (change from baseline in blood-oxygen-level-dependent functional magnetic resonance imaging activation in anterior ventral striatum for the contrast of cue gain>cue no gain on Day 15), no PF-06412562 dose was significantly different from placebo. No doses of PF-06412562 showed a significant difference on two-back task accuracy versus placebo. CONCLUSIONS: Adjunctive treatment with PF-06412562 was safe and well tolerated in patients with schizophrenia. PF-06412562 failed to show clinical benefit relative to placebo on assessments of cognition or reward processing in symptomatically stable patients over a 15-day treatment period. Numerous limitations due to the safety study design warrant further efficacy evaluation for this drug mechanism.

Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.

BACKGROUND: A severe form of encephalitis associated with antibodies against NR1-NR2 heteromers of the NMDA receptor was recently identified. We aimed to analyse the clinical and immunological features of patients with the disorder and examine the effects of antibodies against NMDA receptors in neuronal cultures. METHODS: We describe the clinical characteristics of 100 patients with encephalitis and NR1-NR2 antibodies. HEK293 cells ectopically expressing single or assembled NR1-NR2 subunits were used to determine the epitope targeted by the antibodies. Antibody titres were measured with ELISA. The effect of antibodies on neuronal cultures was determined by quantitative analysis of NMDA-receptor clusters. FINDINGS: Median age of patients was 23 years (range 5-76 years); 91 were women. All patients presented with psychiatric symptoms or memory problems; 76 had seizures, 88 unresponsiveness (decreased consciousness), 86 dyskinesias, 69 autonomic instability, and 66 hypoventilation. 58 (59%) of 98 patients for whom results of oncological assessments were available had tumours, most commonly ovarian teratoma. Patients who received early tumour treatment (usually with immunotherapy) had better outcome (p=0.004) and fewer neurological relapses (p=0.009) than the rest of the patients. 75 patients recovered or had mild deficits and 25 had severe deficits or died. Improvement was associated with a decrease of serum antibody titres. The main epitope targeted by the antibodies is in the extracellular N-terminal domain of the NR1 subunit. Patients' antibodies decreased the numbers of cell-surface NMDA receptors and NMDA-receptor clusters in postsynaptic dendrites, an effect that could be reversed by antibody removal. INTERPRETATION: A well-defined set of clinical characteristics are associated with anti-NMDA-receptor encephalitis. The pathogenesis of the disorder seems to be mediated by antibodies.

In vivo imaging of preferential motor axon outgrowth to and synaptogenesis at prepatterned acetylcholine receptor clusters in embryonic zebrafish skeletal muscle.

Little is known about the spatial and temporal dynamics of presynaptic and postsynaptic specializations that culminate in synaptogenesis. Here, we imaged presynaptic vesicle clusters in motor axons and postsynaptic acetylcholine receptor (AChR) clusters in embryonic zebrafish to study the earliest events in synaptogenesis in vivo. Prepatterned AChR clusters are present on muscle fibers in advance of motor axon outgrowth from the spinal cord. Motor axon growth cones and filopodia are selectively extended toward and contact prepatterned AChR clusters, followed by the rapid clustering of presynaptic vesicles and insertion of additional AChRs, hallmarks of synaptogenesis. All initially formed neuromuscular synapses contain AChRs that were inserted into the membrane at the time the prepattern is present. Examination of embryos in which AChRs were blocked or clustering is absent showed that neither receptor activity or receptor protein is required for these events to occur. Thus, during initial synaptogenesis, postsynaptic differentiation precedes presynaptic differentiation, and prepatterned neurotransmitter clusters mark sites destined for synapse formation.