Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, Mk, R, Xn||ELISA, ICC, IHC, IH(P), RIA||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgG2a liquid in buffer PBS, no preservative.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20ºC from date of receipt.|
|Material Size||100 µg|
|Reference overview||Application||Species||Pub Med ID|
|Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice.|
Planagumà, J; Leypoldt, F; Mannara, F; Gutiérrez-Cuesta, J; Martín-García, E; Aguilar, E; Titulaer, MJ; Petit-Pedrol, M; Jain, A; Balice-Gordon, R; Lakadamyali, M; Graus, F; Maldonado, R; Dalmau, J
Brain : a journal of neurology 138 94-109 2015
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 ?-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.
|Developmental attenuation of N-methyl-D-aspartate receptor subunit expression by microRNAs.|
Corbel, C; Hernandez, I; Wu, B; Kosik, KS
Neural development 10 20 2015
N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors and are expressed throughout the central nervous system (CNS). Their activity is required for excitatory synaptic transmission, the developmental refinement of neural circuits and for the expression of many forms of synaptic plasticity. NMDARs are obligate heterotetramers and the expression of their constituent subunits is developmentally and anatomically regulated. In rodent cortex and hippocampus, the GluN2B subunit is expressed at high levels early in development and decreases to plateau levels later while expression of the GluN2A subunit has a concomitant increase. Regulation of GluN2A and GluN2B expressions are incompletely understood. Here, we showed the influence of miRNAs in this process.Two miRNAs, miR-19a and miR-539 can influence the levels of NMDARs subunits, as they target the mRNAs encoding GluN2A and GluN2B respectively. MiR-539 also modified the expression of the transcription factor REST, a known regulator of NMDAR subunit expression.miR-19a and miR-539, in collaboration with REST, serve to set the levels of GluN2A and GluN2B precisely during development. These miRNAs offer an entry point for interventions that affect plasticity and a novel approach to treat neurodegenerative diseases.
|A novel approach identifies the first transcriptome networks in bats: a new genetic model for vocal communication.|
Rodenas-Cuadrado, P; Chen, XS; Wiegrebe, L; Firzlaff, U; Vernes, SC
BMC genomics 16 836 2015
Bats are able to employ an astonishingly complex vocal repertoire for navigating their environment and conveying social information. A handful of species also show evidence for vocal learning, an extremely rare ability shared only with humans and few other animals. However, despite their potential for the study of vocal communication, bats remain severely understudied at a molecular level. To address this fundamental gap we performed the first transcriptome profiling and genetic interrogation of molecular networks in the brain of a highly vocal bat species, Phyllostomus discolor.Gene network analysis typically needs large sample sizes for correct clustering, this can be prohibitive where samples are limited, such as in this study. To overcome this, we developed a novel bioinformatics methodology for identifying robust co-expression gene networks using few samples (N=6). Using this approach, we identified tissue-specific functional gene networks from the bat PAG, a brain region fundamental for mammalian vocalisation. The most highly connected network identified represented a cluster of genes involved in glutamatergic synaptic transmission. Glutamatergic receptors play a significant role in vocalisation from the PAG, suggesting that this gene network may be mechanistically important for vocal-motor control in mammals.We have developed an innovative approach to cluster co-expressing gene networks and show that it is highly effective in detecting robust functional gene networks with limited sample sizes. Moreover, this work represents the first gene network analysis performed in a bat brain and establishes bats as a novel, tractable model system for understanding the genetics of vocal mammalian communication.
|Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating N-methyl-D-aspartate receptor antibodies in serum using a live cell-based assay.|
Ramberger, M; Peschl, P; Schanda, K; Irschick, R; Höftberger, R; Deisenhammer, F; Rostásy, K; Berger, T; Dalmau, J; Reindl, M
PloS one 10 e0122037 2015
N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1-100.0 and 95.7-100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7-100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4-97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, pless than 0.0001) and correlation (r = 0.697, pless than 0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.
|Mapping synapses by conjugate light-electron array tomography.|
Collman, F; Buchanan, J; Phend, KD; Micheva, KD; Weinberg, RJ; Smith, SJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 35 5792-807 2015
Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex.
|Synaptic and cognitive improvements by inhibition of 2-AG metabolism are through upregulation of microRNA-188-3p in a mouse model of Alzheimer's disease.|
Zhang, J; Hu, M; Teng, Z; Tang, YP; Chen, C
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 14919-33 2014
Abnormal accumulation of ?-amyloid (A?) is the major neuropathological hallmark of Alzheimer's disease (AD). However, the mechanisms underlying aberrant A? formation in AD remain unclear. We showed previously that inhibition of monoacylglycerol lipase (MAGL), the primary enzyme that metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain, robustly reduces A? by inhibiting ?-site amyloid precursor protein cleaving enzyme 1 (BACE1), a key enzyme responsible for A? formation. However, the molecular mechanisms responsible for suppression of BACE1 by inhibition of 2-AG metabolism are largely unknown. We demonstrate here that expression of the noncoding small RNA miR-188-3p that targets BACE1 was significantly downregulated both in the brains of AD humans and APP transgenic (TG) mice, a mouse model of AD. The downregulated miR-188-3p expression was restored by MAGL inhibition. Overexpression of miR-188-3p in the hippocampus reduced BACE1, A?, and neuroinflammation and prevented deteriorations in hippocampal basal synaptic transmission, long-term potentiation, spatial learning, and memory in TG mice. 2-AG-induced suppression of BACE1 was prevented by miR-188-3p loss of function. Moreover, miR-188-3p expression was upregulated by 2-AG or peroxisome proliferator-activated receptor-? (PPAR?) agonists and suppressed by PPAR? antagonism or NF-?B activation. Reducing A? and neuroinflammation by MAGL inhibition was occluded by PPAR? antagonism. In addition, BACE1 suppression by 2-AG and PPAR? activation was eliminated by knockdown of NF-?B. Our study provides a novel molecular mechanism underlying improved synaptic and cognitive function in TG mice by 2-AG signaling, which upregulates miR-188-3p expression through PPAR? and NF-?B signaling pathway, resulting in suppressions of BACE1 expression and A? formation.
|Differential Changes in Postsynaptic Density Proteins in Postmortem Huntington's Disease and Parkinson's Disease Human Brains.|
Fourie, C; Kim, E; Waldvogel, H; Wong, JM; McGregor, A; Faull, RL; Montgomery, JM
Journal of neurodegenerative diseases 2014 938530 2014
NMDA and AMPA-type glutamate receptors and their bound membrane-associated guanylate kinases (MAGUKs) are critical for synapse development and plasticity. We hypothesised that these proteins may play a role in the changes in synapse function that occur in Huntington's disease (HD) and Parkinson's disease (PD). We performed immunohistochemical analysis of human postmortem brain tissue to examine changes in the expression of SAP97, PSD-95, GluA2 and GluN1 in human control, and HD- and PD-affected hippocampus and striatum. Significant increases in SAP97 and PSD-95 were observed in the HD and PD hippocampus, and PSD95 was downregulated in HD striatum. We observed a significant increase in GluN1 in the HD hippocampus and a decrease in GluA2 in HD and PD striatum. Parallel immunohistochemistry experiments in the YAC128 mouse model of HD showed no change in the expression levels of these synaptic proteins. Our human data show that major but different changes occur in glutamatergic proteins in HD versus PD human brains. Moreover, the changes in human HD brains differ from those occurring in the YAC128 HD mouse model, suggesting that unique changes occur at a subcellular level in the HD human hippocampus.
|MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus.|
Qiu, S; Lu, Z; Levitt, P
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 16166-79 2014
The MET receptor tyrosine kinase (RTK), implicated in risk for autism spectrum disorder (ASD) and in functional and structural circuit integrity in humans, is a temporally and spatially regulated receptor enriched in dorsal pallial-derived structures during mouse forebrain development. Here we report that loss or gain of function of MET in vitro or in vivo leads to changes, opposite in nature, in dendritic complexity, spine morphogenesis, and the timing of glutamatergic synapse maturation onto hippocampus CA1 neurons. Consistent with the morphological and biochemical changes, deletion of Met in mutant mice results in precocious maturation of excitatory synapse, as indicated by a reduction of the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of AMPA receptors at synaptic sites. Thus, MET-mediated signaling appears to serve as a mechanism for controlling the timing of neuronal growth and functional maturation. These studies suggest that mistimed maturation of glutamatergic synapses leads to the aberrant neural circuits that may be associated with ASD risk.
|In vitro ischemia triggers a transcriptional response to down-regulate synaptic proteins in hippocampal neurons.|
Fernandes, J; Vieira, M; Carreto, L; Santos, MA; Duarte, CB; Carvalho, AL; Santos, AE
PloS one 9 e99958 2014
Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARP?3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia.
|An Immunological Approach to Increase the Brain's Resilience to Insults.|
Lin, EJ; Symes, CW; Townsend-Nicholson, A; Klugmann, M; Klugmann, CB; Lehnert, K; Fong, D; Young, D; During, MJ
ISRN neuroscience 2014 103213 2014
We have previously demonstrated the therapeutic potential of inducing a humoral response with autoantibodies to the N-methyl D-aspartate (NMDA) receptor using a genetic approach. In this study, we generated three recombinant proteins to different functional domains of the NMDA receptor, which is implicated in mediating brain tolerance, specifically NR1[21-375], NR1[313-619], NR1[654-800], and an intracellular scaffolding protein, Homer1a, with a similar anatomical expression pattern. All peptides showed similar antigenicity and antibody titers following systemic vaccination, and all animals thrived. Two months following vaccination, rats were administered the potent neurotoxin, kainic acid. NR1[21-375] animals showed an antiepileptic phenotype but no neuroprotection. Remarkably, despite ineffective antiepileptic activity, 6 of 7 seizing NR1[654-800] rats showed absolutely no injury with only minimal changes in the remaining animal, whereas the majority of persistently seizing rats in the other groups showed moderate to severe hippocampal injury. CREB, BDNF, and HSP70, proteins associated with preconditioning, were selectively upregulated in the hippocampus of NR1[654-800] animals, consistent with the observed neuroprotective phenotype. These results identify NR1 epitopes important in conferring anticonvulsive and neuroprotective effects and support the concept of an immunological strategy to induce a chronic state of tolerance in the brain.
|Pathways and Biomarkers of Glutamatergic Synapse Flyer|
|Anti-NMDAR1, extracellular loop between transmembrane regions III & IV, clone 54.1 - Data Sheet|