David Ng; Graham M Pitcher; Rachel K Szilard; Andréa Sertié; Marijana Kanisek; Steven J Clapcote; Tatiana Lipina; Lorraine V Kalia; Daisy Joo; Colin McKerlie; Miguel Cortez; John C Roder; Michael W Salter; Roderick R McInnes

“ To determine whether the association of Neto1 with NMDARs was entirely dependent upon the binding of its C-terminal PDZ ligand to PSD-95, we examined the binding of PSD-95 to an hemagglutinin (HA) -tagged Neto1 protein lacking the C-terminal 20 amino acids (Neto1-Δ20HA) . As predicted both by the interaction between PSD-95 and the NR2 subunits of the NMDAR [28] , and by the binding of Neto1 to PSD-95 described above, we found that Neto1 was co-immunoprecipitated by anti-NR1 antibodies from lysates of cells co-expressing Neto1, PSD-95, NR1, and NR2B (Figure 4A, lane 1) . ”

“ To investigate the role of Neto1 in the biology of mammalian excitatory synapses, we determined the molecular basis of the Neto1:NMDAR interaction and defined the nonredundant functions of Neto1 in synaptic plasticity, learning, and memory using Neto1 protein null mice. ”

“ Our identification of Neto1 as a critical auxiliary protein for NR2A-NMDARs raises the possibility that other proteins, perhaps other CUB domain proteins, may be required, like Neto1, to maintain non-NR2A-NMDARs at synapses. Thus, Neto1 represents a new protein that functions to specifically maintain synaptic NMDARs, a protein that has been elusive for NMDARs. ”

“ Representative traces show NMDAR EPSCs (+60 mV) before (black traces) and 20–30 min after CX546 (25 μM; gray traces) administration in hippocampal slices from Neto1+/+ (+/+) and Neto1-null (tlz/tlz) mice. Each EPSC is the average of six consecutive traces. Scale bars: +/+, 100 ms, 75 pA; tlz/tlz, 100 ms, 35 pA. Below: Histogram of pharmacologically isolated NMDAR EPSC amplitude (left) or decay (right) from CA1 neurons in hippocampal slices from +/+ (n = 8 neurons) and tlz/tlz (n = 6 neurons) mice 20–30 min after CX546 administration (25 μM; filled bars) . ”

“ Left: Binding of CX546 to AMPA receptors alters the receptor desensitization kinetics and prolongs membrane depolarization, allowing more influx of sodium ions. Right: During LTP induction, at a concentration of CX546 sufficient to restore the LTP deficit in Neto1-null Schaffer collateral-CA1 synapses, prolonged membrane depolarization extends the temporary relief of the magnesium ion blockade, increasing the sodium and calcium ion influx through NMDARs to levels sufficient to restore LTP to wild-type levels. ”

“ All mice had a similar latency to find the DO (wild-type: 35.3 s ± 5.7 s; Neto1-null: 31.2 s ± 12.7 s) , therefore, excluding a possible influence of anxiety in response to the spatial changes and reaction to the DO. (E) Response to object replacement. Neto1-null mice were not impaired in novel object recognition. ANOVA revealed a significant effect of object novelty (F1,22 = 67.6, p < 0.001) , no main effect of genotype on time spent on familiar object (FO) versus novel object (NO) (F1,22 = 0.04, p > 0.05) or their interactions (F1,21 = 0.04, p > 0.05) . ”

“ In the mammalian central nervous system, excitatory transmission at synapses is mediated primarily by the amino acid glutamate, acting through the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl-D-aspartic acid receptors (NMDARs) [1] . ”

“ Neto1 Binds to NR2, but Not to NR1 Subunits (A–D) Immunoblots of immunoprecipitations from transfected HEK293 cell lysates. The identities of the transfected cDNAs are shown above each lane. Blot, antibodies used for immunoblot analysis; IP, antibodies used for immunoprecipitation. ”

“ JCR and MWS hold Canada Research Chairs; MWS is an International Research Scholar of the Howard Hughes Medical Institute; DN held a CIHR Doctoral Research Award; RKS was supported by postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and in part through the Hospital for Sick Children Research Training Centre ”

“ Frohlich, S & Weber, BH, “A novel gene encoding a putative transmembrane protein with two extracellular CUB domains and a low-density lipoprotein class A module: isolation of alternatively spliced isoforms in retina and brain”, Gene 286: 223–231, pmid:11943477 1 2 3 4 Ng, D; Sertie, A; Szilard, RK; Kalia, LV & Salter, MW, “Neto1, a novel synaptic membrane receptor associated with aberrant axonal guidance and seizures”, Am J Hum Genet 71: 166 ↑ Romero, A ”