| Abstract No.: | C-B3046 |
| Country: | Canada |
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| Title: | THE CUB DOMAIN PROTEIN NETO2 IS A COMPONENT OF THE NMDA RECEPTOR IN THE MAMMALIAN BRAIN |
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| Authors/Affiliations: | 1 Cristina Tang*; 1 David Ng; 2 Marijana Kanisek; 1 Mike Salter; 1 Roderick McInnes;
1 Hospital for Sick Children, Toronto, ON, Canada; 2 Samuel Lunenfeld Research Institute, Toronto, ON, Canada
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| Content: | In an in-silico search for proteins involved in the development and maintenance of the nervous system, we identified two novel homologous transmembrane proteins, Neto1, and Neto2, expressed in both the developing and the mature mouse brain. We have previously shown that the brains and behaviour of Neto1-/-, Neto2-/-, and Neto1/Neto2 double knockout mice are grossly normal, suggesting that these proteins are not required for essential neuronal cell fate decisions and/or survival. However, loss of the Neto proteins results in partial defects in axon guidance during development in several areas of the brain, and in an impaired spatial learning and memory in the adult based on tests in the Morris water maze. Consistent with the impaired learning phenotype, we have shown that Neto1 is a component of the NMDAR complex on the postsynaptic membrane of the hippocampus. Loss of Neto1 results in a 50% reduction in NMDAR-dependent form of LTP at hippocampal CA3-CA1 synapses. Here we report that Neto2 is also a component of the NMDAR. Like Neto1, Neto2 is expressed widely in the CNS, but most strongly in the hippocampus, olfactory bulb, cerebellum, and cortex. Biochemical fractionation of mouse whole brain revealed that Neto2 is present on both presynaptic membranes and the postsynaptic density (PSD), although the majority of Neto2 is intracellular. The colocalization of Neto1 and Neto2 in the PSD suggested that the two proteins may associate (a prediction confirmed by coIPs from whole brain), and that Neto2 may also be part of the NMDAR. By in vitro coexpression experiments in HEK293 cells, I found that on the cell membrane, Neto2 interacts directly with the obligatory NR1 subunit of the NMDA receptor. In addition, coimmunoprecipitation experiments from adult mouse brain crude synaptosomal fractions confirmed that Neto2 associates with the NMDAR in vivo. In behavioural studies of learning and memory, loss of Neto2 alone did not impair the performance of mice in the initial learning (acquisition), reversal, or delayed-matched-to-place tests of the Morris water maze. However, loss of both Neto2 and Neto1 resulted in a severe behavioural deficit in the acquisition phase of the test, which was not observed in Neto1 or Neto2 single knockout mice. We conclude that Neto2 is a novel NMDA receptor interacting protein, and like Neto1, it participates in NMDAR-dependent spatial learning and memory.
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