NMDA subunit antibodies highlight the importance of immunogen selection.

NMDAR2B is implicated in aging-related memory loss.

The NMDA receptor is widely recognized as playing a pivotal role in long term potentiation (LTP) a key cellular substrate of learning and memory. This receptor is composed of NR1 subunits and at least in forebrain 2 homologous NR2 subunits (NR2A and B). Despite their homology, the presence of NR2A or NR2B appears to produce key differences in the functions of the NMDAR. Knockdown of the NR2B but not NR2A produces memory deficits. We have also demonstrated that an NR2B expression deficit is correlated memory impairment in rats. These data suggest an important role for reduced NR2B expression in age-related cognitive decline. Importantly, none of this work would have been possible without the development of antibodies that could specifically assay the NR2B and its related subunits.

We needed an antibody to differentiate the NR2 A and B subunits.

We have always been extremely focused on making sure our antibodies are specific for their targets of interest. We test every antibody to be certain that it labels only a single band of the correct MW in western blots of an appropriate tissue lysate. However even this test may not be sufficient in some rare instances such as described below in the making of antibodies to the NMDA receptor.

Making these NMDA antibodies was difficult due to their close homology.

The development of these antibodies was not straightforward. The difficulty we encountered with the NR2 A and B subunits is due to the fact that the two proteins sequences are quite homologous. Simply selecting the C or N terminal areas as antigens would have been a disaster as these sequences are identical in the two NR2s. To deal with this issue we scoured the sequences and identified unique sequence with both proteins. These sequences encompassed about 130 amino acids. We next generated fusion proteins for the entirety of these two specific sequences and used the fusion proteins as antigens. Fortunately, these antigens yielded highly specific NR2B and NR2A antibodies. We confirmed the specificity of the antibodies by demonstrating that the NR2A or NR2B antibody was able to recognize the corresponding fusion protein but showed no cross reactivity toward the other fusion protein. Moreover, they each recognized only a single band in a brain tissue lysate.

Our challenging project was rewarded with key reagents for memory and aging studies.

Given the importance of the relative expression of NR2B and NR2A for age related memory impairment, these highly specific 2A and 2B antibodies should greatly facilitate future work in this area. Thus, these antibodies make it possible to quantitate the relative expression of these subunits throughout development. Even more importantly, they permit precise evaluation of the effect of drugs that manipulate 2B subunit expression. A better understanding of this functionality could ameliorate age-related memory impairment and advance clinical therapies for dementia patients.

References:

1. Clayton DA, Mesches MH, Alvarez E, Bickford PC, Browning MD. (2002) An hippocampal NR2B deficit can mimic age-related changes in LTP and spatial learning in the Fischer 344 rat. J Neurosci 22(9):3628-3637.

2. Clayton DA, Grosshans DG, Browning MD. (2002). Aging and surface expression of hippocampal NMDA receptors. J Biol Chem (277)17:14367-14369.