Martha Constantine-Paton, Ph.D.
Professor of Biology
Massachusetts Institute of Technology
January 24, 2000
Function and Regulation of the NMDA Receptor During Synaptogenesis in the Superior Coliculus
Local control of protein synthesis through synaptic function is becoming recognized as an important signaling system for producing long-term changes in synaptic function. My talk focused on our studies of a mechanism that controls synthesis of the Ca" Calmodulin dependent protein kinase (CaM Kll). This enzyme has been implicated in increasing the synaptic response to glutamate during cellular models of learning and memory such as LTP. In our studies of developing synapses in the visual pathway we discovered that the normal expression of this enzyme is retarded by blocking the NMDA subtype of glutamate receptor. I discussed in my presentation our studies of a mechanism that might be responsible for this result. The content of this talk is briefly outlined below.
We discovered that eukaryote Elongation Factor 2 (EF2), a major control point in protein translation, became phosphorylated within minutes of activating NMDA receptors in the visual tectal lobes of young frogs. In collaboration with Angus Nairn of Rockefeller University, who developed an antibody specific for phospho-EF2, we demonstrated that EF2 indeed becomes phosphorylated within 30 seconds of NMDAR activation in tadpole tecta maintained in vitro. Furthermore, photopic stimulation of the retina for 30 seconds significantly increased phospho-EF2 in the retinotectal neuropil in intact tadpoles, and this increase required NMDAR function in that tectum. Using light and electron microscopy we documented that this phosphorylation occurs in the immediate post-synaptic process; that compared to levels in the tadpole, NMDA-induced phophorylation of EF2 is much reduced in the adult frog; and that in the frog, phospho-EF2 is retricted to the most distal segments of tectal neuron dendrites in layer 9A. This lamina receives a dense, indirect input from the ipsilateral eye via the nucleus isthmi and is likely to be the site of most of the structural rearrangement of synapses in frogs after metamorphosis. EF2 is phosphorylated by an EF2 specific Ca++ Calmodulin dependent kinase, which is also localized in dendrites.
These experiments demonstrated a direct signaling pathway between the NMDARs and a major control point in protein translation. Furthermore, this control was exerted during physiological stimulation of the visual pathway. It was downregulated when plasticity was downregulated. It occurred with an exceptionally short latency. It could occur in isolated segments of tectal neuron dendrites. These findings in conjunction with our data suggesting that NMDAR function was necessary for the normal maturation of the sSC neuropil in the neonatal rat motivated a new set of studies on NMDAR-induced effects on protein translation in the rat.
Using isolated synaptic fractions (synaptoneurosomes) from PI 3 rat pup sSC, we found that the same AP5 blockable NMDAR stimulation used in the frog tectum caused a rapid < 1 min onset and short-lived five to 10 minute phosphorylation of EF2. Coordinated 35S-methionine pulse-chase labeling experiments showed that the known effect of eEF2 phosphorytation, namely a brief block of protein synthesis, occurred in the synaptoneurosome preparations. However, while the synthesis of the majority of proteins was decreased, some proteins showed increased synthesis. At this point we knew that CaM Kll expression and maturation were linked to NMDAR stimulation in the developing rat sSC neuropil; that CaM Kll transcript was prominent in dendrites; and that the kinase is intimately associated with plasticity at glutamatergic synapses. We developed an immunoprecipitation assay for 35 -labeled CaM Kll and showed that within the short latency time window when phospho-EF2 levels are high following NMDAR stimulation, 35S-labeled CaM Kll significantly increased. Moreover, this brief (30 sec) period of NMDAR activation also increased total CaM Kll protein in synaptoneurosomes by nearly 50 percent. We also showed that a number of proteins do not increase their synthesis in sSC synaptoneurosomes in response to NMDAR stimulation and that the immunoprecipitation of alpha Cam Kll co-precipitates several other 35Slabeled proteins. The 35S-labeled proteins in the immunoprecipitated complex differ between P8 and PI 3, suggesting a developmental change in NMDAR-mediated dendritic protein synthesis. A means of specifically blocking EF2 kinase was not then available. Consequently, to test the prediction that it was the NMDARmediated phosphorylation of EF2 and the slowing of protein translation that phosphorylation of EF2 is known to produce that caused the upregulation of the synthesis of certain proteins, including CaM Kll we used cycloheximide. Cycloheximide blocks protein translation through a mechanism independent of EF2. Low doses of cycloheximide applied to sSC synaptoneurosomes increased CaM Kll synthesis while reducing synthesis of total protein by 90 percent. The actual mechanism of this effect of slowing protein translation is unknown, though it has been seen before for other proteins in fibroblasts. Our favored hypothesis is that the slowing of translation shifts the rate limiting step in translation from initiation to elongation. This situation would favor a significant increase in translation of those transcripts that are highly abundant but poorly initiated over the translation of proteins from fewer transcripts with a high affinity for the initiating complex.
In short, we have documented an extremely rapid means through which activation of the NMDAR at young visual synapses may rapidly and transiently alter the protein content of the post-synaptic process. Although other means of dendritic control of protein synthesis exist, the NR/EF2 pathway has kinetics sufficiently rapid to be titrated by synaptic activity. Moreover, the NR/EF2 pathway exerts an important control over at least one highly significant post-synaptic density protein, CaM KII.