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.
