The neuron is the basic cellular unit of the brain. For neurons to work properly, they must be plastic and constantly capable of changing in response to stimuli, forming and stabilizing new connections. This process requires proteins to be added to the new synaptic contact, and this in turn results from the targeting of mRNA to these sites of activity. This is the mechanistic basis of learning and memory since the synapse is stabilized by the production of proteins in response to stimulation that are important for its structural integrity. How this mRNA is regulated in neurons to make the right protein at the right place and time has been the subject of our investigations over the years of this funding. This proposal exploits the tools we generated during the last funding period to address how mRNA is regulated in dendrites. One of these tools is a mouse where we have tagged the ?-actin gene with a fluorescent marker to follow individual mRNAs in live neurons. We have found that the mRNA is encased in an inert form as it travels around in the dendrite. When it comes into the proximity of a stimulated dendritic spine, it unfurls its RNA payload and makes a burst of protein, but then returns to a dormant state after 16 minutes. The mRNA sits at the place where it was last stimulated for hours, awaiting the next signal, wherein it will initiate another round of proteins. In this way, the synaptic contact is built up, consistent with a learning and memory paradigm that relies on repetitive stimulation. If there are no further activating signals, the mRNA continues its search, moving in short processive movements broken by periods of diffusion. The current proposal follows up on the discovery of the particular protein that binds to the mRNA zipcode responsible for directing it to its site (zipcode binding protein, ZBP1), anchors the mRNA at the site of stimulation. The model we have constructed suggests that the mRNA translates upon a further stimulation and we intend to focus on this point of regulation by describing the kinetics of these events and the proteins that play a role in these events. Up to this point we have investigated ?-actin mRNA because actin is the major structural protein in cells, and in the synapse as well. However the regulatory mechanism leading to a complex structure such as a synapse must orchestrate the expression of many proteins. For this reason, we have constructed another mouse with an mRNA important for learning, Arc, that has been tagged with a different fluorescent marker. In this proposal, we will characterize the mRNAs for ?-actin and Arc in mice together where both mRNAs are individually detectable by different colored fluorochromes. We propose eventually a third hybrid-color mRNA for CaMKII?, an essential protein in synapses. Our goal is to uncover the mechanisms that govern the regulation of different mRNAs in response to stimulatory activations of the neuron: the timing of their synthesis, localization into dendrites to activated spines, their translational repression, activation and eventual degradation, and some proteins associated with each of these events. The tools are now in place to characterize each of these steps at the single molecule level in live neurons.
The mechanism controlling the time and place where messenger RNA is activated to make proteins is unknown, yet this problem is at the heart of all cell function and at the basis of neurological diseases and cancer. We have devised a variety of technologies to answer this question, using transgenic animals, innovative microscopy and unique mRNA reporters to determine where the critical events occur in the cell. We will use this methodology on neurons, where the local activation of protein synthesis at synapses is at the basis of learning and memory.
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