Dissecting the role of Drosophila Orb2 in long-term memory

Long-term memory in Drosophila is critically dependent on the function of the CPEB (cytoplasmic polyadenylation element-binding) protein Orb2. Specifically, in a courtship conditioning paradigm, orb2 mutants form a normal short-term memory but cannot form a long-term memory even after extended training periods. Other CPEB proteins have been shown to regulate mRNA transport or translation and some of them have also been shown to localize to neuronal synapses. Our working hypothesis is therefore that Orb2 regulates synaptic translation. Here, we aim to elucidate the precise mechanism by which Orb2 contributes to long-term memory formation in Drosophila, examining this process at the molecular and cellular levels. To this end, we have established a system to rapidly modify the endogenous orb2 locus. This provides a means to introduce any desired modification to the endogenous Orb2 protein in order to study its function and properties in the relevant neurons in vivo. By attaching epitope tags, we will test whether Orb2 localizes to synapses, and if so whether it is pre- or post-synaptic. Photoconvertible GFP tags will be used in a similar manner to assess the dynamics of Orb2 localization. Other tags will be added to report any self-association of Orb2 proteins. We will also use this strategy to identify the molecular determinants, required for Orb2 function in long-term memory, and ask whether these features are unique to Orb2 or also shared by other members of the CPEB family. This will be assessed by introducing specific mutations in the various protein domains, as well as replacing them individually and collectively with the corresponding domains of other CPEB proteins. Importantly, all these modifications will be made in the endogenous orb2 locus to ensure correct spatial and temporal expression patterns. In each case, we will examine how these modifications effect the molecular and cellular properties of Orb2, as well as its role in long-term memory formation. In parallel, we will also use these reagents to identify other proteins that interact with Orb2 and potentially regulate its function in long-term memory. We will immunoprecipitate endogenously tagged Orb2 protein complexes from fly brain extracts and analyze their composition with mass spectroscopy. Identified candidates will then be tested for a role in courtship long-term memory by knock-down their function specifically in mushroom body neurons with transgenic RNAi. These approaches should provide valuable insight into the molecular and cellular mechanisms by which the CPEB protein Orb2 contributes to long-term memory formation in Drosophila.

Most behaviors can be modified through the process of learning and memory, allowing the individual to adapt its innate behavioral repertoire to the specific contingencies of the local environment. Depending on the duration, intensity and salience of the learning experience, memories can be either short or long lasting. These behavioral modifications are thought to reflect anatomical and functional changes at specific synapses. Long-term synaptic plasticity requires new protein synthesis both at the soma and locally at the synapse. Candidates for such a local protein synthesis regulator are members of the Cytoplasmic Polyadenylation Element Binding (CPEB) family. In Aplysia CPEB is thought to maintain long-term synaptic facilitation, a physiological correlate of the long-term memory due to its putative prion-like properties determined by the glutamine-rich region, the Q-domain. In Drosophila, we have previously shown that long-term memory is mediated by the Drosophila CPEB, Orb2, and is critically dependent on the Q-domain.To further understand the cellular and molecular contributions of Orb2 to learning and memory in Drosophila, we have conducted detailed genetic, biochemical, and behavioral analysis of the endogenous Orb2 protein to assess its role in a naturally occuring form of memory in Drosophila, called courtship conditioning. This is one of the most robust forms of learning in flies, where the males have to learn through experience to selectively court receptive virgin females in order to optimize their future reproductive success. We have found that the two Orb2 isoforms, Orb2A and Orb2B, both contribute to long-term memory formation, albeit by distinct mechanisms. The two isoforms share the same RNA-binding and Q-domains, yet each uniquely requires only one of these domains for its function in long-term memory formation. Specifically, the Q-domain is essential in Orb2A but not Orb2B, whereas the RNA-binding domain is required in Orb2B but not Orb2A. Moreover, we found that Orb2A lacking its RNA-binding domain is able to fully complement Orb2B lacking its Q-domain by formation of the protein complexes, which acutely depends on Orb2A and its Q-domain. Moreover, we have established that Orb2A and B have distinct functions in long-term memory. During memory acquisition Orb2A is activated in the specific synapses by dopamine signal to mark them as a potential site fort the memory storage. Activated Orb2A recruits Orb2B into complex, which upon late postacquisition dopamine signal regulates translation of the protein essential fort the long-term memory to last.