Searching for the engram at the proteome level
Memories of events long past bear on our present motivations and behaviour. Malfunctioning of such long-term mnemonic function has significant clinical consequences. In project B15N, we deal with the ‘where’ and ‘what’ of long-term memory. We aim (i) at localizing a long-term associative engram by visualizing de novo protein synthesis → ‘where’ and (ii) at characterizing this engram’s molecular content by identifying the newly synthesized proteins → ‘what’. Requirement for de novo protein synthesis is a characteristic of long-term memory. The respective proteins should be critical for both memory consolidation and the long-lasting storage. Notwithstanding their importance, a comprehensive, cell-specific inventory of these proteins so far does not exist in any organism. To fill this gap, uniquely in Drosophila, we can combine a well-established long-term memory paradigm with in vivo, cell-specific metabolic protein labeling using click chemistry. Converging evidence point to the mushroom body Kenyon cells as the site of coincidence detection for olfactory associative learning in the fly. Individual Kenyon cell-afferent and -efferent neurons have been identified for reinforcement signaling and memory retrieval, respectively. We will metabolically label proteins that are acutely synthesized in Kenyon cells and their critical post-synaptic partners upon olfactory learning. These proteins will be (i) visualized using fluorescent non-canonical amino acid tagging (FUNCAT) to monitor a ‘systems consolidation’-like process, including its temporal dynamics as reflected in critical time-windows as well as global protein synthesis and turnover rates. The proteins made de novo upon learning will also be (ii) purified using bioorthogonal non-canonical amino acid tagging (BONCAT) and “inventoried” by mass spectrometry. As a side-track, we will apply these two approaches also to the glial cells as these critically contribute to neuronal function, including long-term memory. Finally, we will (iii) validate the emerging candidate proteins in terms of their causal role in long-term memory using reverse genetics. Given that molecular mechanisms of learning and memory are well-conserved across phyla, we can reasonably hope for translational value of our results.