Het ontrafelen van cel- en compartiment specifieke distributie van synaptische eiwitten in somatosensorische circuits

Neurons receive diverse inputs across their dendritic domains, and dendritic compartment-specific input localization is thought to be the foundation of sensory information integration. Growing evidence supports the idea that cell type-specific expression of cell-surface proteins (CSPs) regulates the specification of synaptic circuits and that mutations in CSPs are involved in neurodevelopmental disorders. In an attempt to gain insight into the molecular mechanisms that regulate precise cortical connectivity and sensory processing, I proposed to investigate synaptic protein expression and localization in cortical layer 5 (L5) neurons. L5 neurons are usually categorized in two main populations based on morphology, electrophysiological properties and projection targets. In the somatosensory cortex, these two populations segregate to L5a and L5b respectively, their dendrites occupy the same cortical layers and therefore overlap with the same axonal branches. Nevertheless, their proximal and distal dendritic compartments, receive mostly non-overlapping thalamic inputs. Recent single-cell sequencing data linked gene expression to cortical neuron population heterogeneity, but the molecular players involved in cortical neuron- and compartment specific synapse formation remain unknown. Studies in the hippocampus and retina showed how synaptic proteins are often expressed in a population-specific manner and, through localization at specific dendritic compartments, instruct the formation and function of specific inputs. A similar mechanism is likely involved in the formation of specific synapses in the cortex, and might explain how precise cortical connectivity is achieved in a highly crowded environment. Many studies have relied on proteomics to characterize synaptic protein. However, it has proven difficult to achieve cell-specificity and high spatial resolution in vivo with these approaches. Proximity biotinylation takes advantage of promiscuous biotin-ligases' ability to covalently bind a biotin molecule to proteins in a short-radius around them. However, conventional biotin-ligases are either not efficient to analyze small populations of neurons (BioID) or not compatible with in vivo conditions (APEX2). Because of these limitations, it has been extremely challenging to study cortical neuron-specific synaptic protein composition and localization in vivo. In my PhD, I optimized a cell type-specific proximity biotinylation-based workflow using the engineered biotin ligase turboID to achieve subcellular proteomic resolution in genetically identified cell types in intact cortical circuits. I applied this approach to purify and characterize the excitatory postsynaptic proteomes of cortical L5 neurons at the cell-type and compartment-specific levels. I found that the molecular synaptic signatures of L5 IT and PT neurons are characterized by differential expression of ion channels, neurotransmitter receptors and cell-surface proteins, suggesting that these proteins contribute to the specification of the functional properties and input connectivity patterns of the two L5 neuron types.