Cortical wiring by synapse type–specific control of local protein synthesis

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type–specific and synapse type–specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system. Description Synapse-specific translational control Neurons, with their far-flung branches, cede some translational control to their periphery. In the adult brain, local protein synthesis occurs at synapses. Bernard et al. now show that decentralized control of protein synthesis also affects how developing neurons build synapses and circuits. Excitatory synapses forming on interneurons in the developing mouse cerebral cortex depend on messenger RNA translation that is responsive to which type of cell and which type of synapse is involved. —PJH Local protein synthesis is regulated within connectivity motifs to control synapse formation in the mouse nervous system. INTRODUCTION The function of the cerebral cortex relies on the specificity of synaptic connections among dozens of different types of excitatory glutamatergic pyramidal cells and inhibitory γ-aminobutyric acid-containing (GABAergic) interneurons. Neurons use dedicated transcriptional programs to control synapse specificity during development. Still, it is presently unclear whether regulation of mRNA translation is also involved in promoting the formation of specific synapses. One of the main signaling pathways controlling protein synthesis involves the mechanistic target of rapamycin complex 1 (mTORC1), a molecular complex activated by growth factor signals and inhibited by the proteins Tsc1 and Tsc2 (TSC subunits 1 and 2, respectively). Multiple proteins of the mTORC1 pathway are present in developing axons, and local protein synthesis occurs at excitatory and inhibitory synapses in the adult cerebral cortex. Many genes whose variation has been linked to neurodevelopmental disorders code proteins found at synapses and altered protein synthesis is a plausible pathophysiological mechanism for several of these conditions. For example, mutations in TSC1 and TSC2 cause tuberous sclerosis, a syndrome whose clinical features include seizures and autism spectrum disorder (ASD). RATIONALE Local translation contributes to brain wiring during axon guidance but its direct contribution to synapse formation is unclear. Moreover, to what extent local translation is differentially regulated at the level of specific synaptic connections remains unknown. We used mouse genetics, RNA sequencing, biochemistry, and experimental manipulations in vivo to study local protein synthesis in the development of synaptic connections in the cerebral cortex. We investigated the formation of excitatory synaptic connections between cortical pyramidal cells and two of the main subclasses of cortical interneurons, parvalbumin (PV)- and somatostatin (SST)-expressing cells. These interneurons share similar developmental trajectories, occupy the same layers of the neocortex, and are reciprocally connected with pyramidal cells, but they play very different roles in cortical function. RESULTS We found that loss of Tsc2 during development causes overactivation of mTORC1 in both PV+ and SST+ interneurons, leading to abnormal activation of mTORC1 effector protein S6rp and increased cell size. However, Tsc2 function seems required to establish a normal complement of excitatory synapses only onto PV+ and not SST+ interneurons. Tsc2 also seems dispensable for developing inhibitory PV+ synapses onto pyramidal cells. Thus, although the loss of Tsc2 causes a global disruption of mTOR signaling in both PV+ and SST+ cells, it affects the wiring of interneurons in a cell type–specific and synapse type–specific manner. Our experiments indicate that this specificity is mediated by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which is specifically expressed by PV+ interneurons and is present at postsynaptic densities contacted by excitatory axons. ErbB4 signaling inhibits Tsc2 function to enable mTOR activity at the postsynaptic densities of excitatory inputs contacting PV+ interneurons. ErbB4 activation ultimately leads to the synaptic translation of mRNAs coding cell adhesion molecules (SynCAM1, Nlgn3, TrkC, and Clstn2) and AMPA receptor-related proteins (GluA4 and Stargazin). Loss of function experiments revealed that these molecules are required to develop a normal complement of excitatory synapses on PV+ interneurons. Although a direct link between ErbB4 activation and local protein synthesis has only been established for a few mRNAs, analysis of the synaptic translatome of PV+ interneurons lacking ErbB4 suggests that local translation of more transcripts—including many for which variation has been associated with ASD—might be under the control of this signaling pathway during synapse formation. CONCLUSION Our findings reveal that protein synthesis is regulated in a cell type–specific and synapse type–specific manner during synapse formation. Tsc2, a regulator of mTORC1 signaling in multiple cellular contexts, regulates the development of excitatory synapses onto PV+ cells but not onto SST+ interneurons. This specificity is mediated by activation of ErbB4, which controls excitatory synapse development through inhibition of Tsc2 and subsequent induction of a molecular program of local mRNA translation. Thus, local protein synthesis is regulated at the level of specific connections to control synapse formation in the nervous system. Local protein synthesis in synapse formation. Schematic of a pyramidal cell contacting parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons in the cerebral cortex. The development of excitatory synapses on PV+ interneurons is mediated by (1) activation of ErbB4 through binding to neuregulin 3 (Nrg3) expressed by pyramidal cells, (2) inhibition of Tsc2 through Akt-dependent phosphorylation cascade, (3) inhibition of mTORC1 through Rheb-dependent phosphorylation cascade, (4) activation of protein synthesis of ErbB4 targets at the synapse, and (5) synapse formation through ErbB4 targets.