Subtype- and context-dependent subcellular mRNA regulation in axons and growth cones of neocortical projection neurons

Abstract

Long range projection neurons of the cerebral cortex are crucial for connectivity between distant areas of the brain and are important for sensory integration, motor processing, and higher cognitive function. During development, distinct subtypes of cortical projection neurons send their axons to vastly different areas; this process of axonal guidance is implemented by the leading tip of the axon, the growth cone (GC). Prior work has identified local protein synthesis of axonally-trafficked transcripts to be required for directional responses to at least some guidance cues; however, how axons and growth cones establish, maintain, and regulate their local, subtype-specific molecular machinery over the course of development is essentially unknown. Here, I collaboratively first investigate subcellular transcriptomes of two subtypes of cortical projection neurons with distinct connectivity and function. I find that GC-localized transcriptomes of two distinct subtypes of projection neurons (PN), callosal (CPN) and corticothalamic (CThPN) are only ~60% overlapping and are enriched for genes associated with neurodevelopmental diseases. I compare the subtype-specific portion of the GC-localized transcriptomes and identify known and novel potential regulators of distinct phases of circuit development: growth, gray matter innervation, and synapse formation. Secondly, I find evidence for GC-enriched motifs in 3’UTRs, suggesting initial steps of a mechanism involved in the dynamic regulation of transcript localization and stability, as circuitry is established. Specifically, I identify the cytoplasmic adenylation element binding protein 4 (Cpeb4) as potentially regulating localization and translation of mRNAs encoding molecular machinery important for axonal branching and complexity. Lastly, I identify dynamic changes in GC-localized transcriptomes as CPN transition through different developmental stages to generate circuitry. I identify RNA Binding Motif Single Stranded Interacting Protein 1 (Rbms1) as potential key regulator of RNA stabilization that enables successful CPN circuitry formation. I next investigate the dynamics and stability of mRNAs in CPN at the stage of initial gray matter innervation. I adapt tools for neuron subtype-specific metabolic labeling (4-thiouracil with SLAM-sequencing) to assay temporal dynamics of CPN mRNA turnover in vivo. I find that RNA turnover in neuronal cell bodies (somata) is highly associated with features in the 3’UTR, including long AU- and U-repeats and a propensity to form secondary structure. I detect anterogradely transported mRNA in axons via neuron subtype-specific metabolic labeling in vivo, and find that mRNAs transported with distinct kinetics are associated with distinct gene classes and 3’UTR sequence features. Further, I find that the most rapidly transported mRNAs are enriched for AU-rich motifs in their 3’UTRs, suggesting that the fast turnover in somata is in substantial part due to rapid transport out of the soma, rather than mRNA decay. In summary, using a suite of approaches in my dissertation research, I identify cortical projection neuron subtype-specific and dynamically regulated subcellular molecular processes regulating axonal growth cone circuit formation, and likely later circuit maintenance and function once GCs mature into presynaptic elements. These results enable a substantially new conceptual and mechanistic understanding of development, maintenance, and function of diverse connectivity and circuitry in the brain and will likely provide new insights into potential etiologies and potential therapeutics for neurodevelopmental and neuropsychiatric disorders.