An overarching, central question in all of neuroscience is the specificity, modification, and function of the immense diversity of function-specific circuitry? a question still inaccessible in multiple core aspects. This is what underlies how the brain-nervous system senses, integrates, moves the body, thinks, functions with precision, malfunctions with specificity in disease, degenerates with circuit specificity, might be regenerated, and/or might be modeled in culture. What actually implements and maintains circuit specificity is a key, core issue from developmental specificity of circuits, to developmental abnormalities, to proper function (or dysfunction) and circuit type-specific molecular regulators, to subtype-specific degeneration (e.g. in ALS, Huntington's, Parkinson's diseases), to regeneration (or typical lack thereof) in the CNS for spinal cord injury or with optimal accuracy in the PNS, to mechanistic and therapeutic modeling of disease using iPS/ES-derived neurons. Growth cones (GCs) ?build? circuits and mature into synapses, where human genomic risk associations are showing up in neuropsychiatric diseases such as schizophrenia, autism, bipolar disorder, developmental intellectual disabilities. I propose uniquely enabling, pioneering work on these issues? now possible by our innovative approaches. We are now able to directly investigate molecular machinery of distinct GC subtypes, thus distinct circuits. Despite their importance, we know little about the diversity and specialization of circuit-specific GCs? the subcellular molecular machines that implement specific circuit wiring, mature with precision into presynaptic halves of immensely diverse synapses, and control the long-standing ?sorting problem?. GCs perform these functions over many days of development for each pathway, often 103-105 cell body diameters away from the nucleus and transcriptional control. Remarkably, but rarely considered, one nucleus, with one transcriptional regulatory machinery, can control 2 or more divergent GCs to wire multi-target circuitry. I propose entirely new, highly innovative, pioneering work in development, cell biology, disease, and regeneration (also relevant to modeling) to uniquely address this critical gap in knowledge. We developed new approaches to investigate subtype- and stage-specific GCs directly from brains, with high-depth, quantitative proteomic and RNA analysis, and have already completed proof-of-concept experiments enabling a range of pioneering new work. We selectively purify GCs based on neuron subtype, projection trajectory, and developmental stage using a combination of molecular, anatomic, and genetic labeling strategies; subcellular biochemistry; newly developed small-particle sorting; peptide mass spectrometry; and Next Gen sequencing. Simultaneous isolation of protein and RNA from parent somas and their GCs identifies hundreds of proteins and transcripts enriched orders of magnitude in GCs, essentially not detected in parent somas. This indicates that investigation of GCs might actually be required to understand subtype-specific circuitry. GCs appear to be ?programmed? early, then ?poised? to exert quite autonomous local control. I propose ambitious and venturesome investigations of subtype-, stage-, and target-specific GC proteins and RNAs in multiple specific settings to study mechanisms of development, cell biology, disease, regeneration, & iPS/ES models. These directions range from immediate, to ~5 yrs, to an ~10 yr horizon. Results will generate new hypotheses and investigations.
/ Relevance: An overarching, central question in all of neuroscience is the specificity, modification, and function of the immense diversity of function-specific circuitry? a question still inaccessible in multiple core aspects; this is what underlies how the brain-nervous system senses, integrates, moves the body, thinks, functions with precision, malfunctions with specificity in disease, degenerates with circuit specificity, might be regenerated, and/or might be modeled in culture. What actually implements and maintains circuit specificity is a key, core issue from developmental specificity of circuits, to developmental abnormalities and disease, to proper function (or dysfunction) and circuit type-specific molecular regulators and drugs, to subtype-specific degeneration (e.g. in ALS, Huntington's, Parkinson's diseases), to regeneration (or typical lack thereof) in the CNS for spinal cord injury or with optimal accuracy in peripheral nerves, to mechanistic and therapeutic modeling of disease using iPS/ES-derived neurons; growth cones (GCs) are the subcellular structures that ?build? circuits with specificity and mature into synapses, where human genomic risk associations are showing up in neuropsychiatric diseases such as schizophrenia, autism, bipolar disorder, developmental intellectual disabilities, but we know little about the diversity and specialization of circuit-specific GCs. I propose entirely new, highly innovative, pioneering work to deeply investigate subtype-, stage-, and target-specific GCs in development, cell biology, disease, and regeneration (also relevant to modeling) to uniquely address this critical gap in knowledge; we developed new approaches to investigate subtype- and stage-specific GCs directly from the brain, with high-depth, quantitative protein and RNA analysis, and have already completed proof-of-concept experiments identifying that hundreds of special proteins and RNAs are specifically enriched up to hundreds-fold in GCs, indicating that investigation of GCs might actually be required to understand subtype-specific circuitry, and enabling a range of pioneering new work.
