ABSTRACT
Growth cones at the tips of extending axons navigate through developing organisms by probing extracellular cues, which guide them through intermediate steps and onto final synaptic target sites. Widespread focus on a few guidance cue families has historically overshadowed potentially crucial roles of less well-studied growth factors in axon guidance. In fact, recent evidence suggests that a variety of growth factors have the ability to guide axons, affecting the targeting and morphogenesis of growth cones in vitro. This review summarizes in vitro experiments identifying responses and signaling mechanisms underlying axon morphogenesis caused by underappreciated growth factors.
ABSTRACT
Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.
Subject(s)
Axon Guidance/genetics , Induced Pluripotent Stem Cells/metabolism , Nerve Net/metabolism , Neurogenesis/genetics , Neurons/metabolism , Tuberous Sclerosis Complex 2 Protein/metabolism , Tuberous Sclerosis/metabolism , rhoA GTP-Binding Protein/metabolism , Adolescent , Axon Guidance/drug effects , Biopsy , CRISPR-Cas Systems , Cell Line , Ephrins/pharmacology , Fluorescence Resonance Energy Transfer , Haploinsufficiency , Heterozygote , Humans , Male , Myosins/metabolism , Nerve Net/pathology , Neurogenesis/drug effects , Neurons/drug effects , Organoids/cytology , Organoids/metabolism , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis/genetics , Tuberous Sclerosis Complex 2 Protein/geneticsABSTRACT
The assembly of neuronal circuits during development depends on guidance of axonal growth cones by molecular cues deposited in their environment. While a number of families of axon guidance molecules have been identified and reviewed, important and diverse activities of traditional growth factors are emerging. Besides clear and well recognized roles in the regulation of cell division, differentiation and survival, new research shows later phase roles for a number of growth factors in promoting neuronal migration, axon guidance and synapse formation throughout the nervous system.
Subject(s)
Axon Guidance , Growth Cones , Axons , Nervous System , Neural Networks, Computer , NeuronsABSTRACT
The microenvironment of developing neurons is a dynamic landscape of both chemical and mechanical cues that regulate cell proliferation, differentiation, migration, and axon extension. While the regulatory roles of chemical ligands in neuronal morphogenesis have been described, little is known about how mechanical forces influence neurite development. Here, we tested how substratum elasticity regulates neurite development of human forebrain (hFB) neurons and human motor neurons (hMNs), two populations of neurons that naturally extend axons into distinct elastic environments. Using polyacrylamide and collagen hydrogels of varying compliance, we find that hMNs preferred rigid conditions that approximate the elasticity of muscle, whereas hFB neurons preferred softer conditions that approximate brain tissue elasticity. More stable leading-edge protrusions, increased peripheral adhesions, and elevated RHOA signaling of hMN growth cones contributed to faster neurite outgrowth on rigid substrata. Our data suggest that RHOA balances contractile and adhesive forces in response to substratum elasticity.
Subject(s)
Neurogenesis , Neurons/metabolism , Signal Transduction , rhoA GTP-Binding Protein/metabolism , Axons/metabolism , Cell Culture Techniques , Cells, Cultured , Cerebral Cortex/cytology , Fluorescent Antibody Technique , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mechanotransduction, Cellular , Myosin Type II/metabolism , Nerve Regeneration , Neuronal Outgrowth , Neurons/cytology , Organ Specificity , PhosphorylationABSTRACT
Local protein synthesis directs growth cone turning of nascent axons, but mechanisms governing this process within compact, largely autonomous microenvironments remain poorly understood. In this issue, Wang et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201510107) demonstrate that the calcineurin regulator Down syndrome critical region 1 protein modulates both basal neurite outgrowth and growth cone turning.
