We will use the unique advantages of the lamprey nervous system to determine whether after spinal cord injury (SCI), local protein synthesis in the axon tip plays a role in the mechanism of axon regeneration. It has long been assumed that axon regeneration depends on protein synthesis in the cell body, the proteins then being transported to the growing axon tips. However, based mainly on observations in tissue culture and injured peripheral nerve, where ribosomes and mRNAs have been detected in axons, a role has been proposed for local protein synthesis in axon regeneration. Locally synthesized proteins might be used to form new axon constituents, or be transported retrogradely to the cell body to signal cell survival and growth-associated gene expression. Until now, evidence for local protein synthesis in the central nervous system (CNS) has been limited primarily to growth cones in early stages of neuronal development in vivo and in vitro. In mature mammalian CNS, where axons do not regenerate, the capacity for local translation is maintained primarily in the dendritic tree; the axons have minimal capacity to synthesize proteins. However, work in lamprey spinal cord, where axons do regenerate, suggests that mature CNS axons regenerate without growth cones, i.e., their tips lack filopodia, have relatively little F-actin, are packed with neurofilaments (NFs), and elongate much more slowly than developing axons. This raises the question whether local protein synthesis plays a role in regeneration of mature CNS axons, even though they do not have growth cones. We previously showed that after SCI in lamprey, mRNA and ribosomes accumulate in the axon tips, and that actively growing tips have more mRNA than static or retracting tips. Moreover, the mRNAs include abundant neurofilament transcripts, but much less actin transcripts, the opposite of findings in mammalian axons growing in vitro or in injured peripheral nerve. Now we will use RNAseq to analyze laser microdissected cytoplasm from large, identified reticulospinal neurons, and micro-aspirated axoplasm from their injured axon tips, to identify mRNAs associated with axon regeneration. We will determine how the mRNAs are regulated in the cell body and get to the tip by determining a) the role of the MAP kinase pathway in signaling axotomy and triggering regeneration, and b) the time course of mRNA appearance along the axon and in the growing tip. Then we will determine whether we can enhance regeneration by locally overexpressing some proteins with micro-injected mRNAs, and whether we can inhibit regeneration by a) inhibiting local protein synthesis with locally applied protein synthesis inhibitors, and b) locally applying morpholino antisense oligonucleotides. This study will help us determine where and how to target therapies to enhance axon regeneration and restore function after SCI.
Spinal cord injury causes permanent paralysis, partly because with postnatal development, motor command nerve cells that project their nerve fiber (axon) to the spinal cord lose the ability to manufacture enough of the correct proteins locally at the axon tip to allow the axon to regenerate. If we show that protein synthesis in the growing axon tip is part of the mechanism of regeneration, and if we can identify which proteins are involved, we would target therapy to the site of axons' injury in the spinal cord, rather than to their cell bodies in the brain. Surgeons often decompress the spinal cord at the site of injury anyway, but invasive molecular manipulations in the brain remote from the injury would add risk.
|Zhang, Guixin; Hu, Jianli; Rodemer, William et al. (2018) RhoA activation in axotomy-induced neuronal death. Exp Neurol 306:76-91|
|Hu, Jianli; Zhang, Guixin; Rodemer, William et al. (2017) The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury. Neurobiol Dis 98:25-35|