Following spinal cord transection in the sea lamprey, axons regenerate selectively in their correct paths. Several molecules belonging to the semaphorin/collapsin and netrin families are thought to help guide axons during embryonic development and may also play a role in guiding regeneration in the injured, mature CNS. In previous studies, expression of netrin and semaphorin receptors occurred only in bad-regenerating neurons. Thus these guidance molecules may act as chemorepellents preventing axons from regenerating to their targets in the spinal cord.
Aim 1. We will determine whether inhibiting expression of netrin and semaphorin receptors will enhance the regenerative capacities of identified reticulospinal neurons. Expression in these neurons will be inhibited by retrograde transport of fluoroscein-tagged antisense morpholino oligonucleotides injected at the site of spinal cord transection. The effect on distance and direction of regeneration will be determined by intra-axonal injection of tracer and compared with those in control animals injected with random oligonucleotides.
Aim 2. Because chemorepulsion can be converted to attraction by manipulation of the cyclic guanosine-monophosphate(cGMP) and cyclic adenosine monophosphate (cAMP) signaling pathways, we will determine the effect of altering intracellular cAMP and/or cGMP concentrations by extracellular injections of membrane-permeable analogs on the regenerative capacities and/or pathway specificities of identified reticulo-spinal neurons.
Aim 3. We will continue our search for axonal guidance molecules that are expessed preferentially in good-regenerating neurons. We will determine whether ephrins and their receptors Eph, the new semaphorin receptor CD72, intergins, and RTPR guide regenerating axons in their correct paths. Using primers from partial sequences already cloned and degenerate primers for others, we will clone by RTPCR the lamprey homologs of CD 72, ephrins, and their corresponding receptors, and determine the anatomical distribution of their mRNAs by semiquantitative in situ hybridization. The effect of spinal transection on their expression will be correlated in identified reticulospinal neurons with their regenerative capacities. By elucidating the mechanisms of regeneration in CNS axons, these experiments may lead to improved therapeutic approaches to inducing regeneration and functional recovery in human patients following spinal cord injury, head trauma or stroke.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
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Kleitman, Naomi
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University of Pennsylvania
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Jin, Li-Qing; Pennise, Cynthia R; Rodemer, William et al. (2016) Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey. J Comp Neurol 524:3614-3640
Zhang, Guixin; Jin, Li-qing; Hu, Jianli et al. (2015) Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons. PLoS One 10:e0137670
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Zhang, Guixin; Vidal Pizarro, Ivonne; Swain, Gary P et al. (2014) Neurogenesis in the lamprey central nervous system following spinal cord transection. J Comp Neurol 522:1316-32
Hu, Jianli; Zhang, Guixin; Selzer, Michael E (2013) Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains. J Neurosci Methods 220:92-8
Laramore, C; Maymind, E; Shifman, M I (2011) Expression of neurotrophin and its tropomyosin-related kinase receptors (Trks) during axonal regeneration following spinal cord injury in larval lamprey. Neuroscience 183:265-77
Jin, Li-Qing; Zhang, Guixin; Pennicooke, Brenton et al. (2011) Multiple neurofilament subunits are present in lamprey CNS. Brain Res 1370:16-33
Barreiro-Iglesias, A; Laramore, C; Shifman, M I et al. (2010) The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain. Neuroscience 168:659-69
Jin, Li-Qing; Zhang, Guixin; Jamison Jr, Curtis et al. (2009) Axon regeneration in the absence of growth cones: acceleration by cyclic AMP. J Comp Neurol 515:295-312
Shifman, Michael I; Yumul, Rae Eden; Laramore, Cindy et al. (2009) Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey. Exp Neurol 217:242-51

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