title: An in vitro assay to study induction of the regenerative state in sensory neurons.
abstract: After injury, peripheral neurons activate a pro-regenerative program that facilitates axon regeneration. While many regeneration-associated genes have been identified, the mechanism by which injury activates this program is less well understood. Furthermore, identifying pharmacological methods to induce a pro-regenerative state could lead to novel treatments to repair the injured nervous system. Therefore, we have developed an in vitro assay to study induction of the pro-regenerative state following injury or pharmacological treatment. First, we took advantage of the observation that dissociating and culturing sensory neurons from dorsal root ganglia activates a pro-regenerative program. We show that cultured neurons activate transcription factors and upregulate regeneration-associated genes common to the pro-regenerative program within the first hours after dissection. In a paradigm similar to pre-conditioning, neurons injured by dissociation display enhanced neurite outgrowth when replated as early as 12h after being removed from the animal. Furthermore, stimulation of the pro-regenerative state improves growth on inhibitory substrates and requires DLK/JNK signaling, both hallmarks of the pro-regeneration response in vivo. Finally, we modified this assay in order to identify new methods to activate the pro-regenerative state in an effort to mimic the pre-conditioning effect. We report that after several days in culture, neurons down-regulate many molecular hallmarks of injury and no longer display enhanced neurite outgrowth after replating. Hence, these neurons are functionally naïve and are a useful tool for identifying methods to induce the pro-regenerative state. We show that both injury and pre-treatment with forskolin reactivate the pro-regenerative state in this paradigm. Hence, this assay is useful for identifying pharmacological agents that induce the pro-regenerative state in the absence of injury.
authors.affiliation: Department of Developmental Biology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: erin.frey@wustl.edu.
authors.forename: E
authors.initials: E
authors.lastname: Frey
authors.affiliation: Department of Developmental Biology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: vvalakh@wustl.edu.
authors.forename: V
authors.initials: V
authors.lastname: Valakh
authors.affiliation: Department of Developmental Biology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: karney-grobe@wustl.edu.
authors.forename: S
authors.initials: S
authors.lastname: Karney-Grobe
authors.affiliation: Department of Developmental Biology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: popstone07@gmail.com.
authors.forename: Y
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Genetics, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: jmilbrandt@wustl.edu.
authors.forename: J
authors.initials: J
authors.lastname: Milbrandt
authors.affiliation: Department of Developmental Biology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: diantonio@wustl.edu.
authors.forename: A
authors.initials: A
authors.lastname: DiAntonio
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000502:physiology*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D009416:Nerve Regeneration / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D011984:Sensory Receptor Cells / Q000502:physiology*
keywords: Axon regeneration; Dorsal root ganglion (DRG); Dual leucine zipper kinase (DLK); Replating assay; Superior cervical ganglion 10 (SCG10); cJun N-terminal kinase (JNK)