title: Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f (Sac2) as an Endogenous Suppressor of Repair after Spinal Cord Injury.
abstract: Axonal growth and neuronal rewiring facilitate functional recovery after spinal cord injury. Known interventions that promote neural repair remain limited in their functional efficacy. To understand genetic determinants of mammalian CNS axon regeneration, we completed an unbiased RNAi gene-silencing screen across most phosphatases in the genome. We identified one known and 17 previously unknown phosphatase suppressors of injury-induced CNS axon growth. Silencing Inpp5f (Sac2) leads to robust enhancement of axon regeneration and growth cone reformation. Results from cultured Inpp5f(-/-) neurons confirm lentiviral shRNA results from the screen. Consistent with the nonoverlapping substrate specificity between Inpp5f and PTEN, rapamycin does not block enhanced regeneration in Inpp5f(-/-) neurons, implicating mechanisms independent of the PI3K/AKT/mTOR pathway. Inpp5f(-/-) mice develop normally, but show enhanced anatomical and functional recovery after mid-thoracic dorsal hemisection injury. More serotonergic axons sprout and/or regenerate caudal to the lesion level, and greater numbers of corticospinal tract axons sprout rostral to the lesion. Functionally, Inpp5f-null mice exhibit enhanced recovery of motor functions in both open-field and rotarod tests. This study demonstrates the potential of an unbiased high-throughput functional screen to identify endogenous suppressors of CNS axon growth after injury, and reveals Inpp5f (Sac2) as a novel suppressor of CNS axon repair after spinal cord injury. Significance statement: The extent of axon regeneration is a critical determinant of neurological recovery from injury, and is extremely limited in the adult mammalian CNS. We describe an unbiased gene-silencing screen that uncovered novel molecules suppressing axonal regeneration. Inpp5f (Sac2) gene deletion promoted recovery from spinal cord injury with no side effects. The mechanism of action is distinct from another lipid phosphatase implicated in regeneration, PTEN. This opens new pathways for investigation in spinal cord injury research. Furthermore the screening methodology can be applied on a genome wide scale to discovery the entire set of mammalian genes contributing to axonal regeneration.
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Yixiao
authors.identifier: http://orcid.org/0000-0003-2471-6057
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Massimiliano
authors.identifier: http://orcid.org/0000-0002-5827-902X
authors.initials: M
authors.lastname: Stagi
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Xingxing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Kazim
authors.initials: K
authors.lastname: Yigitkanli
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Chad S
authors.identifier: http://orcid.org/0000-0002-6580-0477
authors.initials: CS
authors.lastname: Siegel
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: Fubito
authors.initials: F
authors.lastname: Nakatsu
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536.
authors.forename: William B J
authors.identifier: http://orcid.org/0000-0001-6263-2167
authors.initials: WB
authors.lastname: Cafferty
authors.affiliation: Cellular Neuroscience, Neurodegeneration and Repair Program, Interdepartmental Neuroscience Program, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06536 stephen.strittmatter@yale.edu.
authors.forename: Stephen M
authors.identifier: http://orcid.org/0000-0001-8188-3092
authors.initials: SM
authors.lastname: Strittmatter
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D007150:Immunohistochemistry; D000072181:Inositol Polyphosphate 5-Phosphatases; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009416:Nerve Regeneration / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D020127:Recovery of Function / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013119:Spinal Cord Injuries / Q000378:metabolism / Q000473:pathology*
chemical_list: D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases
keywords: axon regeneration; inositol phosphate; sac2; siRNA; spinal cord injury