In vivo single branch axotomy induces GAP-43–dependent sprouting and synaptic remodeling in cerebellar cortex

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

[1]  F. Helmchen,et al.  Resting Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma in Vivo , 2005, Science.

[2]  P. Strata,et al.  Impaired Sprouting and Axonal Atrophy in Cerebellar Climbing Fibres following In Vivo Silencing of the Growth-Associated Protein GAP-43 , 2011, PloS one.

[3]  K. Svoboda,et al.  Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience , 2006, Neuron.

[4]  Michael P. Coleman,et al.  Neuronal death: where does the end begin? , 2007, Trends in Neurosciences.

[5]  F. Gage,et al.  Regenerating the damaged central nervous system , 2000, Nature.

[6]  John T. Finn,et al.  Axonal Self-Destruction and Neurodegeneration , 2002, Science.

[7]  F. Rossi,et al.  Degenerative phenomena and reactive modifications of the adult rat inferior olivary neurons following axotomy and disconnection from their targets , 1998, Neuroscience.

[8]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[9]  J. S. McCasland,et al.  Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Strata,et al.  Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. I. Development of new collateral branches and terminal plexuses , 1991, The Journal of comparative neurology.

[11]  F. Barkhof,et al.  Growth‐associated protein 43 in lesions and cerebrospinal fluid in multiple sclerosis , 2006, Neuropathology and applied neurobiology.

[12]  J. Fetcho,et al.  Cyclic AMP-Induced Repair of Zebrafish Spinal Circuits , 2004, Science.

[13]  P. Strata,et al.  Reparative mechanisms in the cerebellar cortex , 2004, Progress in Neurobiology.

[14]  B. Kundu,et al.  The Unusual Response of Serotonergic Neurons after CNS Injury: Lack of Axonal Dieback and Enhanced Sprouting within the Inhibitory Environment of the Glial Scar , 2011, The Journal of Neuroscience.

[15]  M. Kerschensteiner,et al.  Neuroimaging: In vivo imaging of the diseased nervous system , 2006, Nature Reviews Neuroscience.

[16]  J. Kapfhammer,et al.  Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice , 1995, Cell.

[17]  A. Routtenberg,et al.  Can GAP-43 interact with brain spectrin? , 1999, Brain research. Molecular brain research.

[18]  Y Shinoda,et al.  Morphology of single olivocerebellar axons labeled with biotinylated dextran amine in the rat , 1999, The Journal of comparative neurology.

[19]  L. Dekker,et al.  Inhibition of noradrenaline release by antibodies to B-50 (GAP-43) , 1989, Nature.

[20]  C. Bendotti,et al.  Distribution of GAP‐43 mRNA in the adult rat brain , 1993, The Journal of comparative neurology.

[21]  V. De Paola,et al.  Synaptic Elimination and Protection after Minimal Injury Depend on Cell Type and Their Prelesion Structural Dynamics in the Adult Cerebral Cortex , 2013, The Journal of Neuroscience.

[22]  W. Gispen,et al.  Temporal Differences in the Phosphorylation State of Pre- and Postsynaptic Protein Kinase C Substrates B-50/GAP-43 and Neurogranin during Long Term Potentiation (*) , 1995, The Journal of Biological Chemistry.

[23]  A. Holtmaat,et al.  Growth-associated protein GAP-43 and L1 act synergistically to promote regenerative growth of Purkinje cell axons in vivo. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Strata,et al.  Learning-related feedforward inhibitory connectivity growth required for memory precision , 2011, Nature.

[25]  M. Fishman,et al.  Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43 , 1995, Cell.

[26]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[27]  R. Lenox,et al.  Differential Changes in the Phosphorylation of the Protein Kinase C Substrates Myristoylated Alanine‐Rich C Kinase Substrate and Growth‐Associated Protein‐43/B‐50 Following Schaffer Collateral Long‐Term Potentiation and Long‐Term Depression , 1999, Journal of neurochemistry.

[28]  A. Holtmaat,et al.  Targeted Overexpression of the Neurite Growth-Associated Protein B-50/GAP-43 in Cerebellar Purkinje Cells Induces Sprouting after Axotomy But Not Axon Regeneration into Growth-Permissive Transplants , 1997, The Journal of Neuroscience.

[29]  K. Svoboda,et al.  Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window , 2009, Nature Protocols.

[30]  W. Gispen,et al.  B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system , 1997, Progress in Neurobiology.

[31]  Masahiko Watanabe,et al.  Axonal Motility and Its Modulation by Activity Are Branch-Type Specific in the Intact Adult Cerebellum , 2007, Neuron.

[32]  K. Pratt,et al.  The Neuronal Growth-Associated Protein GAP-43 Interacts with Rabaptin-5 and Participates in Endocytosis , 1998, The Journal of Neuroscience.

[33]  W. Snider,et al.  Signaling the Pathway to Regeneration , 2002, Neuron.

[34]  Oswald Steward,et al.  False resurrections: Distinguishing regenerated from spared axons in the injured central nervous system , 2003, The Journal of comparative neurology.

[35]  W. Gan,et al.  ATP mediates rapid microglial response to local brain injury in vivo , 2005, Nature Neuroscience.

[36]  G. Knott,et al.  In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits , 2013, Nature Communications.

[37]  E. Masliah,et al.  Axonopathy and Transport Deficits Early in the Pathogenesis of Alzheimer's Disease , 2005, Science.

[38]  Francesco S. Pavone,et al.  Neuroenergetics Review Article , 2022 .

[39]  Fred A. Hamprecht,et al.  Correlative In Vivo 2 Photon and Focused Ion Beam Scanning Electron Microscopy of Cortical Neurons , 2013, PloS one.

[40]  M. Mosevitsky.  Nerve ending "signal" proteins GAP-43, MARCKS, and BASP1. , 2005, International review of cytology.

[41]  R. Corradetti,et al.  Phosphorylation of the presynaptic protein B-50 (GAP-43) is increased during electrically induced long-term potentiation , 1992, Neuron.

[42]  Y. Misumi,et al.  Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex. , 1997, The Biochemical journal.

[43]  O. Steward,et al.  Concepts and Methods for the Study of Axonal Regeneration in the CNS , 2012, Neuron.

[44]  S. Strittmatter,et al.  Axonal growth therapeutics: regeneration or sprouting or plasticity? , 2008, Trends in Neurosciences.

[45]  P. Strata,et al.  Structural plasticity of climbing fibers and the growth-associated protein GAP-43 , 2013, Front. Neural Circuits.

[46]  Jeff W Lichtman,et al.  In vivo imaging of axonal degeneration and regeneration in the injured spinal cord , 2005, Nature Medicine.

[47]  Leonardo Sacconi,et al.  In vivo multiphoton nanosurgery on cortical neurons. , 2007, Journal of biomedical optics.

[48]  Johannes E. Schindelin,et al.  TrakEM2 Software for Neural Circuit Reconstruction , 2012, PloS one.

[49]  D. Buonomano,et al.  Cortical plasticity: from synapses to maps. , 1998, Annual review of neuroscience.