A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration [Cell Biology]

November 20th, 2019 by Yuichi Sekine, Jane A. Lindborg, Stephen M. Strittmatter

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system (CNS) trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblotting assays, transmission EM of exosomes, and axon-regeneration assays, we explored the secretion and activity of the myelin-associated, neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa, C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961–971. We also detected a Nogo-66 receptor (NgR1)-interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for the Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. The production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that relative to the exosomal marker Alix, a Nogo-immunoreactive 24-kDa protein is enriched in exosomes two-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and represents a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.