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

Abstract

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system 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 immunoblot 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 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. 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 2-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and is a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage

“…In addition, RARβ activation leads to the synthesis of RA in NG2-glia and subsequent exosome-associated further RA release [ 193 , 194 ]. At the same time myelin-associated growth inhibitors are released through exosomes from oligodendrocytes [ 195 ]. Therefore, active production of exosomes in the injuries areas of CNS may have a strong effect on CNS neuroplasticity, including collateral sprouting and unmasking of latent synapses [ 196 , 197 ].…”

The Dichotomous Role of Extracellular Vesicles in the Central Nervous System

“…Recently, a fragment of Nogo-A and its receptor NgR1, which play key roles in neurite outgrowth and branching, were found on exosomal membranes. Furthermore, these exosomes inhibited axonal regeneration of mechanically injured cortical neurons from mice ( Sekine et al., 2020 ). EV-based communication between PC12 and MCG6 microglial cells can trigger rapid phagocytosis of degenerating neurites ( Bahrini et al., 2015 ).…”

Fasudil reduces β-amyloid levels and neuronal apoptosis in APP/PS1 transgenic mice via inhibition of the Nogo-A/NgR/RhoA signaling axis

“…Nogo-A is a well-known myelin-associated protein that inhibits axonal regeneration. Nogo-A was the earliest identified neurite outgrowth inhibitor, which plays a significant role in developing the central nervous system (Chen et al, 2000;Pernet and Schwab, 2012;Sekine et al, 2020). Nogo-A binds to the Nogo-66 receptor (NgR), which forms a complex with the p75 neurotrophin receptor (p75NTR) and a BK channel regulator (LINGO1) to transduce intracellular signals.…”