C‐type synaptic boutons (C‐boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)–related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C‐boutons, including M2 muscarinic receptors, potassium channels, and σ‐1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C‐boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. C‐bouton–mediated regulation of MN excitability has been implicated in MN disease, but NRG1‐mediated functions and the impact of various pathologic conditions on C‐bouton integrity have not been studied in detail. Here, we investigated changes in C‐boutons after electrical stimulation, pharmacological treatment, and peripheral nerve axotomy. SSC‐linked NRG1 clusters were severely disrupted in acutely stressed MNs and after tunicamycin‐induced ER stress. In axotomized MNs, C‐bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal. Activated microglia displayed a positive chemotaxis to C‐boutons. Analysis of transgenic mice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC‐like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals. Moreover, MN‐derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C‐boutons in MN injury and suggest that distinct NRG1 isoform–mediated signaling functions regulate the complex matching between pre‐ and postsynaptic C‐bouton elements.—Salvany, S., Casanovas, A., Tarabal, O., Piedrafita, L., Hernández, S., Santafé, M., Soto‐Bernardini, M. C., Calderó, J., Schwab, M. H., Esquerda, J. E. Localization and dynamic changes of neuregulin‐1 at C‐type synaptic boutons in association with motor neuron injury and repair. FASEB J. 33, 7833–7851 (2019). http://www.fasebj.org

The electric activity of lower motor neurons (MNs) appears to play a role in determining cell-vulnerability in MN diseases. MN excitability is modulated by cholinergic inputs through C-type synaptic boutons, which display an endoplasmic reticulum-related subsurface cistern (SSC) adjacent to the postsynaptic membrane. Besides cholinergic molecules, a constellation of proteins involved in different signal-transduction pathways are clustered at C-type synaptic sites (M2 muscarinic receptors, Kv2.1 potassium channels, Ca2+ activated K+ [SK] channels, and sigma-1 receptors [S1R]), but their collective functional significance so far remains unknown. We have previously suggested that neuregulin-1 (NRG1)/ErbBs-based retrograde signalling occurs at this synapse. To better understand signalling through C-boutons, we performed an analysis of the distribution of C-bouton-associated signalling proteins. We show that within SSC, S1R, Kv2.1 and NRG1 are clustered in highly specific, non-overlapping, microdomains, whereas ErbB2 and ErbB4 are present in the adjacent presynaptic compartment. This organization may define highly ordered and spatially restricted sites for different signal-transduction pathways. SSC associated proteins are disrupted in axotomised MNs together with the activation of microglia, which display a positive chemotactism to C-bouton sites. This indicates that C-bouton associated molecules are also involved in neuroinflammatory signalling in diseased MNs, emerging as new potential therapeutic targets.

Peripheral nerve section with subsequent disconnection of motor neuron (MN) cell bodies from their skeletal muscle targets leads to a rapid reactive response involving the recruitment and activation of microglia. In addition, the loss of afferent synapses on MNs occurs in concomitance with microglial reaction by a process described as synaptic stripping. However, the way in which postaxotomy‐activated microglia adjacent to MNs are involved in synaptic removal is less defined. Here, we used confocal and electron microscopy to examine interactions between recruited microglial cells and presynaptic terminals in axotomized MNs between 1 and 15 days after sciatic nerve transection in mice. We did not observe any bulk engulfment of synaptic boutons by microglia. Instead, microglial cells internalized small membranous‐vesicular fragments which originated from the acute disruption of synaptic terminals involving the activation of the necroptotic pathway. The presence of abundant extracellular vesicles in the perineuronal space after axotomy, together with the increased expression of phospho‐mixed lineage kinase domain‐like protein and, later, of extracellular vesicle markers, such as CD9, CD63, and flotillin, indicate that the vesicles mainly originated in synapses and were transferred to microglia. The upregulation of Rab7 and Rab10 in microglia interacting with injured MNs, indicated the activation of endocytosis. As activated microglia and synaptic boutons displayed positive C1q immunoreactivity, a complement‐mediated opsonization may also contribute to microglial‐mediated synaptic disruption. In addition to the relevance of our data in the context of neuroinflammation and MN disease, they should also be taken into account for understanding functional recovery after peripheral nerve injury.