Sara Salvany

Neuregulins (NRGs) are ligands of tyrosine kinase receptors from the ErbB family and play multiple developmental roles. NRG1–ErbB signaling regulates myelination and has been associated with amyotrophic lateral sclerosis (ALS) pathology. Given the potential therapeutic relevance of this pathway for motor neuron (MN) diseases, we employed a transgenic (TG) mouse with persistent neuronal overexpression of neuregulin type III (NRG1-III) to investigate its impact on the neuromuscular system. We performed an analysis of phenotypic changes in this TG model, including motor behavior, neuropathological evaluation by immunocytochemistry and ultrastructural examination of the spinal cord, peripheral nerves, and neuromuscular junctions (NMJs). Calcium dynamics in cultured MNs were also examined. We found that cholinergic C-boutons on TG MNs, where NRG1-III typically accumulates, exhibited upregulation of C-bouton-associated proteins and expansion of the subsynaptic cistern (SSC)-associated endoplasmic reticulum. Calcium imaging revealed altered homeostasis in TG MNs, accompanied by the upregulation of molecules linked to axonal plasticity. At NMJs, regressive changes involving autophagic dysregulation were observed. These alterations were accompanied by increased motor activity in behavioral tests. Overall, our findings indicate that persistently elevated NRG1-III signaling compromises MN connectivity and long-term health, a factor to consider when developing therapeutic strategies for neurodegenerative diseases such as ALS.

Neuregulins (NRGs) are ligands for tyrosine kinase receptors from the ErbB family and serve multiple developmental functions in the nervous system. NRG1-ErbB signaling regulates myelination and is associated with amyotrophic lateral sclerosis (ALS) pathology. Given the potential therapeutic relevance of this pathway for motor neuron (MN) diseases, we employed a transgenic (TG) mouse model with persistent neuronal overexpression of NRG type III (NRG1-III) to investigate its long-term impact on the neuromuscular system. We examined MNs and their synaptic inputs, including cholinergic C-boutons, at which NRG1-III typically accumulates. MNs from TG mice exhibited persistent upregulation of C-bouton-associated postsynaptic proteins, as well as increased biogenesis of subsynaptic cistern-like ER-plasma membrane contacts. Following nerve injury, NRG1-III overexpression resulted in exacerbated neuroinflammation and disruption of enlarged NRG1 clusters. Calcium imaging revealed altered homeostasis in TG MNs, which upregulated molecules linked to axonal plasticity. At neuromuscular junctions, we observed regressive changes involving autophagic dysregulation. These alterations were accompanied by increased motor activity in behavioral tests. Overall, our findings indicate that persistently elevated NRG1-III signaling diminishes MN connectivity and long-term health, which must be considered when developing therapeutic strategies for neurodegenerative diseases, such as ALS.

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). 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.