Functional regeneration of the murine neuromuscular synapse relies on long-lasting morphological adaptations

Abstract

Background In a broad variety of species, muscle contraction is controlled at the neuromuscular junction (NMJ), the peripheral synapse composed of a motor nerve terminal, a muscle specialization, and non-myelinating terminal Schwann cells. While peripheral nerve damage leads to successful NMJ reinnervation in animal models, muscle fiber reinnervation in human patients is largely inefficient. Interestingly, some hallmarks of NMJ denervation and early reinnervation in murine species, such as fragmentation and poly-innervation, are also phenotypes of aged NMJs or even of unaltered conditions in other species, including humans. We have reasoned that rather than features of NMJ decline, such cellular responses could represent synaptic adaptations to accomplish proper functional recovery. Here, we have experimentally tackled this idea through a detailed comparative study of the short- and long-term consequences of irreversible (chronic) and reversible (partial) NMJ denervation in the convenient cranial levator auris longus muscle. Results Our findings reveal that irreversible muscle denervation results in highly fragmented postsynaptic domains and marked ectopic acetylcholine receptor clustering along with significant terminal Schwann cells sprouting and progressive detachment from the NMJ. Remarkably, even though reversible nerve damage led to complete reinnervation after 11 days, we found that more than 30% of NMJs are poly-innervated and around 65% of postsynaptic domains are fragmented even 3 months after injury, whereas synaptic transmission is fully recovered two months after nerve injury. While postsynaptic stability was irreversibly decreased after chronic denervation, this parameter was only transiently affected by partial NMJ denervation. In addition, we found that a combination of morphometric analyses and postsynaptic stability determinations allows discriminating two distinct forms of NMJ fragmentation, stable-smooth and unstable-blurred, which correlate with their regeneration potential. Conclusions Together, our data unveil that reversible nerve damage imprints a long-lasting reminiscence in the NMJ that results in the rearrangement of its cellular components. Instead of being predictive of NMJ decline, these traits may represent an efficient adaptive response for proper functional recovery. As such, these features are relevant targets to be considered in strategies aimed to restore motor function in detrimental conditions for peripheral innervation.

Regeneration of neuromuscular synapses after acute and chronic denervation by inhibiting the gerozyme 15-prostaglandin dehydrogenase

“…This was achieved by assessing the coincidence of presynaptic motor neuron staining with antibodies to NF and SV2 and postsynaptic AChR marked by BTX in the injured legs compared with uninjured contralateral legs 14 dpi. In accordance with previous studies ( 42 , 43 ), partial recovery of innervation was observed in vehicle-treated controls at 14 dpi. Pharmacological inhibition of 15-PGDH increased innervation in injured EDL muscles from 84.5 ± 2.2% in vehicle-treated controls to 97.4 ± 2.1% in PGDHi-treated mice (Fig.…”

Impaired communication at the neuromotor axis during Degenerative Cervical Myelopathy

“…These findings hold substantial importance in understanding motor deficits associated with DCM. Notably, our model deviates from traditional neuromuscular models characterized by partial or complete denervation, axonal swelling, and significant alterations in NMJ structure ( Valdez et al, 2010 ; Torres-Benito et al, 2011 ; Bermedo-García et al, 2022 ). Partially, these disparities might arise from a more direct mechanism in which motoneurons and their corresponding target myofibers are concurrently lost, leaving the tissue with limited capacity to compensate for the damage.…”

“…This peripheral cholinergic synapse is constituted by an extra-laminar capping of kranocyte cells, a presynaptic motor nerve terminal, a postsynaptic muscle specialization evidenced by clusters of acetylcholine receptors (AChRs), and non-myelinated terminal Schwann cells (tSCs; Sanes and Lichtman, 2001 ; Wu et al, 2010 ; Shi et al, 2012 ; Tintignac et al, 2015 ). Multiple pathologies, nerve or muscular injuries, lack of physical activity, and aging impair the synaptic structure and function at the NMJ, resulting in poly-innervation, partial or total denervation, postsynaptic fragmentation, and muscle atrophy, among other phenotypes ( Valdez et al, 2010 , 2012 ; Tejero et al, 2016 , 2020 ; Woehlbier et al, 2016 ; Bermedo-García et al, 2022 ). However, the behavior of the cellular components of the neuromuscular synapse in the context of DCM has not been studied.…”

Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration

“…Third, we did not study the functional regeneration of muscle fibers after an acute injury in the control and macrophage-depleted groups. The contraction of muscle fibers could be explored by high-speed imaging of second harmony generation (SHG) 91 and the functional repair of the neuromuscular junctions (a synaptic connection between terminal end of a motor nerve and a muscle) 92,93 could also be studied further.…”