Botulinum neurotoxin as a tool to study neuromuscular junction plasticity and remodeling

he neuromuscular junction (NMJ) is crucial for motor control, enabling communication between motor neurons and muscle fibers to initiate contraction. Axonal sprouting, a form of neural plasticity, enables new connections after injury or blocked neurotransmission, such as with Botulinum Neurotoxin type A (BoNT/A). Although BoNT/A causes muscle atrophy, motor neurons remain intact and remodel NMJ innervation through sprouting. To unveil molecular determinants of BoNT/A-induced sprouting, we conducted a targeted transcriptome analysis focused specifically on the NMJ, by performing laser-capture microdissection (LCM) and RNA sequencing (RNA-seq) of mouse soleus (slow-twitch, sproutogenic) and EDL (fast-twitch, non-sproutogenic) muscles after BoNT/A injection. At the transcriptomic level, significant differences in DEGs were observed between the sproutogenic soleus muscle and the nonsproutogenic EDL muscle, especially 18 days post-BoNT/A, when sprouting peaks. GO analysis revealed that the soleus responded more rapidly and robustly, with notable changes in mitochondrial function, ECM composition, and ribosomal activity during sprouting. Further analysis identified a key gene, likely a myokine involved in nervous system development and anatomical morphogenesis, which regulates Wnt signaling and was selected as a candidate in the sprouting process. In vitro and in vivo experiments demonstrated that this released factor promotes NMJ formation by enhancing AChR clustering and expression. Neutralization of this myokine in mice reduced AChR density and satellite NMJ formation, impairing muscle recovery from paralysis. These findings suggest that both the myokine and Wnt signaling are essential for NMJ plasticity, positioning this myokine as a potential therapeutic target for neuromuscular regeneration. Our comprehensive transcriptome analysis provides new insights into NMJ remodeling during sprouting, offering promising therapeutic avenues for neuromuscular disorders. The NMJ undergoes continuous remodeling, balancing denervation and reinnervation, but this balance is lost with aging, leading to motor impairments and muscle atrophy. Fast-twitch muscles, like the EDL, degenerate earlier than slow-twitch muscles like the soleus. To investigate the molecular mechanisms behind age-related motor decline, we performed gene and protein expression analysis using RNA-seq and mass spectrometry (MS) to profile aging NMJ changes. Our integrated transcriptome and proteome analysis of aging NMJs revealed that post-transcriptional regulation plays a crucial role in NMJ biology. Fast-twitch EDL muscles showed more pronounced proteomic changes with age than slow-twitch soleus muscles, particularly in genes with discordant mRNA and protein expression, likely regulated by miRNAs and RNAbinding proteins. This uncoupling was especially significant in EDL muscles, which are more vulnerable to age-related degeneration. Our findings highlight the impact of post-transcriptional regulation on NMJ function and muscle vulnerability, identifying potential biomarkers and therapeutic targets for neurodegenerative diseases.