The Role of AMPK in Neuromuscular Junction Biology
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abstract
The neuromuscular junction (NMJ) is the signaling apparatus between a motoneuron and the corresponding muscle fibers that it innervates. While its stability is vital for the neuromuscular system, the NMJ is susceptible to remodeling across the lifespan. Adenosine monophosphate‐activated protein kinase (AMPK) is a primary modulator of skeletal muscle phenotype, however the precise role of AMPK at the NMJ has yet to be investigated. Thus, the purpose of this study was to examine the role of skeletal muscle AMPK on NMJ biology across the lifespan. We employed 3‐ and 12‐month‐old muscle specific AMPKβ1β2 knock out mice (mKO, n = 3), age‐matched wild‐type (WT, n = 3) mice, as well as 22‐month‐old WT animals. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected to represent fast glycolytic and slow oxidative tissues, respectively. These muscles were then whole‐mounted and immunolabelled for presynaptic neurofilament and synaptic vesicle 2A proteins, as well as postsynaptic acetylcholine receptors (AChR) revealed by □‐bungarotoxin. Approximately 25 NMJ images per muscle were acquired via confocal microscopy. In total, we conducted 3D analysis on over 400 NMJs to determine metrics of NMJ morphology. Postsynaptic analyses revealed an effect of age (p < 0.05) on the total amount of fragmented and ectopic NMJs in the SOL and EDL muscles of WT animals. Interestingly, mKO animals exhibited a significantly greater count of fragmented and ectopic NMJs in the SOL and EDL muscles, compared to their WT littermates. In fact, NMJs positive for postsynaptic abnormalities in EDL muscles of 12‐month‐old mKO animals were significantly greater than 22‐month‐old WT animals (p < 0.05), while SOL muscles were comparable. An effect of age (p < 0.05) on axonal blebbing and sprouting was observed in the EDL and SOL muscles, respectively. Additionally, NMJs with axonal blebbing were observed more frequently (p < 0.05) in the mKO animals compared to their age‐matched WT littermates. Quantitative real‐time PCR was also utilized to examine genes important for NMJ development and maintenance in the tibialis anterior muscles of 3‐month‐old WT and mKO animals (n = 10). Specifically, AChR□/ɣ, low‐density lipoprotein receptor‐related protein 4, rapsyn, and muscle‐specific kinase mRNA expression was significantly higher in the mKO animals relative to their WT counterparts. Our results suggest that AMPK is critical to the determination and maintenance of a healthy pre‐ and post‐synaptic apparatus in motoneurons and muscles of disparate phenotypes. Additionally, the loss of muscle‐specific AMPK exacerbates age‐induced postsynaptic alterations in faster, and more glycolytic muscle fibers.Support or Funding InformationCanadian Institutes of Health Research, Canada Research Chairs, and Ontario Graduate Scholarship.
