Regulation of nicotinic acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/lysosomal and atrophy pathways

Abstract

Muscle atrophy is a process of muscle wasting induced under a series of catabolic stress conditions, such as denervation, disuse, cancer cachexia, heart and renal failure, AIDS, and aging. Neuromuscular junctions (NMJs), the synapses between motor neurons and muscle fibers undergo major changes in atrophying muscles, ranging from mild morphological alterations to complete disintegration. In this study, we hypothesized that remodeling of NMJs and muscle atrophy could be linked together. To test this, we examined if a major atrophy-promoting E3 ubiquitin ligase, MuRF1, is involved in the maintenance of NMJs. Immunofluorescence revealed that MuRF1 is highly enriched close to the NMJ. Affinity precipitation and in vivo imaging showed that MuRF1 interacts in endocytic structures with both, acetylcholine receptor, the primary postsynaptic protein of the NMJ, as well as with Bif-1, an autophagy- and endocytosis-regulating factor. In vivo imaging, radio labeling, and weighing approaches demonstrated that metabolic destabilization of acetylcholine receptors and muscle atrophy induced by denervation were significantly rescued in MuRF1-KO animals. Notably, interaction with Bif-1, and the rescue of AChR lifetime and muscle atrophy were specific to MuRF1 but not MuRF2. Our data demonstrate an involvement of MuRF1 in membrane protein-turnover, including the degradation of AChRs at the NMJ under atrophying conditions where MuRF1 also interacts and associates with Bif-1.

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

“…44 We have recently shown that endogenous TRIM63 is highly enriched at the nerve-muscle synapse, the neuromuscular junction, and heterologous expression of TRIM63-GFP is detected by in vivo microscopy in puncta colocalizing with internalized CHRN, the major postsynaptic ion channel of the NMJ. 28 Furthermore, since CHRN half-life upon denervation is significantly stabilized in trim63 −/ − mice and TRIM63 coprecipitates with CHRN, a crucial role of TRIM63 for CHRN turnover was claimed. 28 Despite the enormous physiological relevance of CHRN, deplorably little is known about the mechanisms driving its turnover.…”

“…Our previous extensive work on the mechanisms underlying CHRN recycling 25,34,35 and CHRN degradation identified an interaction between CHRN and the E3 ligase TRIM63. 28 Furthermore, the previously reported direct interaction between TRIM63 and SQSTM1 29 prompted us to speculate that TRIM63 may induce selective autophagy as a principal route for CHRN removal. To address this issue, we first transfected the autophagic marker GFP-MAP1LC3A into live mouse muscle.…”

“…Starvation-induced destabilization of CHRN is absent in trim63 −/− mice Given the recently described role of the atrogene and E3 ubiquitin ligase TRIM63, in regulating the turnover of CHRN 28 and the known importance of TRIM63 in fasting-induced muscle remodeling, 19,30,31 we then asked if this atrogene also participates in starvation-induced regulation of CHRN turnover. Therefore, trim63 −/ − mice were starved for 48 h and the radioiodine experiment was performed as before.…”

“…28 To address, whether SH3GLB1 might be involved in autophagy of CHRN in skeletal muscle, we cotransfected muscles with GFP-MAP1LC3A and SH3GLB1-mCherry and performed in vivo imaging after BGTAlexaFluor 647 labeling of CHRN. As expected, SH3GLB1-mCherry nicely colocalized with endocytic CHRN vesicles and was capped by GFP-MAP1LC3A (Fig.…”

“…26,27 We have recently reported that the E3-ubiquitin ligase TRIM63 plays an important role in the turnover of CHRN. 28 TRIM63 was found to be highly enriched in the perisynaptic region and to coprecipitate with CHRN that was in vivo-labeled with α-bungarotoxin (BGT)-biotin.…”