Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

Abstract

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.cAMP | muscle | neuromuscular junction | microdomain | synaptic plasticity R ecently, myosin V motor proteins were found to be crucial for the plasticity of central and peripheral synapses by facilitating, respectively, the recycling of AMPA (1-3) and acetylcholine receptors (AChRs) (4). The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle fibers. Even though it is formed during ontogenesis and then maintained over the years (5), half-lives (t 1/2 ) of a few days are typical for its protein constituents, such as the AChR (6). The high enrichment of AChRs in the postsynapse is regulated by accurate targeting and turnover of this receptor. Vesicular transport is employed for this purpose, using different routes of exocytosis, endocytosis, and recycling (7-9). We identified myosin Va as the first motor protein involved in AChR trafficking and have found it to facilitate its recycling (4). Down-regulation of myosin Va led to a reduced t 1/2 of AChRs (4) similar to pathological conditions, such as denervation (6, 10-13) or dystrophy (14). A shortened t 1/2 of AChRs accompanies morphological changes of the NMJ: while NMJs in mouse muscles are normally elliptic and pretzel-shaped (15), they elongate and fragment under pathological conditions (16,17).Previous studies have revealed a major role of cAMP signaling in controlling the t 1/2 of AChRs (12, 18). cAMP is formed upon activation of G protein-coupled receptors and mediates its effects mainly through protein kinase A (PKA). Notably, stimulation of cAMP synthesis rescued AChR lifetime in denervated (12,19) and dystrophic muscles (14). Several groups have described antagonistic functions of protein kinase C and PKA on AChR persistence and synaptic strength (18,20,21): although activation of protein kinase C destabilizes AChRs, PKA activity protects this receptor from such destabilization (18). In this context, the neuropeptide and cAMP agonist, α-calcitonin gene-rel...

Cardiac troponin T and fast skeletal muscle denervation in ageing

“…Because NMJ deteriorates in the fast skeletal muscle more severely with ageing than in slow skeletal muscle, while PKA RIα plays a key role in nAChR stabilization at the NMJ postsynaptic membrane,12, 15 we postulate that the amount of PKA RIα at the NMJ will decrease with ageing and possibly mainly in the fast skeletal muscles. To test this, we first performed double immunofluorescence staining of PKA RIα and NMJ in SOL and TA muscle cryosections from young and old mice.…”

“…Therefore, it is important to determine the subcellular localization of cTnT in the skeletal muscle for a better understanding of its potential biological role in old skeletal muscle. Given that cTnT functions as a D‐AKAP that interacts with PKA RI and RII regulatory subunits,10 both of which are enriched at the NMJ in skeletal muscle,11, 12, 13, 14, 15 we next wanted to determine if cTnT is also localized at the NMJ. To test this, we performed double immunofluorescence staining using 1C11 anti‐cTnT antibody to label endogenous cTnT and Alexa 568‐conjugated α‐bungarotoxin to label nicotinic acetylcholine receptors (nAChRs) at the postsynaptic NMJ membrane.…”

“…Although PKA RIα, RIIα, and RIIβ are all localized at the postsynaptic NMJ, their patterns differ 11. The role of RIα in mediating nAChRs stabilization at the postsynaptic NMJ is known, but the role of RIIα and RIIβ at this site is still unknown 12, 13, 14, 15. Our data show for the first time that enrichment of PKA RIα and RIIα at NMJ is muscle type‐specific and age‐dependent, with PKA RIα mainly abundant at the NMJ in young muscle fibres and old slow‐twitch fibres, while PKA RIIα was predominantly enriched at the NMJ in old fast‐twitch fibres.…”

“…Therefore, cTnT enrichment at the NMJ in old skeletal muscle may competitively bind more PKA RII but less RI at the NMJ, subsequently inhibiting the PKA RIα signalling that mediates nAChRs stabilization at the postsynaptic NMJ. Given that myosin Va12 and rapsyn15 are involved in PKA RIα signalling at the NMJ, we examined both of them in this study. However, we saw no differences in rapsyn enrichment at the NMJ in old TA muscle, indicating that cTnT mediates PKA RI and RII binding at the NMJ in a rapsyn‐independent manner.…”

“…Although PKA RIα, RIIα, and RIIβ are all localized at the postsynaptic neuromuscular junction (NMJ), their patterns differ 11. The role of RIα in mediating nicotinic acetylcholine receptor (nAChR) stabilization at the postsynaptic NMJ is known, but the role of RIIα and RIIβ at this site is still not clear 12, 13, 14, 15. In addition, whether PKA RIα plays a role in regulation of NMJ stability in a fibre type‐specific and age‐dependent manner is unknown.…”