Abstract
BackgroundDevelopment of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs.ResultsWe have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein.ConclusionOur data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.
Highlights
Development of neural networks requires that synapses are formed, eliminated and stabilized
protein tyrosine phosphatases (PTPs) inhibition by pervanadate reduces the stability of agrin-induced acetylcholine receptors (AChRs) clusters To address the role of the activity of PTPs in the stabilization of agrin-induced AChR clusters, we made use of the potent PTP inhibitor sodium pervanadate [23,24] and cultured C2C12 myotubes, in which AChRs were visualized by treatment with fluorescent α-bungarotoxin
When pervanadate was added at the time of agrin withdrawal, significantly less AChR clusters were observed after a 16 h withdrawal period than in controls lacking pervanadate (Figure 1)
Summary
Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs. Neural networks are shaped through the formation, stabilization and elimination of synapses that connect neurons with their targets. The neuromuscular junction (NMJ), a model synapse in the peripheral nervous system, forms by the contact of motor neurons and muscle fibers These interactions lead to a polyinnervated synapse at birth, in which acetylcholine receptors (AChRs) are clustered in a flat, plaque-like postsynaptic membrane. A short pulse of agrin leads to long-lasting MuSK phosphorylation and normal AChR clustering much later, implying that, once activated, a balance of downstream protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) keeps postsynaptic clustering mechanisms activated [7]
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