Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse

Abstract

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.

Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo

“…There is a wealth of studies showing a direct correlation between the amount of rapsyn present at NMJs and the abundance or the metabolic stability of synaptic AChRs (Brockhausen et al, 2008;Gervasio et al, 2007;Gervasio and Phillips, 2005;Luo et al, 2008;Martinez-Martinez et al, 2009;Wang et al, 1999). Furthermore, mutations in the RAPSN gene are amongst the most frequent causes for myasthenic syndromes, which are characterised by activity-dependent muscle weakness (Beeson et al, 2008;Müller et al, 2007).…”

ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

“…First, developmental regulation induces changes in rapsyn-to-AChR ratio linked to higher packing and stability of AChRs in the postsynaptic membrane (Frail et al, 1989;Schaeffer et al, 2001;Gervásio et al, 2007). Second, agrin stimulates rapsyn-AChR interactions in vitro and in vivo, a process that is also correlated with tighter AChR packing in clusters (Moransard et al, 2003;Gervásio et al, 2007;Brockhausen et al, 2008). Our observation that clusters of AChRs are more densely packed in ColQ-deficient clusters could thus be related to higher expression of rapsyn with an increased ratio of rapsyn-toAChR in vivo.…”

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

“…However, the background (extrasynaptic) fluorescence is generally lower compared to en face zprojection images. Thus, it can be easier to discriminate 'real' (specific) immunostaining and establish fixed confocal gain and offset values using transverse optical sections [13][14][15]18 . For example, in a mouse model of myasthenia gravis (where endplate AChR staining is markedly reduced) endplates were clearly delineated in transverse optical sections 18,21 .…”

“…The protocols described here were refined through a series of studies that investigated developmental, physiological and pathophysiological changes to the NMJ. Such studies require measurement of the area of synaptic specializations at the mouse motor endplate and the relative density of packing of synaptic proteins within postsynaptic specializations [13][14][15] .…”