Nerve-independent formation of a topologically complex postsynaptic apparatus

As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)-rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity.     

Lithium inhibits a late step in agrin‐induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle‐specific kinase MuSK, activation of Src‐family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) β subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high‐density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin‐induced change in AChR distribution without affecting the number of AChRs or the agrin‐induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin‐induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 346–357, 2003     

Regulation of muscle acetylcholine receptor turnover by β subunit tyrosine phosphorylation

At the neuromuscular junction (NMJ), the postsynaptic localization of muscle acetylcholine receptor (AChR) is regulated by neural signals and occurs via several processes including metabolic stabilization of the receptor. However, the molecular mechanisms that influence receptor stability remain poorly defined. Here, we show that neural agrin and the tyrosine phosphatase inhibitor, pervanadate slow the degradation of surface receptor in cultured muscle cells. Their action is mediated by tyrosine phosphorylation of the AChR β subunit, as agrin and pervandate had no effect on receptor half‐life in AChR‐β^3F/3F muscle cells, which have targeted mutations of the β subunit cytoplasmic tyrosines. Moreover, in wild type AChR‐β^3Y muscle cells, we found a linear relationship between average receptor half‐life and the percentage of AChR with phosphorylated β subunit, with half‐lives of 12.7 and 23 h for nonphosphorylated and phosphorylated receptor, respectively. Surprisingly, pervanadate increased receptor half‐life in AChR‐β^3Y myotubes in the absence of clustering, and agrin failed to increase receptor half‐life in AChR‐β^3F/3F myotubes even in the presence of clustering. The metabolic stabilization of the AChR was mediated specifically by phosphorylation of βY390 as mutation of this residue abolished β subunit phosphorylation but did not affect δ subunit phosphorylation. Receptor stabilization also led to higher receptor levels, as agrin increased surface AChR by 30% in AChR‐β^3Y but not AChR‐β^3F/3F myotubes. Together, these findings identify an unexpected role for agrin‐induced phosphorylation of β^Y390 in downregulating AChR turnover. This likely stabilizes AChR at developing synapses, and contributes to the extended half‐life of AChR at adult NMJs. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 399–410, 2013     

Rapsyn‐mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high‐density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the α subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn‐induced clustering of the AChR β, γ, and δ subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into αδ dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild‐type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn‐mediated clustering of an assembly intermediate, the αδ dimer. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 486–501, 2003     

Laminin-1 redistributes postsynaptic proteins and requires rapsyn,tyrosine phosphorylation,and Src and Fyn to stably cluster acetylcholine receptors

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.     

Population density regulates Drosophila synaptic morphology in a Fasciclin‐II‐dependent manner

Genetic analysis of the Drosophila larval neuromuscular junction has identified some of the key molecules that regulate synaptic plasticity. Among these molecules, the expression level of Fasciclin II (FasII), a homophilic cell adhesion molecule, is critically important for determining the final form of the neuromuscular junction. Genetic reduction of FasII expression by 50% yields more elaborate nerve terminals, while a greater reduction in expression, to 10% of wild‐type, yields a substantial reduction in the nerve terminal morphology. Importantly, regulation of FasII expression seems to be the final output for several genetic manipulations that transform NMJ morphology. In an effort to understand the importance of this regulatory pathway in the normal animal, we have undertaken studies to identify environmental cues that might be important for initiating FasII‐dependent changes in synaptic plasticity. Here we report on the relationship between larval population density and synaptic morphology, synaptic strength, and FasII levels. We raised Drosophila larvae under conditions of increasing population density and found an inverse exponential relationship between population density and the number of synaptic boutons, the number of branches, and the length of branches. We also observed population‐dependent alteration in FasII levels, with lower densities having less FasII at the synapse. The correlation between density and morphological change was abrogated in larvae constitutively expressing FasII, and in wild‐type larvae grown on soft culture medium. Together these data show that environmental cues can induce regulation of FasII. Interestingly, however, the quantal content of synaptic transmission was not different among the different population densities, suggesting that other factors contribute to maintaining synaptic strength at a defined level. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Matrix metalloproteinase‐3 removes agrin from synaptic basal lamina

Agrin, a heparin sulfate proteoglycan, is an integral member of the synaptic basal lamina and plays a critical role in the formation and maintenance of the neuromuscular junction. The N‐terminal region of agrin binds tightly to basal lamina, while the C‐terminal region interacts with a muscle‐specific tyrosine kinase (MuSK) to induce the formation of the postsynaptic apparatus. Although the binding of agrin to basal lamina is tight, the binding of agrin to MuSK has yet to be shown; therefore, basal lamina binding is critical for maintaining the presentation of agrin to MuSK. Here we report evidence that supports our hypothesis that matrix metalloproteinase‐3 (MMP‐3) is responsible for the removal of agrin from synaptic basal lamina. Antibodies to the hinge region of human MMP‐3 recognize molecules concentrated at the frog neuromuscular junction in both cross sections and whole mounts. Electron microscopy of neuromuscular junctions stained with antibodies to MMP‐3 reveals that staining is found in the extracellular matrix surrounding the Schwann cell. Treatment of sections from frog anterior tibialis muscle with MMP‐3 results in a clear and reproducible removal of agrin immunoreactivity from synaptic basal lamina. The same MMP‐3 treatment does not alter anti‐laminin staining. These results support our hypothesis that synaptic activity results in the activation of MMP‐3 at the neuromuscular junction and that MMP‐3 specifically removes agrin from synaptic basal lamina. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 140–149, 2000     

Pre‐synaptic localization of the γ‐secretase‐inhibiting protein p24α2 in the mammalian brain

Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α₂ reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α₂ in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α₂ in the mouse brain. Immunostaining showed that p24α₂ is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α₂ in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α₂ is condensed at active zone‐docked SV. During development, p24α₂ expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α₂ in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α₂ in the regulation of Aβ generation and secretion in the brain.

     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

Age‐related differences in synaptic plasticity following muscle unloading

The objective of the present investigation was to determine the effects of muscle unloading—a form of subtotal disuse— on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n = 8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT‐4 expression. Quantitative analyses revealed that aged controls displayed significant (p < 0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls > young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT‐4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 246–256, 2003     

Pre‐ and postsynaptic mechanisms in Hebbian activity‐dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down‐regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity‐dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity‐dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output. Published 2002 Wiley Periodicals, Inc. J Neurobiol 52: 241–250, 2002     

Assembly,trafficking and function of α1β2γ2 GABAA receptors are regulated by N‐terminal regions,in a subunit‐specific manner

GABA_A receptors are pentameric ligand‐gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand‐gated ion channels structures, sequence analysis predicts an α‐helix near the N‐terminus of each GABA_A receptor subunit. Preceding each α‐helix are 8–36 additional residues, which we term the N‐terminal extension. In homomeric GABA_C receptors and nicotinic acetylcholine receptors, the N‐terminal α‐helix is functionally essential. Here, we determined the role of the N‐terminal extension and putative α‐helix in heteromeric α1β2γ2 GABA_A receptors. This role was most prominent in the α1 subunit, with deletion of the N‐terminal extension or further deletion of the putative α‐helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N‐terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α‐helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α‐helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N‐terminal extensions and α‐helices make key subunit‐specific contributions to assembly, consistent with both regions being involved in inter‐subunit interactions.

     

Arctic mutant Aβ40 aggregates on α7 nicotinic acetylcholine receptors and inhibits their functions

Amyloid β protein (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Point mutations within the Aβ sequence associated with familial AD (FAD) are clustered around the central hydrophobic core of Aβ. Several types of mutations within the Aβ sequence have been identified, and the ‘Arctic’ mutation (E22G) has a purely cognitive phenotype typical of AD. Previous studies have shown that the primary result of the ‘Arctic’ mutation is increased formation of Aβ protofibrils. However, the molecular mechanism underlying this effect remains unknown. Aβ42 binds to a neuronal nicotinic acetylcholine receptor subunit, neuronal acetylcholine receptor subunit alpha‐7 (CHRNA7), with high affinity and, thus, may be involved in the pathogenesis of AD. Therefore, to clarify the molecular mechanism of Arctic mutation‐mediated FAD, we focused on CHRNA7 as a target molecule of Arctic Aβ. We performed an in vitro binding assay using purified CHRNA7 and synthetic Arctic Aβ40, and demonstrated that Arctic Aβ40 specifically bound to CHRNA7. The aggregation of Arctic Aβ40 was enhanced with the addition of CHRNA7. Furthermore, the function of CHRNA7 was detected by measuring Ca²⁺ flux and phospho‐p44/42 MAPK (ERK1/2) activation. Our results indicated that Arctic Aβ40 aggregation was enhanced by the addition of CHRNA7, which destabilized the function of CHRNA7 via inhibition of Ca²⁺ responses and activation of ERK1/2. These findings indicate that Arctic Aβ mutation may be involved in the mechanism underlying FAD. This mechanism may involve binding and aggregation, leading to the inhibition of CHRNA7 functions.