Synapses form in skeletal muscles lacking neuregulin receptors.

The formation of the neuromuscular junction (NMJ) is directed by reciprocal interactions between motor neurons and muscle fibers. Neuregulin (NRG) and Agrin from motor nerve terminals are both implicated. Here, we demonstrate that NMJs can form in the absence of the NRG receptors ErbB2 and ErbB4 in mouse muscle. Postsynaptic differentiation is, however, induced by Agrin. We therefore conclude that NRG signaling to muscle is not required for NMJ formation. The effects of NRG signaling to muscle may be mediated indirectly through Schwann cells.

An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy.

Congenital muscular dystrophy is a heterogeneous and severe, progressive muscle-wasting disease that frequently leads to death in early childhood. Most cases of congenital muscular dystrophy are caused by mutations in LAMA2, the gene encoding the alpha2 chain of the main laminin isoforms expressed by muscle fibres. Muscle fibre deterioration in this disease is thought to be caused by the failure to form the primary laminin scaffold, which is necessary for basement membrane structure, and the missing interaction between muscle basement membrane and the dystrophin-glycoprotein complex (DGC) or the integrins. With the aim to restore muscle function in a mouse model for this disease, we have designed a minigene of agrin, a protein known for its role in the formation of the neuromuscular junction. Here we show that this mini-agrin-which binds to basement membrane and to alpha-dystroglycan, a member of the DGC-amends muscle pathology by a mechanism that includes agrin-mediated stabilization of alpha-dystroglycan and the laminin alpha5 chain. Our data provides in vivo evidence that a non-homologous protein in combination with rational protein design can be used to devise therapeutic tools that may restore muscle function in human muscular dystrophies.

Neural agrin controls acetylcholine receptor stability in skeletal muscle fibers.

At mammalian neuromuscular junctions (NMJs), innervation induces and maintains the metabolic stability of acetylcholine receptors (AChRs). To explore whether neural agrin may cause similar receptor stabilization, we injected neural agrin cDNA of increasing transfection efficiencies into denervated adult rat soleus (SOL) muscles. As the efficiency increased, the amount of recombinant neural agrin expressed in the muscles also increased. This agrin aggregated AChRs on muscle fibers, whose half-life increased in a dose-dependent way from 1 to 10 days. Electrical muscle stimulation enhanced the stability of AChRs with short half-lives. Therefore, neural agrin can stabilize aggregated AChRs in a concentration- and activity-dependent way. However, there was no effect of stimulation on AChRs with a long half-life (10 days). Thus, at sufficiently high concentrations, neural agrin alone can stabilize AChRs to levels characteristic of innervated NMJs.

Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.

In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin.

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

The zinc finger domain of the 43-kDa receptor-associated protein, rapsyn: role in acetylcholine receptor clustering.

We injected rat myotubes with proteins and antibodies to assess the importance of the zinc finger (ZnF) domain of the 43-kDa receptor-associated protein, rapsyn, in clustering acetylcholine receptors (AChR). Injection of rat myotubes with a fusion protein containing the ZnF domain of rapsyn disrupted AChR clusters. Clusters were unaffected by a fusion protein containing a double mutant that does not bind zinc. Similar results were obtained with the purified wild type and mutant ZnF domains. The ZnF of HIV-1 nucleocapsid protein had no effect. AChR clusters were also disrupted in myotubes injected with antibodies to the ZnF domain, followed by injection of anti-antibodies. Injection of antibodies directed against a different rapsyn epitope or against the cytoplasmic domain of the AChR had no effect. In transfection experiments with HEK 293 cells, the ZnF domain failed to associate with membrane aggregates containing full-length rapsyn, AChR, or rapsyn and AChR together. We conclude that the ZnF domain of rapsyn provides a binding site essential for AChR clustering, but that this site is unlikely to be involved in high affinity binding of rapsyn to itself or to AChR.

A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle.

We have presented ultrastructural and semiquantitative immunofluorescence evidence to support the idea that AChR are clustered in rat myotubes by virtue of their ability to associate with a spectrin-based membrane skeleton. Many of the interactions postulated to be involved in the formation of this skeleton, and in the anchoring of AChR to it, must still be examined at the biochemical level, but the overall similarity of this structure to that of the human erythrocyte is already clear. The ability of different members of the spectrin superfamily to associate in various combinations to form distinct plasmalemmal domains provides some exciting hints as to how the surface membrane can be organized efficiently to subserve multiple purposes. One of the challenges of future research will be to learn how innervation regulates the assembly of the membrane skeleton at the developing NMJ, and how this structure is altered as the junction matures. Another will be to learn if the principles of neuromuscular synaptogenesis are relevant to interactions between neurons in the brain, where cells must distinguish between multiple synaptic inputs and assemble synaptic structures at thousands of distinct sites on the neurolemma. Members of the spectrin superfamily have been identified in synaptic structures in the central nervous system (e.g., Carlin et al., 1983; LeVine and Sahyoun, 1986; Malchiodi-Albedi et al., 1993), so much of what we have learned at the neuromuscular junction may be applicable to central synapses.

[Interaction of cholesterol and sphingomyelins in membranes of the sarcotubular system after denervation]. / Interakcia cholesterolu a sfingomyelínov v membránach sarkotubulárneho systému po denervácii.

We investigated the content of cholesterol and phospholipids in the sarcotubular membranes from the rabbit fast-twitch muscle in various time intervals after the section of sciatic nerve. Along with the unchanged content of total phospholipids we found an increase of cholesterol in all investigated intervals. Significant elevation of cholesterol by about 33.8% following 14 days of denervation is associated with a three-times higher level of sphingomyelins. The elevated level of sphingomyelins persists after 28 days and the molar ratio cholesterol/phospholipids is 2.6 times higher in comparison with the control group. Investigation of cholesterol synthesis on muscle slices from the precursor 14C-mevalonic acid as well as of the activity of HMG-CoA reductase have shown that the cholesterol increase following denervation is not in correlation with its synthesis de novo. Similarly it has been shown that an increase of membrane cholesterol is not linked with the change of membrane fluidity (Tab. 3, Fig. 3, Ref. 30.)

Intracellular Ca2+ store from rabbit brain: kinetic aspects of Ca(2+)-stimulated ATPase.

The endoplasmic reticulum of the rabbit brain manifests low but distinguishable Ca(2+)-stimulated ATPase activity with some functional peculiarities compared to the muscle isoforms. The activity is highly sensitive to the presence of detergents and optimal activatory concentration was shown to be close to the critical micellar concentration. Kinetic properties, such as (i) activation by low (K0.5 = 0.45 microM) and inhibition by high (more than 100 microM) concentration of calcium, (ii) biphasic activation with ATP and (iii) inhibition by vanadate clearly showed that the protein of Ca(2+)-ATPase is endowed with properties typical of the family of sarco/endoplasmic reticulum calcium pumps.

Alteration in rabbit brain endoplasmic reticulum Ca2+ transport by free oxygen radicals in vitro.

The involvement of free oxygen radicals in ischemia-reperfusion injury is generally accepted. We describe here the loss of efficiency of reticular membranes to sequester Ca2+ due to free oxygen radical damage in vitro. Based on experimental results we suggest that the primary effect of free oxygen radicals is alteration of the lipid component of the membrane manifested in the increase of passive Ca2+ leak. Prolonged treatment of microsomes with free oxygen radical generating systems also led to the decrease of Ca(2+)-ATPase activity which is caused as we suppose by modulation of the lipid-Ca2+ pump interactions. A protective effect of butylated hydroxytoluene on the depression in the Ca2+ uptake and Ca(2+)-ATPase activities supports our suggestions.

Distribution of Ca(2+)-modulating proteins in sarcoplasmic reticulum membranes after denervation.

The early response to the loss of motor innervation to the muscle is connected with an altered Ca(2+)-homeostasis. Our study, based on Western blotting, indicates that denervation influenced expression of some sarcoplasmic Ca(2+)-modulating proteins. Evidence has been brought for an increase of the level of calsequestrin and of the putative ryanodine receptor paralleled with a slight decrease of the total amount of Ca(2+)-pump protein. The expression of unchanged Ca(2+)-pump isoform and unaltered quantities of other non-junctional Ca(2+)-binding proteins support the hypothesis that changed cellular Ca2+ homeostasis include also an alteration of Ca(2+)-modulating systems, mainly from the junctional region of sarcoplasmic membranes.

[Properties of skeletal muscle sarcoplasmic reticulum membranes prepared in the presence of dithiothreitol]. / Vlastnosti membrán sarkoplazmatického retikula kostrového svalu pripravených za prítomnosti ditiotreitolu.

The authors studied the effect of dithiothreitol (DTT), an agent modulating the redox state of SH-groups, on functional properties of sarcoplasmic reticulum membranes of skeletal muscle. DTT (5 mmol.l-1) exerted a protective effect not only on (Ca2 + Mg2)-ATPase activity but also on the activity of Ca2+ uptake by vesicles, while the most pronounced effect was recorded at low levels of ionized Ca2+. The presence of DTT in the isolation medium decreased also passive permeability of membranes for Ca2+. In the light of the results obtained, modulation of the redox state is recommended for each isolation of membranes designed for the study of ion transport.