Membraneless condensates by Rapsn phase separation as a platform for neuromuscular junction formation.

Our daily life depends on muscle contraction, a process that is controlled by the neuromuscular junction (NMJ). However, the mechanisms of NMJ assembly remain unclear. Here we show that Rapsn, a protein critical for NMJ formation, undergoes liquid-liquid phase separation (LLPS) and condensates into liquid-like assemblies. Such assemblies can recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation. Rapsn LLPS requires multivalent binding of tetratricopeptide repeats (TPRs) and is increased by Musk signaling. The capacity of Rapsn to condensate and co-condensate with interaction proteins is compromised by mutations of congenital myasthenic syndromes (CMSs). NMJ formation is impaired in mutant mice carrying a CMS-associated, LLPS-deficient mutation. These results reveal a critical role of Rapsn LLPS in forming a synaptic semi-membraneless compartment for NMJ formation.

Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.

In 15-d-old chick latissimi dorsi muscles, the nicotinic acetylcholine receptor (AChR) alpha-subunit mRNA is densely accumulated at the level of subsynaptic nuclei of the motor endplate (Fontaine et al., 1988). In this paper, using in situ hybridization with genomic probes, we further show that the expression of the AChR alpha-subunit gene in the embryo, revealed by the accumulation of mature mRNAs, starts in myotomal cells and persists during the first stages of muscle development in a majority of muscle nuclei. Subsequently, the distribution of AChR alpha-subunit mRNAs becomes restricted to the newly formed motor endplates as neuromuscular junctions develop. To assess the transcriptional activity of individual nuclei in developing muscles, a strictly intronic fragment of the AChR alpha-subunit gene was used to probe in situ the level of unspliced transcripts. AChR alpha-subunit unspliced transcripts accumulate around a large number of sarcoplasmic nuclei at embryonic day 11, but can no longer be detected at their level after embryonic day 16 in the embryo. A similar decrease in the accumulation of AChR alpha-subunit transcripts is observed between day 4 and day 6 in primary cultures of muscle cells. On the other hand, in vivo denervation and in vitro blocking of muscle electrical activity by the sodium channel blocker tetrodotoxin results in an increase in the labeling of muscle nuclei. Yet, only 6% of the muscle nuclei appear labeled by the strictly intronic probes after denervation. The possible significance of such heterogeneity of muscle nuclei during motor endplate formation in AChR gene expression is discussed.

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.-P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.

Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCßI and nPKCε) in Neuromuscular Developmental Synapse Elimination.

BACKGROUND: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. METHODS: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5-P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. RESULTS: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCßI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. CONCLUSIONS: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

The Neuromodulator Adenosine Regulates Oligodendrocyte Migration at Motor Exit Point Transition Zones.

During development, oligodendrocyte progenitor cells (OPCs) migrate extensively throughout the spinal cord. However, their migration is restricted at transition zones (TZs). At these specialized locations, unique glial cells in both zebrafish and mice play a role in preventing peripheral OPC migration, but the mechanisms of this regulation are not understood. To elucidate the mechanisms that mediate OPC segregation at motor exit point (MEP) TZs, we performed an unbiased small-molecule screen. Using chemical screening and in vivo imaging, we discovered that inhibition of A2a adenosine receptors (ARs) causes ectopic OPC migration out of the spinal cord. We provide in vivo evidence that neuromodulation, partially mediated by adenosine, influences OPC migration specifically at the MEP TZ. This work opens exciting possibilities for understanding how OPCs reach their final destinations during development and identifies mechanisms that could promote their migration in disease.

Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle.

We used specific antibodies to gamma, delta, and epsilon subunits to characterize acetylcholine receptor (AChR) in extracts and at endplates of developing, adult, and denervated rat muscle. The AChRs in normal adult muscle were immunoprecipitated by anti-epsilon and anti-delta, but not by anti-gamma antibodies, whereas AChRs in denervated and embryonic muscles were precipitated by anti-gamma and anti-delta, but showed little or no reactivity to anti-epsilon antibodies. In immunofluorescence experiments, AChRs at neonatal endplates bound antibodies to gamma or delta, but not epsilon, subunit, whereas those in adult muscles bound antibodies to epsilon or delta, but not gamma, subunit. AChRs at denervated endplates and at developing endplates between postnatal days 9 and 16 bound all three antibodies. We conclude that the distribution of gamma and epsilon subunits of the AChR parallels the distribution of AChRs with embryonic and adult channel properties, respectively.

Prolongation of evoked and spontaneous synaptic currents at the neuromuscular junction after activity blockade is caused by the upregulation of fetal acetylcholine receptors.

It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

Human ontogenesis. II. Development of the human neuromuscular junction.

The ultrastructural pattern of the early stages of end-plate formation is described in the quadriceps femoris muscle of 9- to 20-week-end human fetuses. Features of the neuromuscular junction are first observed in the ninth week of fetal life. The primitive motor end-plate contains a few axons always covered by one Schwann cell and does not seem to change either in number or in structure from the tenth through the twentieth week. Continuous modification of the post-synaptic apparatus is observed from the tenth to the twentieth weeks of human fetal life.

Quantal endplate currents from newborn to adult mice and the switch from embryonic to adult channel type.

Quantal endplate currents (qEPCs) were recorded extracellularly by a macropatch electrode from excised diaphragms of mice. During the first 3 days after birth, the mean rise time t(r) was 0.5 ms (0.1-0.9 peak, 20 degrees C). Double-exponential, amplitude-weighted fits of the decay discerned almost equally abundant components tau(1)' approximately equal to 6 ms and tau(2)' approximately equal to 9 ms. Beginning on day 3, on days 4 and 5 after birth both t(r) and the tau' dropped. Further decreasing slowly, adult values were reached at day 8, with t(r) approximately equal to 0.3 ms, tau(1)' approximately equal to 2 ms and a very weak tau(2)' approximately equal to 6 ms component. When compared to the kinetics of fetal channels, the tau(1)' and tau(2)' of up to 3 day qEPCs could correspond to the short and long splice variants of the fetal channel type. The tau(1)' of adult muscles of 2 ms agrees well with the burst durations of adult channels while a weak longer tau(2)' component may represent 'extrasynaptic' channels. The long t(r) of very young mice may correspond to the relatively slow rise of channel currents elicited by ACh pulses in mouse myoballs.

Retarded myelination in the lumbar spinal cord of piglets born with spread-leg syndrome.

Piglets born with spread-leg syndrome, a congenital weakness of the hindlimb adductors, were investigated to determine the site of lesion leading to limb impairment. Histological and immunohistochemical studies of the motor neuron unit showed no alterations but quantitative analysis revealed a reduction of axonal diameter and myelin sheath-thickness of the fibres innervating the adductors of the affected limbs. In the lumbar spinal cord a lack of myelination was observed in the tracts descending to the lower motor neurons. Recovery from the syndrome was accompanied by a catching-up of myelination with that of the controls. The spread-leg syndrome is due to a nutritional deficiency in the sow; thus it is assumed that the deficient maternal substances, mainly choline and methionine, are essential for the normal myelin production by spinal white matter oligodendrocytes of the fetus.

Regression of polyneural innervation in the human psoas muscle.

During the early stages of mammalian ontogeny muscle fibres are innervated by more than one axon. This polyneural innervation is replaced by mononeural innervation in the course of development. The regression of polyneural innervation in the psoas muscle in the human is the topic of the present study. Innervation patterns were studied in fetuses from 15 1/2 weeks of post menstrual age (PMA) and in babies until 80 weeks PMA (40 weeks after term age) and compared to data from two adults. Motor endplates were stained by a combined acetylcholinesterase stain. Innervation patterns and motor endplate morphology were studied and the sizes of endplates were measured. As a main result of our study polyneural innervation of the psoas muscle remains at a level of about 2 endings per endplate (range 1-5 terminals) until 18-25 weeks PMA and decreases thereafter. From 52 weeks PMA (12 weeks post term) onwards, muscle fibres are predominantly mononeurally innervated. During development the morphology of the terminal patterns of the nerve endings becomes more complex and the size of endplates increases, implying that the adult pattern of muscle innervation is reached at the age at which a major functional transformation in the neurobehavioural repertoire occurs (i.e. the end of the second and the beginning of the third month.

Three-dimensional architecture and development of lumber intervertebral discs.

The three-dimensional architecture of the collagen framework of human lumbar intervertebral discs was studied with scanning electron microscopy and polarized light microscopy concentrating on the fibrillar interconnection between the intervertebral disc and the vertebral bodies. The fibrillar framework of the annulus fibrosus and the cartilage end-plates encircled the nucleus pulposus as a closed-pack system in the adult. This closed-pack system developed in the seventh embryonic month and was completed by the tenth month. In general, fibrils composing the framework in the fetus were thinner than in the adult. There was no fibrillar interconnection between the cartilage end-plate and subchondral bone. In the inner one-third of the annulus, obliquely oriented fibrillar lamellae interconnected with the cartilage end-plate. In the outer two-thirds, the fibrillar bundles of the lamellae were firmly anchored into the vertebral bodies. This fibrillar anchoring system was already present in the full-term fetus although the vertebral rim was not ossified.

Neuromuscular junctions of the posterior cricoarytenoid muscle in the human adult, human fetus and cat. Histochemical and electron microscopic study.

The motor end plate of the posterior cricoarytenoid muscle (PCA muscle) in the human adult, human fetus and cat was examined by using electron microscopy and histochemical methods. In the present study, we observed the single-type motor end plate and en plaque type neuromuscular junction. At the neuromuscular junction of the fetal PCA muscle, the primary synaptic cleft, the basement membrane and the postsynaptic density could already be observed; however, there was no secondary synaptic cleft. Histochemically, the subneural apparatus was filled with electron-dense products, indicating acetylcholinesterase (AChE) activity. The primary and secondary synaptic clefts in the adult PCA muscle were well developed and intense AChE activity was present. The appearance of the neuromuscular junction and its localization of AChE activity was similar to that in the cat PCA muscle.

HP1-beta is required for development of the cerebral neocortex and neuromuscular junctions.

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-beta isotype, and show that the Cbx1(-/-) -null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1(-/-) mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1(-/-) mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.

The muscle protein Dok-7 is essential for neuromuscular synaptogenesis.

The formation of the neuromuscular synapse requires muscle-specific receptor kinase (MuSK) to orchestrate postsynaptic differentiation, including the clustering of receptors for the neurotransmitter acetylcholine. Upon innervation, neural agrin activates MuSK to establish the postsynaptic apparatus, although agrin-independent formation of neuromuscular synapses can also occur experimentally in the absence of neurotransmission. Dok-7, a MuSK-interacting cytoplasmic protein, is essential for MuSK activation in cultured myotubes; in particular, the Dok-7 phosphotyrosine-binding domain and its target in MuSK are indispensable. Mice lacking Dok-7 formed neither acetylcholine receptor clusters nor neuromuscular synapses. Thus, Dok-7 is essential for neuromuscular synaptogenesis through its interaction with MuSK.

Dynamics of soleplate formation.

Immunohistochemical techniques with anti-desmin, anti-acetylcholine receptor and anti-fibronectin antisera and autohistoradiography were used to determine the dynamics of neuromuscular synaptogenesis. Fast twitching muscles were taken from chick embryos at 5 to 14 days of incubation. "Primitive eminences" at terminal arborizations of motor neurons were composed of Karnowsky positive, anti-desmin and anti-acetylcholine receptor positive cells which contained sites bound to alpha-bungarotoxin. These cells, characterized as myoblasts, fused with the myotubes during formation of neuromuscular junctions in the sites of contact with terminal arborizations of motor neurons. Their nuclei and cytoplasmic organelles become the nuclei and organelles in the soleplate.

Development and motor innervation of a distal pair of fast and slow wing muscles in the chick embryo.

The chick wrist muscle ulnimetacarpalis dorsalis (umd) has two heads. Using myosin ATPase and acetylcholinesterase (ACh.E) staining it was shown that one of the heads is composed almost entirely of acid-stable muscle fibres with multiple end plates (slow muscle fibres) and the other of acid-labile fibres with single end plates (fast muscle fibres). The development of the muscle was traced from E7 (Stage 32-33) when it is a relatively homogeneous mass, to E18. The two heads of the muscle are first distinguishable, by ATPase staining, at E8 (Stage 33-34) prior to their cleaving. Both heads of the muscle are innervated by motoneurons positioned laterally in the lateral motor column in spinal segments 15 and 16. There is no observable difference in the positions of the motoneuron pools to the two heads. At E18 the motoneurons innervating the fast head tend to be slightly larger than those innervating the slow head.

Morphogenesis of motor endplates along the proximodistal axis of the mouse hindlimb.

The morphogenesis of motor endplates along the proximodistal hindlimb axis is described for the mouse using nonspecific cholinesterase histochemistry and electron microscopy. There is a two day lag in relative stages of development between a proximal muscle (rectus femoris, RF) and a distal muscle (flexor hallucis brevis, FHB). Cholinesterase activity first appears in the RF on embryonic day 15 and the FHB on embryonic day 17. In the following days, faint wisps of reaction product thicken, form small ovals on myotubes, and finally enlarge with internal ramifications as the muscle fibers increase in diameter. Axons first enter the RF between embryonic days 12 and 13, and contact both embryonic cells (most likely myoblasts) and cells assumed to be Schwann cells. Myotubes are present in the RF the following day. The first signs of synapse formation-appearance of symmetrical electron opaque membrane patches, and dense cored and synaptic vesicles--occur between axons and myotubes in the RF on embryonic day 15. During the following days basal lamina material accumulates in the synaptic cleft, coated vesicles and postjunctional folds appear in the myotubes, and synaptic vesicles accumulate in the axon terminals. By postnatal day 42 the axon terminals lay in primary gutters opposite deep secondary postjunctional folds, and are separated and capped by Schwann cell processes.