Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron–Myotube Interactions Required for Functional Synapse Formation

Abstract

The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation. Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.

Identifying gene expression programs of cell-type identity and cellular activity with single-cell RNA-Seq

“…An additional activity program which we labeled Synaptogenesis (Syn) was characterized by expression of genes that play a crucial role in synapse formation, including the transcriptional regulator Mef2c (Barbosa et al, 2008; Flavell et al, 2008; Harrington et al, 2016), synaptic adhesion molecules Ncam1 (Hata et al, 2018) and Cadm1 (Biederer et al, 2002; Robbins et al, 2010), membrane vesicle traffickers Syt1 and Syt4 , Actb which constitutes the predominant synapse cytoskeletal protein, and others with a strong connection to synapse biology such as Ywhaz (Foote et al, 2015; Ramser et al, 2010; Xu et al, 2015), Bicd1 (Aguirre-Chen et al, 2011; Li et al, 2010). It was also significantly enriched for relevant Gene Ontology sets including postsynapse, glutamatergic synapse, postsynaptic density, and dendrite morphogenesis (p<=3.25×10 −6 , Supplementary file 3) which further suggests its interpretation as a program involved in the formation or regulation of synapses.…”

Smoke‐induced neuromuscular junction degeneration precedes the fibre type shift and atrophy in chronic obstructive pulmonary disease

“…Sections were dehydrated in an ethanol series and cleared in xylene (Malicdan et al 2009). We also assessed neural cell adhesion molecule (NCAM; aka CD56) labelling in tissue cross-sections, noting that NCAM is a cytokine produced by denervated muscle fibres and motor neurons which comes in three distinct isoforms (180, 140 and 120 kDa) and which is essential for reinnervation and formation of stable motor neuron-muscle contacts (Hata et al 2018). Briefly, sections were post-fixed in 4% paraformaldehyde (ChemCruz, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 20 min at room temperature, washed with 1× TBS, permeabilized with 0.1% Triton-X for 15 min and washed with 1× TBS-0.05% Tween 20 (TBST).…”

Identifying Gene Expression Programs of Cell-type Identity and Cellular Activity with Single-Cell RNA-Seq

“…An additional activity program which we labeled Synaptogenesis (Syn) was characterized by expression of genes that play a crucial role in synapse formation, 13 including the transcriptional regulator Mef2c [31][32][33] , synaptic adhesion molecules Ncam1 34 and Cadm1 35,36 , membrane vesicle traffickers Syt1 and Syt4, Actb which constitutes the predominant synapse cytoskeletal protein, and others with a strong connection to synapse biology such as Ywhaz [37][38][39] , Bicd1 40,41 . It was also significantly enriched for relevant Gene Ontology sets including postsynapse, glutamatergic synapse, postsynaptic density, and dendrite morphogenesis (P<=3.25x10 -6 Supplementary Table 3) which further suggests its interpretation as a program involved in the formation or regulation of synapses.…”