Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission

Abstract

Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shownDrosophilacofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics byDmCFLknockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However,DmCFLknockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin’s roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology.Summary statementCofilin regulates muscle postsynaptic actin organization, structural maintenance, glutamate receptor composition, and neuromuscular junction function in aDrosophilanemaline myopathy disease model.

Postsynaptic BMP signaling regulates myonuclear properties inDrosophilalarval muscles

“…To confirm that the core BMP pathway components (Fig. 2A) are expressed in larval muscles, we analyzed publicly available RNA sequencing data of larval muscle-enriched preps (Christophers et al, 2023). We found moderate expression of genes encoding the BMP type II receptor put (Punt), type I receptors tkv (Thickveins) and sax (Saxophone), BMP transcriptional effector Mad and its co-factor Med , and BMP ligand gbb (Glass bottom boat) (Fig.…”

“…We hypothesize that synaptic myonuclei (magenta) are exposed to higher pMad levels than non-synaptic myonuclei (gray). (B) Normalized mRNA counts for Drosophila BMP pathway genes in third instar control Mhc>mCherry-RNAi larvae from a published bulk RNA sequencing dataset (Christophers et al, 2023). (C) Confocal image (sum slice projection) of VL3-VL4 muscle pair from a Dmef2>mCherry-RNAi control larva immunolabeled for pMad.…”

“…In Imaris 10.0 (Bitplane), confocal z-stacks of GluRIIA and HRP immunostainings were processed as previously done (Christophers et al, 2023). A three-dimensional surface for HRP was generated with a surface detail grain level of 0.355 µm, smoothing enabled, and auto-thresholding.…”

“…Membrane receptors within the SSR, including glutamatergic neurotransmitter receptors, participate in synaptic signaling which leads to muscle contraction (DiAntonio et al, 1999; Nguyen and Stewart, 2016; Zou and Pan, 2022). The SSR develops following new bouton formation (Vasin et al, 2019), and critical regulators of SSR development include scaffolding proteins (Lahey et al, 1994), adhesion proteins (Banovic et al, 2010; Xing et al, 2018), and actin modulators (Blunk et al, 2014; Christophers et al, 2023). Like at the vertebrate postsynapse, Drosophila myonuclei can support SSR development.…”