Biology subjects

Wharton, K. A.

Publications and source records attributed to Wharton, K. A..

4 recordsLinked to original sources

bioRxiv2023

The MuSK-BMP pathway regulates synaptic Nav1.4 localization and muscle excitability

The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ( {Delta}Ig3-MuSK). Synapses formed normally in {Delta}Ig3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in {Delta}Ig3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at {Delta}Ig3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging. Significance StatementThe neuromuscular junction (NMJ) is required for nerve-evoked muscle contraction and movement, and its function is compromised during aging and disease. Although the mechanisms underlying neurotransmitter release and cholinergic response at this synapse have been studied extensively, the machinery necessary for nerve-evoked muscle excitation are incompletely characterized. We show that MuSK (Muscle-specific kinase), in its role as a BMP co-receptor, regulates NMJ structure as well as the localization of the voltage-gated sodium channels necessary for full nerve-evoked muscle fiber excitation and force production. This novel function of MuSK is structurally and mechanistically distinct from its role in organizing cholinergic machinery. The MuSK-BMP pathway thus presents a new opportunity to understand mechanisms that may preserve or enhance neuromuscular excitability in the face of aging and disease.

neuroscience↗

bioRxiv2022

Heterodimerization-dependent secretion of BMPs in Drosophila

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis and disease, Bone Morphogenetic Proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homo- or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homo- and heterodimers in the Drosophila wing imaginal disc. This approach revealed in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb, despite being produced by all the cells of the wing imaginal disc, is only secreted by the cells in which Dpp is expressed. Dpp and Gbb form a gradient of heterodimers whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution in the developing wing. These results indicate that Dpp/Gbb heterodimers are the active signal required for epithelial patterning and growth.

developmental biology↗

bioRxiv2021

Glia-neuron signaling mediated by two different BMP ligands impacts synaptic growth

The nervous system is a complex network of cells whose interactions provide circuitry necessary for an organism to perceive and move through its environment. Revealing the molecular basis of how neurons and non-neuronal glia communicate is essential for understanding neural development, behavior, and abnormalities of the nervous system. BMP signaling in motor neurons, activated in part by retrograde signals from muscle expressed Gbb (BMP5/6/7) has been implicated in synaptic growth, function and plasticity in Drosophila melanogaster. Through loss-of-function studies, we establish Gbb as a critical mediator of glia to neuron signaling important for proper synaptic growth. Furthermore, the BMP2/4 ortholog, Dpp, expressed in a subset of motor neurons, acts by autocrine signaling to also facilitate neuromuscular junction (NMJ) growth at specific muscle innervation sites. In addition to signaling from glia to motor neurons, autocrine Gbb induces signaling in larval VNC glia which strongly express the BMP type II receptor, Wit. In addition to Dpps autocrine motor neuron signaling, Dpp also engages in paracrine signaling to adjacent glia but not to neighboring motor neurons. In one type of dorsal midline motor neuron, RP2, dpp transcription is under tight regulation, as its expression is under autoregulatory control in RP2 but not aCC neurons. Taken together our findings indicate that bi-directional BMP signaling, mediated by two different ligands, facilitates communication between glia and neurons. Gbb, prominently expressed in glia, and Dpp acting from a discrete set of neurons induce active Smad-dependent BMP signaling to influence bouton number during neuromuscular junction growth.

neuroscience↗

bioRxiv2017

Alternative cleavage of a bone morphogenetic protein (BMP) produces ligands with distinct developmental functions and receptor preference

TGF-{beta} and Bone Morphogenetic Protein (BMP) family proteins are made as proprotein dimers, which are cleaved by proprotein convertases to release the active C-terminal ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct forms of active ligand. Cleavage at the S1 or atypical S0 site produces Gbb15, the conventional small BMP ligand, while cleavage at the NS site produces the larger Gbb38 ligand (1, 2). Here, we found that blocking NS cleavage increased association of the full length prodomain with Gbb15 resulting in a concomitant decrease in signaling activity. NS cleavage is required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not in cell culture to enable Gbb15-Dpp het-erodimer activity. Gbb NS cleavage is also required in vivo for the regulation of pupal ecdysis and viability that is dependent on the type II receptor Wishful thinking (Wit). We found that the ability of Gbb38 to signal requires the expression of either Wit or the type I receptor, Saxophone (Sax). Finally, we discovered that the production of Gbb38 in 3rd instar larvae results when processing at the S1/S0 site is blocked by O-linked glycosylation. Our findings demonstrate that BMP prodomain cleavage can ensure that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligand types that signal preferentially through different receptors and exhibit specific developmental functions.

biochemistry↗