Biology subjects

Stewart, B. A.

Publications and source records attributed to Stewart, B. A..

5 recordsLinked to original sources

bioRxiv2023

ESCRT disruption provides evidence against signaling functions for synaptic exosomes

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

cell biology↗

bioRxiv2023

Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues

3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples or from stem cells can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity and appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD and iPSC derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the trophic mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.

cell biology↗

bioRxiv2021

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery

Neuronal extracellular vesicles (EVs) can be locally released from presynaptic terminals, carrying cargoes that are important in intercellular signaling and disease. EVs are derived from endosomes, but it remains unclear how synaptic cargoes are directed to the EV pathway, rather than undergoing conventional retrograde endosomal transport and degradation. Here, we find that the clathrin-mediated endocytic machinery plays an unexpected role in maintaining a release-competent pool of synaptic EV cargoes. Endocytic mutants, including nervous wreck (nwk), Shibire/Dynamin, and AP-2, exhibit local depletion of multiple cargoes in EV donor terminals. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo Synaptotagmin-4, and suppress lethality upon overexpression of the EV cargo Amyloid Precursor Protein. These EV defects are genetically separable from canonical functions of endocytic proteins in synaptic vesicle recycling and synaptic growth. This endocytic pathway opposes the endosomal retromer complex to regulate EV cargo levels, and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that protects EV cargoes from local depletion at synapses.

cell biology↗

bioRxiv2020

Electrophysiological analysis of healthy and dystrophic 3D bioengineered skeletal muscle tissues

Recently, methods for creating three-dimensional (3D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study we provide an electrophysiological analysis of healthy and dystrophic 3D bioengineered skeletal muscle tissues. We focus on Duchenne muscular dystrophy (DMD), a fatal muscle disorder involving the skeletal muscle system. The dystrophin gene, which when mutated causes DMD, encodes for the Dystrophin protein, which anchors the cytoskeletal network inside of a muscle cell to the extracellular matrix outside the cell. Here, we enlist a 3D in vitro model of DMD muscle tissue, to evaluate an understudied aspect of DMD, muscle cell electrical properties uncoupled from presynaptic neural inputs. Our data shows that electrophysiological aspects of DMD are replicated in the 3D bioengineered skeletal muscle tissue model. Furthermore, we test a block co-polymer, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle.Therefore, this study serves as the baseline for a new in vitro method to examine potential therapies directed at muscular disorders.

cell biology↗

bioRxiv2018

The neuronal ceroid lipofuscinosis protein, Cln7, regulates neural development from the post-synaptic cell.

The neuronal ceroid lipofuscinoses (NCLs) are a group of fatal, monogenic neurodegenerative disorders with an early onset in infancy or childhood. Despite identification of the genes disrupted in each form of the disease, their normal cellular role and how their deficits lead to disease pathology is not fully understood. Cln7, a major facilitator superfamily domain-containing protein, is affected in a late infantile-onset form of NCL. Using the Drosophila larval neuromuscular junction as a model to study neural development, we demonstrate that Cln7 is required for the normal growth of synapses. In a Cln7 mutant, synapses fail to develop fully leading to reduced function and behavioral changes with dysregulation of TOR activity. Cln7 expression is restricted to the post-synaptic cell and the protein localizes to vesicles immediately adjacent to the post-synaptic membrane. Our data suggest an involvement for Cln7 in regulating trans-synaptic communication.

developmental biology↗