Dataset Information

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

ABSTRACT: Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

SUBMITTER: Dar GH

PROVIDER: S-EPMC8602309 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Dar Ghulam Hassan GH Mendes Cláudia C CC Kuan Wei-Li WL Speciale Alfina A AA Conceição Mariana M Görgens André A Uliyakina Inna I Lobo Miguel J MJ Lim Wooi F WF El Andaloussi Samir S Mäger Imre I Roberts Thomas C TC Barker Roger A RA Goberdhan Deborah C I DCI Wilson Clive C Wood Matthew J A MJA

Nature communications 20211118 1

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal tha ...[more]

PMID: 34795295

Similar Datasets

Project description:Mesenchymal stromal cells (MSCs) are multipotent stromal cells with regenerative, immunomodulatory, and anti-inflammatory properties, making them a promising tool in cell-based therapies. Increasing evidence suggests the therapeutic effects of MSCs are largely mediated by secreted paracrine factors and extracellular vesicles (EVs), which facilitate extracellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids to target cells. The lipid composition of EVs is critical for their stability, uptake, and functional activity. Sphingomyelinase (SMase), an enzyme that hydrolyzes sphingomyelin into ceramide, plays a key role in EV biogenesis by influencing membrane curvature allowing for invagination and budding of multivesicular bodies (MVBs). However, the impact of SMase treatment on the characteristics and therapeutic potential of MSC-derived EVs remains largely unexplored. In this study, human MSCs were cultured with SMase for 24 hours and EVs were isolated via ultracentrifugation. EV size distribution was analyzed using nanoparticle tracking analysis (NTA), microRNA (miRNA) profile was determined using RNA-sequencing, while lipidomic and proteomic profiling were assessed using mass spectrometry. Functional assays were conducted to assess changes in bioactivity. NTA revealed a shift in EV production, with a higher vesicle concentration in SMase induced MSCs compared to untreated cells. SMase treatment significantly altered MSC-EV composition, leading to increased ceramide and glycosphingolipids. In addition, the modifications in protein and miRNA EV cargo were found to be associated with pathways involving TNF-α signaling, vesicle trafficking, wound healing, and angiogenesis. Functional assays indicated greater suppression of TNF-α in activated macrophages and greater tubular network formation in HUVECS. These findings provide new insights into distinct biophysical and biochemical changes in MSC-derived EVs following SMase stimulation, potentially enhancing their therapeutic properties and may inform strategies for optimizing future EV-based therapies.

Project description:BackgroundThe blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the United States. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied when compared to other arthropod vectors. I. scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility.MethodsUsing BLAST, an in-silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. I. scapularis nymphs were injected with small interfering RNAs to knock down rab27 then fed on naïve and A. phagocytophilum infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition.ResultsOur BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis.ConclusionsRab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

Dataset Information

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Publications

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets