RESUMEN
Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.
Asunto(s)
Vesículas Extracelulares , Medios de Cultivo Condicionados/análisis , Medios de Cultivo Condicionados/farmacología , Vesículas Extracelulares/química , Citometría de Flujo/métodos , Manejo de Especímenes , Ultracentrifugación/métodosRESUMEN
Gangliosides are glycosphingolipids highly abundant in the nervous system, and carry most of the sialic acid residues in the brain. Gangliosides are enriched in cell membrane microdomains ("lipid rafts") and play important roles in the modulation of membrane proteins and ion channels, in cell signaling and in the communication among cells. The importance of gangliosides in the brain is highlighted by the fact that loss of function mutations in ganglioside biosynthetic enzymes result in severe neurodegenerative disorders, often characterized by very early or childhood onset. In addition, changes in the ganglioside profile (i.e., in the relative abundance of specific gangliosides) were reported in healthy aging and in common neurological conditions, including Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), stroke, multiple sclerosis and epilepsy. At least in HD, PD and in some forms of epilepsy, experimental evidence strongly suggests a potential role of gangliosides in disease pathogenesis and potential treatment. In this review, we will summarize ganglioside functions that are crucial to maintain brain health, we will review changes in ganglioside levels that occur in major neurological conditions and we will discuss their contribution to cellular dysfunctions and disease pathogenesis. Finally, we will review evidence of the beneficial roles exerted by gangliosides, GM1 in particular, in disease models and in clinical trials.
RESUMEN
Brain cholesterol homeostasis is altered in Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of a CAG nucleotide repeat in the HTT gene. Genes involved in the synthesis of cholesterol and fatty acids were shown to be downregulated shortly after the expression of mutant huntingtin (mHTT) in inducible HD cells. Nuclear levels of the transcription factors that regulate lipid biogenesis, the sterol regulatory element-binding proteins (SREBP1 and SREBP2), were found to be decreased in HD models compared to wild-type, but the underlying causes were not known. SREBPs are synthesized as inactive endoplasmic reticulum-localized precursors. Their mature forms (mSREBPs) are generated upon transport of the SREBP precursors to the Golgi and proteolytic cleavage, and are rapidly imported into the nucleus by binding to importin ß. We show that, although SREBP2 processing into mSREBP2 is not affected in YAC128 HD mice, mSREBP2 is mislocalized to the cytoplasm. Chimeric mSREBP2-and mSREBP1-EGFP proteins are also mislocalized to the cytoplasm in immortalized striatal cells expressing mHTT, in YAC128 neurons and in fibroblasts from HD patients. We further show that mHTT binds to the SREBP2/importin ß complex required for nuclear import and sequesters it in the cytoplasm. As a result, HD cells fail to upregulate cholesterogenic genes under sterol-depleted conditions. These findings provide mechanistic insight into the downregulation of genes involved in the synthesis of cholesterol and fatty acids in HD models, and have potential implications for other pathways modulated by SREBPs, including autophagy and excitotoxicity.