title: Protein Characterization of Extracellular Microvesicles/Exosomes Released from Cytotoxin-Challenged Rat Cerebrocortical Mixed Culture and Mouse N2a Cells.
abstract: A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142 nm for CTX and 49-77 nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.
authors.affiliation: Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, Gainesville, FL, 32611, USA.|The Departments of Psychiatry, University of Florida, Gainesville, FL, 32611, USA.
authors.forename: Dhwani
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, Gainesville, FL, 32611, USA. mrach8@ufl.edu.|The Departments of Psychiatry, University of Florida, Gainesville, FL, 32611, USA. mrach8@ufl.edu.|The Departments Neuroscience, University of Florida, Gainesville, FL, 32611, USA. mrach8@ufl.edu.
authors.forename: Rachna
authors.initials: R
authors.lastname: Manek
authors.affiliation: Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, Gainesville, FL, 32611, USA.|The Departments of Psychiatry, University of Florida, Gainesville, FL, 32611, USA.|Schizophrenia Research Foundation, R/7A, North Main Road, Anna Nagar West Extension, Chennai, Tamil Nadu, 600101, India.
authors.forename: Vijaya
authors.initials: V
authors.lastname: Raghavan
authors.affiliation: Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, Gainesville, FL, 32611, USA. kwang@ufl.edu.|The Departments of Psychiatry, University of Florida, Gainesville, FL, 32611, USA. kwang@ufl.edu.|The Departments Neuroscience, University of Florida, Gainesville, FL, 32611, USA. kwang@ufl.edu.
authors.forename: Kevin K
authors.initials: KK
authors.lastname: Wang
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D055252:Cell-Derived Microparticles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018920:Coculture Techniques; D003603:Cytotoxins / Q000633:toxicity*; D055354:Exosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D003603:Cytotoxins; D008565:Membrane Proteins
keywords: Astroglial injury; Calpain; Caspase; Exosome; Microvesicles; Neuronal injury