Sphingolipidomic approach to define cytokine-mediated oligodendrocyte cell death signaling.
Sphingolipids are particularly abundant in the central nervous system (CNS) as part of the membrane components of both neurons and glia including astrocytes, microglia and oligodendrocytes, the myelin forming cells of the CNS. Several neurological disorders are associated with deregulation in their metabolism due to genetic defects and metabolic alterations thereby suggesting the importance of therapeutic targeting of specific pathways/enzymes of sphingolipid metabolism. As an example, FTY720, an activator of sphingosine-1-phosphate (S-1-P) receptor is now an approved drug to treat multiple sclerosis due to its immunosuppressive actions. It may also elicit direct neuro- and glio-protective actions by countering toxicity due to abnormal ceramide accumulation. In particular, oligodendrocytes are known to be susceptible to ceramide-mediated cytokine toxicity relevant to MS pathogenesis. Previously, we had reported that the two cytokines i.e., TNF-alpha and IFN-gamma, known to play critical pathogenic roles in MS, in combination induce a marked apoptotic death of oligodendrocyte progenitors (OPC), the cells required for remyelination in MS. The mechanism underlying the synergistic cytotoxic effects of the cytokines is still not understood. Although exogenous ceramide has been shown to be toxic to OPC, its intracellular levels in response to cytokines could not explain synergistic toxicity. We considered the possibility that the cytokine-treated OPC may release ceramide-laden exosomes that may promote the synergistic actions of TNF-alpha and IFN-gamma. In preliminary studies using an oligodendrocyte cell line as a model, we have in fact found that secreted vesicles or exosomes act as a paratropic mediator of synergistic cytotoxicity of the two cytokines. Thus, the exosomal preparations obtained from TNF-alpha-treated ‘donor' cells, while being mildly toxic to fresh cultures, induced enhanced cell death when added to IFN-gamma-primed target cultures in a fashion resembling the effect of cytokine combination. Further, we determined the basic sphingolipid profiles of the secreted exosomes by HPLC-MS/MS (Lipidomics Core, MUSC). The treatment with the cytokines time-dependently induced the formation and (preferential) release, in particular of C24:1-, C24- and C18-Cer species; C24:1-, C24-, and C16-dehydroCer species; and C16-, C24:1- and C24-SM species. That exosome-associated Cer contributes to the cytotoxic activity was supported by the observation that exogenous C6-Cer replicated, albeit partially, the cytotoxic effect of Cer-enriched exosomes from TNF-alpha-treated cells on IFN-gamma-primed cultures.
The goal of this pilot project is to test the hypothesis that exosomes may be formed and released from stressed oligodendrocytes or their progenitors in vivo under an inflammatory environment and/or in response to triggers of demyelination and, by ‘spreading' the cell death signal, interfere with the process of remyelination. We will first confirm our preliminary observations made in the immortalized oligodendrocyte cell line on the role of exosomes in cytokine-induced synergistic cell death using primary oligodendrocyte cultures to support their physiological significance. Then, to test the in vivo validity of exosome formation, we will set up cuprizone model of demyelination and isolate exosomes and determine their sphingolipid profile and cytotoxic activity in cultured oligodendrocytes in the presence and absence of individual cytokines. The outcome of the proposed studies on exosome-mediated oligodendrocyte cell death could lead to novel lipid signaling-based treatment strategies for MS and other demyelinating diseases and conditions.