SPK AND STROKE · Sphingosine kinase 2-mediated preconditioning in stroke

Sphingosine kinase 2-mediated preconditioning in stroke

Description of Work Performed and Major Findings Stroke is the 3rd leading cause of death in the Western World. However, the only available drug, recombinant tissue Plasminogen Activator (tPA), is only used in ~4% of patients. Effective therapies are therefore needed. Our studies focused on preconditioning (in which a stimulus below the threshold of damage is applied, leading to tissue resistance to the same, or different stimuli given beyond damage threshold), because it is an attractive new strategy to identify endogenous protective mechanisms that could be therapeutically implemented, particularly in patients at high risk for stroke (e.g., before carotid endarterectomy or coronary artery bypass). Using two preconditioning stimuli to explore the role of sphingosine kinase 2 (SPK2) in preconditioning, we found that a protective intracellular degradation process termed autophagy is induced by hypoxia and ischemic preconditioning in neurons or brain, and that SPK2 is a key mediator of these effects. We then examined the molecular pathways involved in the process, and demonstrated that SPK2 can directly activate autophagy to protect neurons from injury caused by oxygen and glucose deprivation (an in vitro model of stroke). At the molecular level, we found that SPK2, via its BH3 domain, could dissociate a protein complex made of the proteins Beclin-1 and Bcl-2 by interacting with Bcl-2 to activate autophagy. In order to design potential therapeutic agents exploiting this mechanism, we designed a Tat-SPK2 peptide based on the BH3 domain of SPK2. Our studies show that this Tat-SPK2 peptide promotes neuronal survival through induction of autophagy, implying that SPK2 inducers, Tat-SPK2 peptide or agents acting on Beclin-1/autophagy pathway may provide candidate therapeutic agents against ischemic stroke. Our studies are now investigating whether Tat-SPK2 peptide is effective in animal models of ischemic stroke. Preliminary results are encouraging, and suggest that this approach would constitute a novel therapeutic approach for the management of stroke. Socio-economic impact and wider societal implications of the project Despite an expanding understanding of the cellular and molecular mechanisms underlying ischemia/reperfusion injury, 1000+ experimental compounds have failed at some stage of development. There is a lack of effective treatment for most stroke patients. Thus, innovative therapeutic approaches are needed to develop an effective treatment for stroke. Our studies have identified a key molecular interaction underlying endogenous protective mechanisms induced by preconditioning and its potential to be exploited therapeutically. Autophagy is a process of cytoplasmic component degradation via lysosomes. It plays a role in the pathophysiology of many disorders, including neurodegeneration, heart disease, and cancer. Our findings support the concept that autophagy is a novel therapeutic approach of promoting tolerance to brain ischemia, which will lead to novel therapeutic strategies for stroke and other disorders associated with neuronal death. Training and Integration of Marie Curie CIG Researcher During the finding period, the funded researcher has fully integrated the teaching and research community at his institution, initiating various new research collaboration projects (e.g., on the role of a putative bacterial sphingosine kinase in regulating the interaction between host and microbiome in the gut; on the role of sphingolipid signalling in promoting bone repair; on the development of novel sphingosine kinase inhibitors; on the establishment of a novel stroke model in pigs. This model, combining imaging, histological and functional outcome measures is unique in the world and has the potential to attract significant Pharma industry funding in the future). The researcher is currently co-supervising 5 PhD students and a research assistant. In addition to his research project, the funded researcher has taken on a leadership role in the Animal Experimentation Ethics Committee of his institution, streamlining the application review process and expanding on the educational mission of the committee. The support provided by the CIG funding and the Researcher’s host department were instrumental in helping the Researcher successfully compete for additional grant funding, in particular an HRB Health Research Award focused on the preclinical characterization of fingolimod as a potential therapeutic agent for stroke, and an Irish Research Council Award focused on the study of protective regulatory T lymphocytes and the role in stroke protection by fingolimod. Finally, an application by the Cork NeuroScience Centre, including the researcher, has led to the Cork NeuroScience Centre being designated as a “Centre of Excellence in Neurodegeneration” (CoEN) after a national and international review process. This is a highly significant achievement for the Cork NeuroScience Centre giving international recognition to the high quality integrative multidisciplinary research in neurodegeneration in UCC. By gaining CoEN status, the Cork NeuroScience Centre has successfully applied for research funding awarded through the international CoEN initiative.

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