032 – Development of drug-screening platform with murine and human neurons for multiple sclerosis (MS) to identify compounds with a neuroprotective potential and assess disease-specific dysfunctions.
Svetlana Bezukladova (1) – Annamaria Cafarella (1) – Francesca Ruffini (1) – Elena Brambilla (1) – Sergio Baranzini (2) – Linda Ottoboni (1) – Gianvito Martino (1)
San Raffaele Scientific Institute, Division of Neuroscience – INSPE – Institute of Experimental Neurology, Neuroimmunology Unit, Milano, Italy (1) – UCSF Weill Institute for Neurosciences, Department of Neurology, University of California, San Francisco, United States (2)
Background: Progressive Multiple Sclerosis is a debilitating disease that still remains untreatable, thus discovery of new agents promoting neuroprotection, blocking further neurodegeneration and supporting myelin differentiation is critically needed. Drug repurposing (or repositioning) is a favorable alternative to the development of a new drug, with a significantly lower economic burden. The possibility of generating patient-specific human induced pluripotent stem cells (hiPSC) allow testing and validation of a large platform of drugs on disease-specific human cells. The project aims are: 1) to identify the HIT compounds using in silico approach; 2) to perform drug toxicity test on primary murine cortical neurons; 3) to perform cytokine-, glutamate- and reactive oxygen species-mediated stressor assays on primary murine and hiPSC-derived cortical neurons; 4) to evaluate neuroprotective properties of the HIT compounds on cells exposed to stressors with functional and morphological outcome.
Methods: Repurposed drugs with predictive neuroprotective functions have been pre-selected (n=270) using the bioinformatic tool SPOKE among more than 1500 compounds. To assess their neural toxicity properties E17.5 murine cortical neurons were cultured in 96 well plate and exposed to two doses of the compounds (1uM and 10uM) for 24h, followed by CCK8 cytotoxicity assay. Metabolic (tetrazolium salt), mitochondrial (JC1) and calcium signaling (FLUO4-AM) are functional outcomes. Cerebral organoids are differentiated using STEMCELL proprietary commercial medium.
Results: Neural toxicity of 270 compounds has been evaluated: 146/270 (54%) at 1 uM and 97/270 (36%) at 10 uM promotes cell metabolic activity, while 43/270 (16%) compounds were cytotoxic at both concentrations based on tetrazolium results. Optimized concentration of ex-cytotoxic agents impairs 30-40% of neuronal metabolic activity.
Conclusion: 160/270 HIT compounds will be tested for metabolic and functional neuroprotective role in preventive regimen on both mouse and hiPSC-derived neurons. A selected number of compounds will be tested on cerebral organoids.
33 – RORgammaT is overexpressed in iNKT and gamma-delta T cells during relapse in relapsing-remitting multiple sclerosis
Micha_ Zarobkiewicz (1) – Wioleta Kowalska (1) – Pawe_ Halczuk (2, 3) – Justyna Wo_ (1) – Barbara Jod_owska-J_drych (3) – Konrad Rejdak (2) – Jacek Roli_ski (1) – Agnieszka Bojarska-Junak (1)
Department of Clinical Immunology, Medical University of Lublin, Lublin, Poland (1) – Department of Neurology, Medical University of Lublin, Lublin, Poland (2) – Department of Histology, Medical University of Lublin, Lublin, Poland (3)
Background and objectives: The invariant NKT (iNKT) type I cells and gamma-delta T lymphocytes are both small subsets of T cells expressing specific TCR. They are capable of cytotoxic activity and rapid cytokine production with IL-17A among them. IL-17 seems to be one of the crucial factors involved in the pathogenesis of multiple sclerosis. Its expression is regulated by RORgammaT and also indirectly by IL-23R. The aim of the current study is to evaluate IL-17 production and RORgammaT, and IL-23R expression by iNKT, Th17 and gamma-delta T cells in the peripheral blood of relapsing-remitting multiple sclerosis patients. Materials and Methods: Samples of peripheral blood were taken from 19 patients in relapse and 5 patients in remission as well as 10 healthy volunteers. Specimens were stained with anti-CD3 PE-Cy5, anti-CD4 FITC, anti-IL-17A PE, anti-IL-17A Pacific Blue, anti-TCRgamma-delta FITC, anti-RORgammaT PE, anti-IL-23R PE, anti-iNKT FITC monoclonal antibodies. The standard whole blood assay with erythrocyte cell lysis was followed. Next, flow cytometry was performed. The statistical analysis was performed in GraphPad Prism 8. Results: No significant intergroup differences in iNKT and Th17 percentages were noted. The percentage of gamma-delta T cells was, however, significantly decreased in both relapse and remission compared to the healthy control. The significant overexpression of RORgammaT was observed in all three studied subpopulations. Conclusions: The role of iNKT and gamma-delta T cells in IL-17 production seems to be of minor importance in the already established relapse. It seems, however, that it is far more important shortly prior to the relapse. This is supported by the significantly up-regulated expression of RORgammaT in all three subpopulations.