Maria Teresa PELLECCHIA | Progetti

INSULIN RESISTANCE IN NEURONS AND OLIGODENDROCYTES DERIVED FROM MSA IPSCS

iPSCs from MSA-P and HC donors will be differentiated into DA neurons and oligodendrocytes usingpublished protocols standardized in our laboratories (Kriks et al. 2011; Fedele et al. 2014; Piao et al. 2015).DA neurons and oligodendrocytes from MSA-P and HC donors will be characterized and compared atbaseline.For DA neurons we will evaluate: expression of markers of differentiation and maturation including NURR-1, TH, DAT, GIRK2, VMAT2 by immunofluorescence analysis and qPCR, cell death and changes inmorphology of dendrites and soma (Fedele et al. 2017) at early (day 30) and late (day 70-80) differentiationstages.For oligodendrocytes, we will study the number of progenitors expressing markers of early differentiation,i.e., PDGFRα, Olig2 and NKX2.2, and their morphology at early differentiation stages (day 50). Similarassessments will be performed at late differentiation stage (day 90-100) for oligodendrocytes expressing thematuration markers O1, O4, SOX10 and MBP.Analytic cytometric analysis by FACS will be performed at early and late differentiation stages to evaluatethe numbers of DA neurons and oligodendrocytes that differentiate from MSA-P or HC iPSCs.α-synuclein aggregates/inclusions will be studied in DA neurons and oligodendrocytes byimmunofluorescence and western blot analysis using antibodies specific for human α-synuclein (Recasens etal. 2014; Bassil et al. 2017). Quantification of α-synuclein aggregate/inclusions within the DA neurons andoligodendrocytes will be performed with ImageJ software. Expression of α-synuclein will also be assessed atRNA level by qPCR.The functional integrity of the insulin/IGF-1 pathway will be evaluated by measuring the expression of IRS-1pS312 and pS616 in basic conditions (Talbot et al. 2012; Bassil et al. 2017). Insulin/IGF-1 resistance of DAneurons and oligodendrocytes of MSA-P will be compared to HC donors by evaluating the expression ofIRS-1 pS312 and pS616 by immunofluorescence in basic conditions. Multiplex immunofluorescence will beperformed to quantify the number of TH+ DA neurons, Sox10+ oligodendrocyte progenitors, O4/MBP+mature oligodendrocytes expressing IRS-1 pS312 and pS616. Fluorescence intensity and intracellularlocalization in nucleus or cytoplasm of IRS-1 pS312 and pS616 will be evaluated (Bassil et al. 2017).Western blot analysis of IRS-1 pS312 and pS616 will be performed by Meissner laboratory, in parallelexperiments on cell lysates prepared from DA and oligodendrocytes cultures respectively. The level ofphosphorylation of IRS-1 pS312 and pS616 will be quantified. Difference in levels of IRS-1 pS312 and pS616 between MSA-P and controls will be indicative of insulin/IGF-1 resistance.Once the baseline conditions are established, we will expose DA neurons and oligodendrocytes of MSA-Ppatients and HC donors at early and late stages of differentiation to α-synuclein fractions derived fromMSA and/or PD patients. α-synuclein species will be provided by Meissner laboratory (Recasens et al.2014). Preliminary investigation will be performed to assess the optimal testing dose. The neuropathologyinduced by α-synuclein will be evaluated in time course experiments; cells will be processed at: 24 hrs, 1week, 2 weeks and, for the treatment performed at early differentiation stages, 1 month.Immunofluorescence, morphological analysis (loss of DA neurons and oligodendrocytes, changes in DAneuron and oligodendrocytes morphology, presence and number of α-synuclein intracellular inclusions)will be performed. Cell loss will be evaluated by analytic cytometric analysis by FACS. Western blotanalysis to study high molecular weight and monomeric α-synuclein (Bassil et al. 2017) will be performed.The functional integrity of the insulin/IGF-1 pathway will be further studied in DA neurons andoligodendrocytes following exposure to α-synuclein species. Cells will be processed for western blotanalysis of IRS-1 pS312 and pS616 and immunofluorescence analysis.