Field: Neuroinflammation and Neurodegeneration. Title: Molecular Determinants of Neurodegeneration in Progressive Multiple Sclerosis.

A - During the first year, we will characterise and compare microglial proteomes from formalin-fixed paraffin-embedded (FFPE) material from the motor cortex of 'high' and 'low' fibrin(ogen) MS and control cases in HLA-DRB1*15 positive/negative groups, via laser-capture microdissection (LCM) and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS), relating protein networks to cortical neuronal loss6. B - The second year will be used to identify proteins via LCM and LC-MS/MS that are secreted by fibrin(ogen)-stimulated HLA-DRB1*15 positive/negative human iPSC-derived microglial monocultures and microglial-neuronal co-cultures that overlap with the post-mortem microglial proteome (A)7,8. C - Year three will begin by evaluating and quantifying the distribution and extent of selected proteins (B), and their relationship to neuronal loss via the use of commercial antibodies and double-labelled immunofluorescent analysis9. FFPE material from the motor cortex of independent post-mortem MS and control cohorts, in which quantitative measures of fibrin(ogen), demyelination, inflammation, HLA-DRB1*15 status, and cortical neuronal loss have already been obtained4 will be used for this.D - The remainder of year three will be used to assess the functional impact of microglial proteins associated with cortical neuronal loss (C) on human iPSC-derived glutamatergic cortical neurons, via the assessment of a variety of optimised readouts including neurite outgrowth, live/dead assay, and calcium imaging.