Background. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) can guide a more comprehensive knowledge in terms of identification, abundance, and distribution of biomolecules within tissues. The hyphenation of such ionization technique with the High Resolution Orbitrap MS analyzer improved performance and made the discovery phase more robust and reliable. Furthermore, the non-destructiveness of MALDI source enables the recycling of intact tissue slices for staining techniques to drive a comparison of structural features with the MS data. The Marinesco-Sjogren Syndrome (MSS) is an infantile-onset disease mainly characterized by ataxia, cerebellum atrophy and muscle weakness linked to a loss-of-function mutation of the gene Sil1 encoding for a nucleotide exchange factor of BiP3, a crucial Endoplasmic Reticulum (ER) chaperone. Although Sil1 being widely expressed in the brain, the mutation cause protein accumulation, chronic activation of the Unfolded Protein Response (UPR) and cell death only in the cerebellum Purkinje cells and even more specific it spares the vestibulocerebellum which include lobule X and the caudal region of lobule IX. Given that MALDI-MSI can play a crucial role to understand the reason of the mutation cell-specific effect showing eventual metabolic and signaling rewiring allowing some Sil1-deficient cells to avoid the neurodegeneration. We carried out a high resolution spatial lipidomics on the Marinesco-Sjogren Syndrome (MSS) mouse model woozy (wz) cerebellum.

Methods. The MassTech™ Atmospheric-Pressure MALDI source with Ultra-High spatial Resolution (UHR) has been coupled with an Orbitrap Exploris 120 mass spectrometer for high resolution in situ analysis on cerebellum slices collected from homozygote and heterozygote mice for wz mutation, the latter considered the control. The analysis was conducted on both positive and negative ionization mode and the data were processed with LipostarMSI software.

Results. An evaluation of the optimal sample preparation of mouse cerebellum tissues for MALDI-MSI-based lipidome analysis was conducted, in terms of embedding polymer, size of the raster, matrix and impact of aqueous wash before matrix deposition. Then, we applied the optimized method to compare the lipid signature of cerebellum between the two conditions for both ionization modes. The analysis of the cerebellar arbor vitae revealed a significative difference in the spatial distribution of many lipids between the two conditions and among the different areas of the same cerebellum coherent with the literature and the phenotype observed. These preliminary data pave the way for a better comprehension of the metabolic adaptation that cells pursue to ease the ER stress and avoid the neurodegeneration.