IN-TISSUE MULTISCALE 3D ELECTRON MICROSCOPY TO EXPLORE SUBCELLULAR ARCHITECTURE DISTURBANCES ASSOCIATED TO HUNTINGTON'S DISEASE

In situ analysis of the subcellular architecture and molecular organization of cells and tissues, under physiological conditions, is critical for understanding fundamental biological processes, gaining insights into pathophysiological basis of diseases, enabling identification of new therapeutic targets and for the identification of imaging biomarkers. Cryofixation (vitrification) and cryogenic electron microscopy (cryo-EM) ensure structural analysis in near-native conditions. Several cryo-EM modalities are emerging as major players for in situ structural cell biology. First, cryo-volume imaging with serial cryo-FIB/SEM (Focused Ion Beam/Scanning Electron Microscopy) operates by cyclically (i) milling a thin layer of the specimen using the FIB, followed by (ii) SEM imaging of the exposed surface. Cryo-FIB/SEM thus collects information from large vitrified volumes (up to tens of microns thick) at intervals of, typically, 10-30 nm. Second, cryo-volume imaging with cryo-electron tomography (cryo-ET) relies on transmission electron microscopy (TEM) and the volume is computed by combining a series of projection images acquired at different tilted views from a sample thin enough for TEM (typically < 300 nm).
We present here our approach, combining tissue cryofixation, multiscale 3DEM and image processing, applied to understanding disruptions in subcellular architecture associated to the progression of Huntington’s disease (HD) neurodegeneration. In-tissue studies of both central and peripheral tissues from animal models and human post-mortem samples have facilitated understanding disruptions in the protein synthesis machinery and the mitochondrial network associated to HD.
Acknowledgements: Work supported through grants PID2022-139071NB-I00 and PID2023-153013OB-I00, funded by MCIN/AEI/10.13039/501100011033, “ERDF A way of making Europe” and by the “European Union NextGenerationEU/PRTR.