Journal: Acta Neuropathol Commun / Year: 2021 Title: Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease. Authors: Ya Zhou / Thomas R Peskett / Christian Landles / John B Warner / Kirupa Sathasivam / Edward J Smith / Shu Chen / Ronald Wetzel / Hilal A Lashuel / Gillian P Bates / Helen R Saibil / Abstract: Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited ...Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.
Brain slices were high pressure frozen and freeze substituted
High pressure freezing
Instrument: OTHER Details: Tissue sections were high-pressure frozen in 100 um deep wells of aluminium carriers in the presence of bovine serum albumin using a Leica HPM100.. The value given for _emd_high_pressure_ ...Details: Tissue sections were high-pressure frozen in 100 um deep wells of aluminium carriers in the presence of bovine serum albumin using a Leica HPM100.. The value given for _emd_high_pressure_freezing.instrument is Leica HPM100. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM PACT', 'LEICA EM HPM100', 'LEICA EM PACT2', 'BAL-TEC HPM 010'} so OTHER is written into the XML file.
Cryo protectant
BSA
Sectioning
Ultramicrotomy - Instrument: Leica EM UC7 / Ultramicrotomy - Temperature: 20 K / Ultramicrotomy - Final thickness: 100 nm
Fiducial marker
Manufacturer: EMS / Diameter: 10 nm
-
Electron microscopy
Microscope
FEI TECNAI 12
Image recording
Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beam
Acceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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