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- EMDB-12374: Tomogram of mutant huntingtin inclusion (recruitment foci) in the... -

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Entry
Database: EMDB / ID: EMD-12374
TitleTomogram of mutant huntingtin inclusion (recruitment foci) in the cytoplasm of hippocampal CA1 neuron in 6 month old zQ175 mice
Map dataMutant HTT containing organelle identified in hippocampal CA1 neuron in 6 month old zQ175 mice
Sample
  • Tissue: Mutant huntingtin containing organelle in hippocampal CA1 neuron from zQ175 knock-in mice.
Biological speciesMus musculus (house mouse)
Methodelectron tomography / negative staining
AuthorsZhou Y / Saibil HR
Funding support United Kingdom, United States, 4 items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust079605/2/06/2 United Kingdom
The CHDI Foundation United States
UK Dementia Research Institute United Kingdom
CitationJournal: 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.
History
DepositionFeb 16, 2021-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateMay 5, 2021-
Current statusMay 5, 2021Processing site: PDBe / Status: Released

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Structure visualization

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Supplemental images

emd_12374.png

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Map

FileDownload / File: emd_12374.map.gz / Format: CCP4 / Size: 1.1 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationMutant HTT containing organelle identified in hippocampal CA1 neuron in 6 month old zQ175 mice
Voxel sizeX=Y=Z: 3.683 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)21.627302 (±23.202778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-368-462109
Dimensions33353035120
Spacing30353335120
CellA: 11177.905 Å / B: 12282.806 Å / C: 441.96002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z3.6833.68300029985013.683
M x/y/z30353335120
origin x/y/z0.0000.0000.000
length x/y/z11177.90512282.806441.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-462-368109
NC/NR/NS30353335120
D min/max/mean-128.000127.00021.627

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Supplemental data

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Sample components

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Entire : Mutant huntingtin containing organelle in hippocampal CA1 neuron ...

EntireName: Mutant huntingtin containing organelle in hippocampal CA1 neuron from zQ175 knock-in mice.
Components
  • Tissue: Mutant huntingtin containing organelle in hippocampal CA1 neuron from zQ175 knock-in mice.

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Supramolecule #1: Mutant huntingtin containing organelle in hippocampal CA1 neuron ...

SupramoleculeName: Mutant huntingtin containing organelle in hippocampal CA1 neuron from zQ175 knock-in mice.
type: tissue / ID: 1 / Parent: 0
Details: Cytoplasmic inclusion sites identified by correlative fluorescence/EM on freeze substituted brain sections.
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6J / Organ: Brain / Tissue: hippocampus CA1

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7 / Details: MilliQ water was used.
StainingType: NEGATIVE / Material: Uranyl acetate
Sugar embeddingMaterial: Lowicryl HM20
Details: High pressure frozen samples were freeze substituted in a Leica AFS
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
DetailsBrain slices were high pressure frozen and freeze substituted
High pressure freezingInstrument: 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 protectantBSA
SectioningUltramicrotomy - Instrument: Leica EM UC7 / Ultramicrotomy - Temperature: 20 K / Ultramicrotomy - Final thickness: 100 nm
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 56

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