Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington’s disease

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 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.

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

“…Altered mitophagy may also potentially contribute to the bioenergetic deficits observed in various HD models 13, 96 and at least in part to the excessive weight loss that is characteristic of late-stage human HD 97, 98 . Consistent with our findings, recent work 31 has identified non-fibrillar mHTT within single-membrane-bound organelles in cortical and striatal tissue from zQ175 HD mice, including multivesicular bodies (MVB) and amphisomes, using correlative light and conventional electron microscopy of samples fixed by freeze substitution. The localization of non-fibrillar mHTT changed depending on the disease stage, with presymptomatic stages showing localization within MVBs/amphisomes and late stage disease showing localization to the autolysosomes or residual bodies 31 .…”

“…For example, advances in cryogenic electron microscopy (cryoEM) and tomography (cryoET) have recently elucidated the structure of soluble HTT in complex with HAP40 at near atomic resolution 25 , the topology of mHTT-exon 1 and polyQ in vitro aggregates at nanometer resolution 26 , and have enabled visualization of the interactions between mHTT-exon 1 aggregates and other proteins and cellular compartments in transfected yeast 27 and HeLa 28 cells, and with molecular chaperones in vitro 29, 30 . Correlative light and traditional EM microscopy has also been used to image recruitment of mHTT-exon 1 to cytoplasmic aggregates within single membrane, vesicle-rich endolysosomal organelles 31 .…”

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

“…Consistent with our findings, recent work 31 has identified non-fibrillar mHTT within single-membrane-bound organelles in cortical and striatal tissue from zQ175 HD mice, including multivesicular bodies (MVB) and amphisomes, using correlative light and conventional electron microscopy of samples fixed by freeze substitution. The localization of non-fibrillar mHTT changed depending on the disease stage, with presymptomatic stages showing localization within MVBs/amphisomes and late stage disease showing localization to the autolysosomes or residual bodies 31 . Our findings here in intact, cryo-preserved human patient and mouse model-derived neurons are suggestive of even earlier events and thus are likely complementary.…”

“…For example, advances in cryogenic electron microscopy (cryoEM) and tomography (cryoET) have recently elucidated the structure of soluble HTT in complex with HAP40 at near atomic resolution 25 , the topology of mHTT-exon 1 and polyQ in vitro aggregates at nanometer resolution 26 , and have enabled visualization of the interactions between mHTT-exon 1 aggregates and other proteins and cellular compartments in transfected yeast 27 and HeLa 28 cells, and with molecular chaperones in vitro 29,30 . Correlative light and traditional EM microscopy has also been used to image recruitment of mHTT-exon 1 to cytoplasmic aggregates within single membrane, vesicle-rich endolysosomal organelles 31 .…”

Vesicle trafficking and pathways to neurodegeneration

“…As we develop an ever more detailed understanding of population-wide genetic risk, through large scale sequencing, association studies, and expression analyses, there is an ever longer list of potential risk genes to investigate and comprehend [10]. With regard to this, it was striking that a majority of the presentations at the meeting involved investigating monogenic aspects of neurodegenerative disease, whether that be the ultrastructure of Huntington disease intracellular inclusions and how these disrupt endolysosomal function [11], or the function of Leucine Rich Repeat Kinase 2 in responding to lysosomal damage [12] in Parkinson disease (to cite two examples of topics covered by short talks at the meeting). Moving from a monogenic-centric approach to the cell biology of neurodegeneration to making sense of the complexities of common genetic risk for neurodegeneration at a functional level is a gargantuan task, and one that is only just beginning to be confronted.…”