In situ architecture of neuronal α-Synuclein inclusions
Preprint posted on 7 August 2020 https://www.biorxiv.org/content/10.1101/2020.08.07.234138v1
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-22108-0
Many human diseases are accompanied by the formation of fibrillar protein aggregates in the brain or in other tissues. In the case of Parkinson’s disease, the protein α-synuclein has been found to accumulate in insoluble inclusions termed Lewy Bodies. However, a recent study questioned the fibrillar nature of α-synuclein-positive inclusions, finding that the majority of Lewy Bodies in the human brain consist predominantly of membranous material1. α-Synuclein is known to interact with lipid bilayers, raising the possibility that it might lead to the aberrant clustering of vesicles and organelles, thereby forming the Lewy Body. In contrast, another recent study proposed that fibril formation by α-synuclein is the first step in a series of events that result in the recruitment of vesicular components and the formation of a Lewy Body-like structure2.
In this preprint, the authors aim to resolve the controversy surrounding the nature of Lewy Bodies by using cryo-electron tomography (cryo-ET) to visualise the molecular architecture of α-synuclein inclusions in cultured neurons. To perform cryo-ET, the sample is rapidly frozen and subsequently milled down to a thin slice that allows the electron beam to penetrate. The structural features of the sample are thus preserved without the need for any fixation or staining procedures that may result in artefacts.
The authors induce the formation of inclusions in mouse primary neurons by adding pre-formed α-synuclein fibrils, which act as seeds for the aggregation of the GFP-tagged α-synuclein expressed in the cells. In the electron tomograms, they identify inclusions that indeed contain large amounts of GFP-α-synuclein fibrils. In untransfected cells, they confirm that also the endogenous (mouse) α-synuclein appears fibrillar in the inclusions, although at lower density presumably due to the lower protein levels. Consistent with earlier reports, the authors furthermore identify mitochondria, autophagosomes, ER membranes and vesicles as components of the inclusions, interspersed between the fibrils. When the authors use patient-derived rather than recombinant fibrils as seeds, they obtain essentially the same results, although the properties of the fibrils – which are preserved via seeding – are somewhat different.
The authors next aim to investigate the seeding mechanism. They label the fibrils used as seeds with gold beads, which are easily recognisable in the tomograms. Using this procedure, they observe fibrils in the inclusions that have only a small (3-10) number of gold beads on one end, suggesting that the fibrils grow unidirectionally from a very short oligomeric fibril seed.
Finally, the authors ask whether α-synuclein induces the clustering of membranous material in their system. They find that the α-synuclein fibrils do not have a preferred interaction with membranes, and are thus unlikely to mediate clustering. Moreover, they discover that the inter-membrane distances within the inclusions are actually very similar to those in control cells. This result leads the authors to also exclude any effects of soluble α-synuclein species that may not be observable by cryo-ET. Altogether, the authors conclude that seeded α-synuclein inclusions in this system are of a fibrillar nature, and are not particularly enriched for vesicles and organelles.
Why I chose this preprint
The molecular mechanisms leading to Parkinson’s disease and other disorders associated with Lewy Bodies are still completely unclear. Until recently, only very few structural investigations of Lewy Bodies existed. The introduction of new and improved electron and light microscopy techniques has changed this situation, but has also led to controversy in the field surrounding the molecular architecture of Lewy Bodies. The conflicting results may not be surprising given the complexity of protein aggregation in a cellular environment, where many additional factors play a role. This is not only the case for synuclein-related disorders, but holds true for all protein aggregation diseases, ranging from Alzheimer’s to type II diabetes. It will be very exciting to see more structural studies emerge to further unravel the rules of protein aggregation in vivo.
It is interesting that you do not find evidence for an enrichment in membranous structures within the inclusion. Does this result suggest that the vesicles and organelles inside the inclusion get trapped non-specifically, just because they happen to reside at the location where inclusion formation is initiated?
From the experiment using the gold-labelled seeds, can you estimate how many seeds are typically taken up by a single cell? Is this representative for the spreading of α-synuclein in the human brain?
What are the prospects of applying this methodology to more complex (tissue) samples?
- Shahmoradian, S. H. et al. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nat. Neurosci. 22, 1099–1109 (2019).
- Mahul-Mellier, A.-L. et al. The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 117, 4971–4982 (2020).
Posted on: 14 August 2020 , updated on: 17 August 2020Read preprint
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