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In situ architecture of neuronal α-Synuclein inclusions

Victoria A. Trinkaus, Irene Riera-Tur, Antonio Martínez-Sánchez, Felix J.B. Bäuerlein, Qiang Guo, Thomas Arzberger, Wolfgang Baumeister, Irina Dudanova, Mark S. Hipp, F. Ulrich Hartl, Rubén Fernández-Busnadiego

Preprint posted on August 07, 2020 https://www.biorxiv.org/content/10.1101/2020.08.07.234138v1

Seeing is believing – cryo-electron tomography reveals a fibrillar nature of protein inclusions associated with Parkinson’s disease

Selected by Tessa Sinnige

Categories: cell biology

Background

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.

 

Results

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.

 

Figure 1a-d from the preprint showing a tomogram of a neuronal α-synuclein inclusion. α-Synuclein fibrils are shown in red. Green arrows in b point at GFP decorating the α-synuclein fibrils; orange and black arrows in c point at an actin filament and a microtubule, respectively. Reproduced under a CC BY-NC-ND 4.0 international license.

 

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.

 

Questions

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?

 

References

  1. Shahmoradian, S. H. et al. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nat. Neurosci. 22, 1099–1109 (2019).
  2. 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: 14th August 2020 , updated on: 17th August 2020

doi: https://doi.org/10.1242/prelights.24083

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Author's response

Rubén Fernández Busnadiego shared

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?

It is possible that organelles are trapped non-specifically within the fibrillar network. However, the majority of those organelles appear to be endolysosomal/autophagic structures, which suggests selective interactions with these organelles that we are currently exploring.

 

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?

This is difficult to estimate, as our tomograms represent only a tiny fraction of cellular volume. We believe that these experiments may be first steps towards understanding aggregate spread within the human brain, but of course further work is needed. It is also necessary to address how α-Synuclein aggregates are present in the extracellular space to allow uptake by neighbouring cells.

 

What are the prospects of applying this methodology to more complex (tissue) samples?

Pilot experiments have already demonstrated the feasibility of cryo-ET analysis of tissue (see e.g. PMID 31363205). However, further technical developments are needed to streamline this approach in more user-friendly ways. Ultimately, imaging patient-derived tissues will also require advances in sample preparation procedures. It will be difficult to analyse postmortem samples by high resolution cryo-ET, as the unavoidable delay prior to freezing may lead to substantial cellular deterioration.

 

The authors would like to state that the paper has not yet undergone peer review, that the findings are provisional and that the conclusions may change.

1 comment

1 year

Hilal Lashuel

It is unfortunate that the authors did not cite and discuss our paper, which we published in PNAS early this year and mentioned by Tessa. This paper presents the first demonstration of the formation of LB-like inclusions in an aSyn neuronal seeding model. Using an integrative omics, biochemical and imaging approach, we dissected the molecular events associated with the different stages of LB formation and their contribution to neuronal dysfunction and degeneration. In addition, we demonstrated that the formation of LB-like inclusions involves a complex interplay between aSyn fibrillization, posttranslational modifications, and interactions between aSyn aggregates and membranous organelles, including mitochondria, the autophagosome, and endolysosome. Finally, we showed that the process of LB formation, rather than simply fibril formation, is one of the major drivers of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, and synaptic dysfunctions. The results by Trinkaus confirms many of our data and findings using a complementary technique.
https://www.pnas.org/content/117/9/4971
I encourage the readers to review the data in the supporting information section as well. https://www.pnas.org/content/pnas/suppl/2020/02/18/1913904117.DCSupplemental/pnas.1913904117.sapp.pdf

Please note that this study shows the formation of fibrillar aggregates rather than LB-like inclusions. The fibrils appear to be dispersed throughout the cytoplasm rather than accumulating in round LB-like inclusions, as we reported in our paper.

Another important point to highlight is that the fibrils are derived from aSyn fused to GFP. While the GFP does not seem to influence aSyn ability to form fibrils, it will dramatically impact the interactome of the fibrils as it extensively coats the surface of the fibrils. Please review this recent PNAS, which reports that Fluorescent proteins (FPs) ” have an intrinsic high binding propensity for the core of amyloid fibrils and suggest that caution should be taken when FPs are used as a fusion to visualize the cellular localization of proteins of interest. https://www.pnas.org/content/early/2020/08/20/2001457117

For more information about the “controversy in the field surrounding the molecular architecture of Lewy Bodies,” I urge the readers to read this review which present historical overview of LBs and critical analysis of the paper by Shahmoradian, S. H et al. in the context of the body of work on LBs in the literature.
Do Lewy bodies contain alpha-synuclein fibrils? and Does it matter? A brief history and critical analysis of recent reports https://www.sciencedirect.com/science/article/pii/S0969996120301510?via%3Dihub

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