In situ architecture of neuronal α-Synuclein inclusions

The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution. We show that inclusions seeded by α-Syn aggregates produced recombinantly or purified from patient brain consist of α-Syn fibrils crisscrossing a variety of cellular organelles. Using gold-labeled seeds, we find that aggregate seeding is predominantly mediated by small α-Syn fibrils, from which cytoplasmic fibrils grow unidirectionally. Detailed analysis of membrane interactions revealed that α-Syn fibrils do not contact membranes directly, and that α-Syn does not drive membrane clustering. Altogether, we conclusively demonstrate that neuronal α-Syn inclusions consist of α-Syn fibrils intermixed with membranous organelles, and illuminate the mechanism of aggregate seeding and cellular interaction.


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Sample sizes were selected based on previous experience to obtain statistical significance and reproducibility (see e.g. Table S1 in Collado et al., Dev Cell 2019) Tomograms with insufficient signal-to-noise-ratio were excluded for all conditions.
Replicates were conducted for all experiments quantified as described in the Figure legends and Supplementary Table 1 Mouse embryos of both sexes were chosen randomly for neuronal cell cultures. The MSA patient was selected based on histopathological brain analyses (ramdomization did not apply). Experiments on SH-SY5Y cells were performed comparing different experimental treatments on the same cell line (ramdomization did not apply).
No blinding was applied. All experiments were performed comparing various treatments on otherwise comparable samples, and as such it was necessary for the researchers to be aware of the treatment applied (e.g. PBS, PFFs or MSA aggregates). Appropriate controls were included in each experimental replication.

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Primary cortical neurons were prepared from E15.5 CD-1 wild type mouse embryos of both sexes (breeding line MpiCrlIcr:CD-1). Mice were housed in a specific pathogen free facility at 22 ± 1,5°C, 55 ± 5% humidity, 14-hour light / 10-hour dark cycle. no no All experiments involving mice were performed in accordance with the relevant guidelines and regulations of the Government of Upper Bavaria (Germany).
A brain sample was collected from a male patient who died at the age of 54, 6 years after being diagnosed with a cerebellar type of MSA The patient donated brain tissue to the Neurobiobank Munich. This sample was selected due to the abundant alphasynuclein inclusions in the pons region MSA patient brain tissue was obtained from Neurobiobank Munich. All autopsy cases of the Neurobiobank Munich are collected on the basis of an informed consent according to the guidelines of the ethics commission of the Ludwig-Maximilians-University Munich, Germany. The experiments performed in this paper were approved by the Max Planck Society's Ethics Council.