α-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to ß-spectrin

α-synuclein plays a key role in the pathogenesis of Parkinson’s disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. We show that α-synuclein binds directly to ß-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders we demonstrate that ß-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of ß-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na+/K+ ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. We examine the same pathway in human neurons and find that Parkinson’s disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na+/K+ ATPase, and membrane potential depolarization. Our findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson’s disease and related α-synucleinopathies leads to neuronal dysfunction and death.


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Parkinson's disease is the most common neurodegenerative movement disorder, affecting 1% of

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023.   For examination of the adult fly brain, animals were fixed in formalin and embedded in paraffin. 4 126 μm serial frontal sections were prepared through the entire brain and placed on a single glass 127 slide. Hematoxylin staining was performed on paraffin sections to assess total neuronal density.

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For immunostaining of paraffin sections, slides were processed through xylene, ethanol, and 131 into water. Antigen retrieval by boiling in sodium citrate, pH 6.0, was performed prior to blocking.

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In some studies, whole mount Drosophila brain preparations were alternatively used. Blocking 133 was performed in PBS containing 0.3% Triton X-100 and 2% milk for 1 hour and followed by 134 incubation with appropriate primary antibodies overnight. Primary antibodies used were: 135 anti-tyrosine hydroxylase (Immunostar) at 1:500; anti-α-synuclein (5G4, Millipore) at 1:1,000,000,    Wild type Drosophila and human spectrins were cloned into a histidine tagged vector (2BC-T 153 cloning vector, Addgene # 31070) using ligation independent cloning to obtain C-terminally 154 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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(1:10,000, LICOR) was applied. Images were taken using a LICOR Odyssey DLx imaging system 192 (LICOR). Blots were repeated at least three times, and a representative blot shown.

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.02.543481 doi: bioRxiv preprint 9 For quantification of ankyrin-B and Na + /K + ATPase localization, the fluorescence intensity of 206 staining in the plasma membrane as well as in the cytoplasm was measured in the soma. Plasma

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The spectrin cytoskeleton is anchored to the cytoplasmic face of the plasma membrane and 273 is believed to form a network that contributes to maintaining the structure and shape of the cell        was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.02.543481 doi: bioRxiv preprint 14 membrane ( Fig. 5A-C). The distribution of ankyrin was significantly altered in the α -synuclein 335 transgenic flies, with an increase in cytosolic staining (Fig. 5A,B). Elevated expression of 336 β -spectrin significantly restored the wild type distribution of ankyrin ( Fig. 5A-C). Similarly, Na + /K +

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Immunostaining for β II-spectrin revealed the characteristic subplasmalemmal staining pattern in 359 control neurons (Fig. 6A). In contrast, β II-spectrin immunostaining did not consistently show 360 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.02.543481 doi: bioRxiv preprint 15 subplasmalemmal distribution in triplication neurons (Fig. 6A, arrows, B). We confirmed that 361 human β II-spectrin co-immunoprecipitated with α -synuclein in homogenates from human neurons 362 (Fig. 6C). We next examined ankyrin in human neurons. There are three mammalian ankyrin 363 isoforms, ankryin-B, ankyrin-R and ankyrin-G. Ankryin-B is expressed in a widespread pattern in 364 the nervous system, while ankyrin-R and ankyrin-G have more specific cellular and subcellular 365 localization (Kordeli and Bennett, 1991; Lorenzo, 2020). When we stained for ankryin-B we 366 observed increased cytoplasmic staining compared to isogenic controls (Fig. 6D, arrows, E), 367 similar to findings in α -synucleinopathy model fly brains (Fig. 5A,B). We next used an antibody 368 recognizing the β I subunit of human Na + /K + ATPase to examine localization of Na + /K + ATPase. As 369 in α -synuclein transgenic Drosophila brains (Fig. 5D,E), we found increased cytosolic staining for 370 human Na + /K + ATPase in α -synuclein triplication neurons (Fig. 6F, arrows, G). Alteration of 371 Na + /K + ATPase localization was accompanied by plasma membrane depolarization as monitored 372 by DiBAC4(3) fluorescence in human triplication neurons compare to isogenic controls (Fig. 6H).

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In addition to anchoring plasma membrane proteins such as Na + /K + ATPase, ankyrins also 374 organize discrete membrane domains of neurons. In particular, ankyrin-G localizes to and 375 organizes the axon initial segment (Leterrier, 2018). We thus examined the axon initial segment in 376 triplication and control neurons. Immunofluorescence using an antibody to ankyrin-G, identified a 377 well-defined axon initial segment in most control neurons (Fig. 6I, arrows, K). In contrast, many  ßIV-spectrin. Similar to ankyrin-G, immunostaining for ßIV-spectrin revealed decreased numbers 382 of intact axon initial segments in triplication neurons (Fig. 6J, arrows, L), with fragmentation of 383 many axon initial segments (Fig. 6J, insets, arrowheads, L).       was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.02.543481 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023.         was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.02.543481 doi: bioRxiv preprint