Ubiquitin Ligase SCarFs Up Internalized α-Synuclein, Prevents Seeding

Can researchers speed the degradation of aggregated, toxic proteins without harming physiological forms? In the June 5 Science Translational Medicine, researchers led by Juan Gerez and Paola Picotti at the Swiss Federal Institute of Technology in Zurich suggest this can work for aggregated α-synuclein, which accumulates in Parkinson’s disease and dementia with Lewy bodies. They identify a specific ubiquitin ligase, SCFFBXL5, that stimulates the degradation of α-synuclein fibrils, but not monomers or oligomers, taken up from the environment by cultured human cells. In wild-type mice, SCFFBXL5 was essential for clearing injected fibrils. Though it remains to be seen if the ligase works the same way in human brain, its components are found in Lewy bodies. The findings suggest that boosting SCFFBXL5 activity could help arrest the spread of toxic α-synuclein forms from cell to cell through the brain, the authors propose. It is unclear if the same strategy could also work for other types of aggregated protein.

  • The ubiquitin ligase SCFFBXL5 tags internalized α-synuclein fibrils for destruction.
  • Without the ligase, cultured human cells cannot clear this material.
  • In mice, the ligase prevents injected fibrils from spreading through brain.

Lary Walker at Emory University in Atlanta agreed this has potential for synucleinopathies. “The authors make a good case that this mechanism is fairly selective for a toxic form of the protein. This is still a long way from a therapy, but these data may guide our thinking on how to get there,” he told Alzforum.

Previous research has found that neurons can take up α-synuclein from their surroundings, and that this material accumulates in Lewy bodies (Aug 2009 news; Oct 2011 news). Gerez and colleagues undertook a detailed study of what happens to this internalized α-synuclein. They treated human neuroblastoma SH-SY5Y cells with synthetic α-synuclein monomers, oligomers, or fibrils for 16 hours, then quantified the amount of exogenous protein retained inside the cells by mass spectrometry. Cells exposed to monomers or oligomers contained no more α-synuclein than control cells, indicating that these forms do not accumulate. Mature fibrillar forms, however, led to a 10-fold increase in α-synuclein content.

Clearance Failure. After being injected with α-synuclein fibrils, wild-type mice accumulate α-synuclein inclusions (brown) in the hemisphere of the cortex lacking SCFFBXL5 ubiquitin ligase (right). The other hemisphere remains unscathed (left). [Courtesy of Gerez et al., Science Translational Medicine, 2019.]

What does this accumulation do to cells? The authors analyzed the proteome of cells exposed to fibrils, and found alterations in 150 proteins related to vesicle trafficking, ribosome metabolism, or ubiquitination. A jump in SKP1/Cul1/F-box protein (SCF) ubiquitin ligases drew the researchers’ attention, because it suggested these ligases were activated in response to fibrils.

SCF ligases are complexes made up of several subunits (see image below). The scaffold protein cullin-1 acts as the backbone. On one end, this backbone binds the RING protein Rbx1, which recruits the ubiquitin donor. On the other, Cul1 binds the adaptor protein SKP1, which in turn partners with various F-box proteins that bind substrates for ubiquitin tagging. Cells contain dozens of different F-box proteins that match up with distinct substrates, conferring specificity on the whole complex.

Modular Assembly. SCF ubiquitin ligases contain numerous subunits, including an F-box protein that recognizes distinct substrates and confers target specificity. [Courtesy of Bosu and Kipreos, 2008, Cell Division.]

To find out what the ligase does, Gerez and colleagues knocked down either SKP1 or Cul1 in SH-SY5Y cells exposed to α-synuclein fibrils. More fibrils accumulated in response. When the researchers transfected the cells with a dominant negative version of Cul1 that blocks the endogenous protein, the effect was even stronger; five times more fibrils built up than in wild-type SH-SY5Y cells. To examine longer-term effects of α-synuclein exposure, the researchers allowed cells to recover for 16 hours after fibril treatment. Wild-type SH-SY5Y cells cleared the deposits during this time, but those containing dominant-negative Cul1 did not.

Ubiquitin ligases do not directly degrade anything; instead, they add ubiquitin tags to proteins, marking them for destruction by a cellular disposal system. The authors separately inhibited proteasome and lysosome activity in SH-SY5Y cells exposed to fibrils. Both manipulations mimicked the effect of dominant-negative Cul1, suggesting that the SCF ubiquitin ligase targets internalized α-synuclein fibrils for degradation by both systems, the authors note.

Which F-box protein recognized α-synuclein fibrils? Knocking down 31 different F-box proteins in cultured cells, the authors found that only absence of FBXL5 caused α-synuclein to accumulate. They verified a physical interaction by showing that FBXL5 and α-synuclein fibrils co-immunoprecipitate, and confirmed that SCFFBXL5 can ubiquitinate these fibrils in vitro.

Does SCF Shut Down Seeding?
Next, the authors wondered if SCFFBXL5 could prevent seeding of α-synuclein aggregates. For these experiments, they used SH-SY5Y cells that expressed fluorescent α-synuclein. Exogenous fibrils recruited the endogenous fluorescent protein into cytoplasmic aggregates. When the cells expressed dominant-negative Cul1, four times as many cells developed such foci, suggesting that SCFFBXL5 indeed helps keep seeding in check (see image below).

Clearance Problem. Cells without SCF function (bottom) accumulate more inclusions (arrows) when exposed to α-synuclein fibrils (green), than do control cells (top). Red indicates dominant-negative Cul1. [Courtesy of Gerez et al., Science Translational Medicine, 2019.]

At what point in the uptake and seeding process does this ligase act? The authors believe fibrils access the cytoplasm when endolysosomal vesicles that import the proteins from the cell surface break down. Two hours after cells were exposed to α-synuclein fibrils, they appeared only in endosomes, but by six hours, they were in the cytoplasm. The fibrils associated with Gal3, a marker of ruptured vesicles, and with SCFFBXL5, suggesting the ligase intercepts these broken vesicles. Fibrils also correlated with lysosome damage. This fit with previous research proposing that α-synuclein ruptures vesicles to gain entry to cells (Freeman et al., 2013). 

Other types of protein aggregate, such as SOD1, tau, and huntingtin, are reported to enter cells through endosomes and access the cytosol by damaging them (Zeineddine et al., 2015; Calafate et al., 2016; Flavin et al., 2017). Whether SCFFBXL5 might tag them for destruction remains to be tested. “This might be a general mechanism by which prion-like proteins get into the cytoplasm and propagate themselves,” Walker noted. “These data provide some mechanistic insight into cytopathology.”

Does SCFFBXL5 work this way in vivo? The authors knocked down SKP1 in the hippocampi of mice that overexpressed wild-type human α-synuclein, then injected α-synuclein fibrils in the same spot five weeks later (Rockenstein et al., 2002). Ten weeks after injection, α-synuclein inclusions were up to four times more abundant in the hippocampi and striata of animals lacking SKP1 than in transgenic controls, suggesting SCFFBXL5 protects the animals against α-synuclein seeding.

The ligase protected wild-type mice. When the researchers knocked down SKP1 or FBXL5 on one side of the cortex, then injected α-synuclein fibrils into both sides five weeks later, the side lacking SCFFBXL5 activity had up to four times as many inclusions as the control side (see image above).

And what about people? The authors examined postmortem brain samples from 10 patients who had died with PD or DLB, and found both SKP1 and Cul1 in Lewy bodies, hinting at a role in human disease. Notably, researchers in Zurich had previously implicated SKP1 as a PD risk gene, linking low levels to neuronal death (Grünblatt et al., 2004; Mandel et al., 2009; Grünblatt et al., 2018). 

Even if SCFFBXL5 performs the same task in human as in mouse, exploiting this therapeutically will be challenging, the authors noted. Cul1 and SKP1 participate in a plethora of biological processes, making them unsuitable targets, Gerez told Alzforum. FBXL5 could be promising, but this subunit also regulates iron metabolism in the cell, so it would have to be modulated carefully. Iron stabilizes the subunit and would boost its activity, but iron also triggers oxidative stress, which is bad for the brain, Gerez noted. An alternative would be to find a small molecule that works like iron does to stabilize the subunit. The authors are looking for one, and are also trying to raise FBXL5 levels with gene therapy.

As for this ubiquitin ligase clearing other types of protein aggregate, silencing SCF did not change clearance of Aβ42 fibrils, but the authors have not yet tested other toxic proteins.—Madolyn Bowman Rogers
 

Comments

  1. This work by Gerez et al. helps us fill out the molecular machinery that controls uptake and degradation of α-synuclein from the extracellular space. Consistent with other literature, the authors seem to see a signature around endosomal pathways for uptake based on their differential proteome analysis. They then show convincingly that knockdown of the E3 ligase complex leads to greater accumulation of p-syn. There are some interesting aspects to this story that should be followed up. One important question is how material used to "seed" pathology and p-synuclein in the inclusions are related. Which material is the primary client of the E3 ligase is ambiguous based on available data and this might be resolved by identifying lysines for ubiquitin addition on seeds and endogenous synuclein. In principle, one could then determine whether the E3 ligase complex promotes degradation of the fibrils, the endogenous material, or both.

    It seems likely to me that there are going to be additional components of the endocytic machinery that are required for protein uptake into neurons in the brain. Many pathogens enter cells by "hijacking" the endocytosis pathways and it has been productive in other fields (e.g., virology) to use unbiased approaches such as the one used here to map out entry pathways. It would be important to continue searching for required molecular components for the transmission of α-synuclein and other protein assemblies.

    View all comments by Mark Cookson

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References

News Citations

  1. Research Brief: α-synuclein Spoils the Neural Neighborhood
  2. Modeling Sporadic PD in a Dish?

Paper Citations

  1. Freeman D, Cedillos R, Choyke S, Lukic Z, McGuire K, Marvin S, Burrage AM, Sudholt S, Rana A, O'Connor C, Wiethoff CM, Campbell EM. Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis. PLoS One. 2013;8(4):e62143. PubMed.
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  3. Calafate S, Flavin W, Verstreken P, Moechars D. Loss of Bin1 Promotes the Propagation of Tau Pathology. Cell Rep. 2016 Oct 18;17(4):931-940. PubMed.
  4. Flavin WP, Bousset L, Green ZC, Chu Y, Skarpathiotis S, Chaney MJ, Kordower JH, Melki R, Campbell EM. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins. Acta Neuropathol. 2017 May 19; PubMed.
  5. Rockenstein E, Mallory M, Hashimoto M, Song D, Shults CW, Lang I, Masliah E. Differential neuropathological alterations in transgenic mice expressing alpha-synuclein from the platelet-derived growth factor and Thy-1 promoters. J Neurosci Res. 2002 Jun 1;68(5):568-78. PubMed.
  6. Grünblatt E, Mandel S, Jacob-Hirsch J, Zeligson S, Amariglo N, Rechavi G, Li J, Ravid R, Roggendorf W, Riederer P, Youdim MB. Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes. J Neural Transm. 2004 Dec;111(12):1543-73. PubMed.
  7. Mandel SA, Fishman-Jacob T, Youdim MB. Modeling sporadic Parkinson's disease by silencing the ubiquitin E3 ligase component, SKP1A. Parkinsonism Relat Disord. 2009 Dec;15 Suppl 3:S148-51. PubMed.
  8. Grünblatt E, Ruder J, Monoranu CM, Riederer P, Youdim MB, Mandel SA. Differential Alterations in Metabolism and Proteolysis-Related Proteins in Human Parkinson's Disease Substantia Nigra. Neurotox Res. 2018 Apr;33(3):560-568. Epub 2017 Dec 7 PubMed.

Further Reading

Papers

  1. Bosu DR, Kipreos ET. Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div. 2008 Feb 18;3:7. PubMed.

Primary Papers

  1. Gerez JA, Prymaczok NC, Rockenstein E, Herrmann US, Schwarz P, Adame A, Enchev RI, Courtheoux T, Boersema PJ, Riek R, Peter M, Aguzzi A, Masliah E, Picotti P. A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body-like pathology. Sci Transl Med. 2019 Jun 5;11(495) PubMed.

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