More than a decade has passed since researchers led by Dennis Selkoe of Brigham and Women’s Hospital in Boston first reported that when α-synuclein has its druthers, it prefers the company of three others. Now, a new study led by Selkoe reports that disease-associated mutations in another PD gene, LRRK2, destabilize this tetrameric form of α-synuclein. Published September 16 in the journal Parkinson’s Disease, the study found that in indued neurons from LRRK2 mutation carriers who had PD, the ratio of α-synuclein tetramers to monomers was low compared to control cells, while phosphorylation of α-synuclein was high. Treating the cells with LRRK2 kinase inhibitors restored the tetramers and lessened the pathological phosphorylation. So did an inhibitor of stearoyl-CoA desaturase, an enzyme that nixes lipids thought to stabilize α-synuclein tetramers.
- PD-linked variants in LRRK2 reduced the proportion of α-synuclein in tetrameric form.
- The mutations also boosted α-synuclein phosphorylation at serine-129.
- LRRK2 kinase inhibitors, or an inhibitor of a fatty-acid-desaturating enzyme, prevented these effects.
“This is an interesting study, providing additional evidence on the potential pathogenic interplay between LRRK2 and α-synuclein,” wrote Huaibin Cai, NIA, Bethesda, Maryland. “The exact mechanisms of how LRRK2 modulates the oligomerization and phosphorylation of α-synuclein, however, remains to be determined.”
In 2011, Selkoe and colleagues caused a stir in the PD field when they reported that the most stable, aggregation-resistant, physiological form of α-synuclein is a tetramer (Aug 2011 news; Feb 2012 news). They later reported that disease-associated mutations in α-synuclein destabilized the tetrameric form, and that transgenic mice expressing a tetramer-reticent form of α-synuclein developed a PD-like disorder (Jun 2015 news). Most recently, researchers at Johns Hopkins University, Baltimore, extended these findings to mutations in glucocerebrosidase (GBA1), which also discouraged the formation of α-synuclein tetramers (Kim et al., 2018).
Might variants in LRRK2—after all, the most common genetic cause of PD—do the same? To investigate, first author Luis Fonseca-Ornelas and colleagues obtained induced pluripotent stem cells from four people with PD; two carried the G2019S LRRK2 variant, two had the R1441C variant. These mutations reside in the kinase and GTPase domains of this large protein, respectively. The scientists generated mutation-corrected versions of each iPSC line to serve as wild-type controls, then differentiated the iPSC lines into induced neurons for study.
Next, the scientists deployed cross-linking agents to stabilize existing multimers of α-synuclein within intact cells. They found that both LRRK2 mutations significantly dampened the ratio of tetramers to monomers. The LRRK2 variants also upped phosphorylation at serine-129, a hotspot for disease-associated hyperphosphorylation of α-synuclein. Across the cell lines, a negative correlation emerged, such that the more α-synuclein existed as a loner, the more of it was phosphorylated at serine-129.
Reasoning that revved-up kinase activity might be to blame for the effect of the LRRK2 variants, the researchers treated the cell lines with two inhibitors of the kinase, PF-06447475 and MLi-1, for several days while the neurons were maturing. Strikingly, either inhibitor prevented the dip in tetramerization caused by the mutations and also thwarted serine-129 phosphorylation.
“These results add to the collective body of data that LRRK2 kinase activity appears to (albeit subtly) push the normal biology of α-synuclein toward states associated with pathological conformations,” commented Andrew West of Duke University School of Medicine in Durham, North Carolina. “Next steps will be to determine how LRRK2 directs α-synuclein away from tetrameric states, whether acting directly, or indirectly through Rab phosphorylation or mitochondrial interactions, for example.”
Separately, the scientists also investigated how stearoyl-CoA desaturase inhibitors affect α-synuclein tetramerization. The SCD enzyme catalyzes the rate-limiting step in the production of monounsaturated fatty acids from saturated fatty acid precursors, and studies from Selkoe’s and other labs have shown that SCD inhibitors can relieve α-synuclein toxicity (Dec 2018 news). Recently, they also showed that an SCD inhibitor supported tetramer formation in transgenic mice that expressed tetramer-destabilizing form of α-synuclein (Oct 2020 news). Now, they report the same was true in the context of LRRK2 mutations. Although the SCD inhibitor did not influence LRRK2 kinase activity, it did restore the tetramer to monomer ratio in LRRK2 mutant neurons, and reduced the hyperphosphorylation of α-synuclein.
The researchers obtained similar results from the same series of experiments when they differentiated the patient iPSCs into dopaminergic neurons instead of cortical neurons.
How do mutations in LRRK2 discourage the formation of α-synuclein tetramers? Selkoe does not know, but he noted that mutations in α-synuclein itself, GBA1, and now, LRRK2, all seem to destabilize -synuclein tetramers, casting α-synuclein homeostasis as a central disease mechanism. That SCD inhibitors restore the tetramer-to-monomer ratio in LRRK2 mutant cells suggests that lipid metabolism can act as a downstream mediator of genetic insults, disrupting α-synuclein’s ability to oligomerize, Selkoe proposed.
Despite some converging evidence, not everyone is convinced that shifts in -αsynuclein multimerization are the central mechanism driving PD. “I don’t think we know at this stage whether loss of tetramers is damaging to neurons, although the association with dominant mutations of both LRRK2 and SNCA would imply so,” wrote Mark Cookson of the National Institutes of Health in Bethesda, Maryland. Cookson added that multiple studies suggest that LRRK2 plays an important role in lysosomal function in non-neuronal cells, which were not examined in the current study.—Jessica Shugart
- An α-Synuclein Twist—Native Protein a Helical Tetramer
- Synuclein—Researchers Out of Sync on Structure
- Form and Function: What Makes α-Synuclein Toxic?
- Does Oleic Acid Hold the Key to α-Synuclein Toxicity?
- Curbing Fatty Acids Means No Parkinson’s—If You Are a Mouse
- Kim S, Yun SP, Lee S, Umanah GE, Bandaru VV, Yin X, Rhee P, Karuppagounder SS, Kwon SH, Lee H, Mao X, Kim D, Pandey A, Lee G, Dawson VL, Dawson TM, Ko HS. GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers. Proc Natl Acad Sci U S A. 2018 Jan 23;115(4):798-803. Epub 2018 Jan 8 PubMed.
No Available Further Reading
- Fonseca-Ornelas L, Stricker JM, Soriano-Cruz S, Weykopf B, Dettmer U, Muratore CR, Scherzer CR, Selkoe DJ. Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons. NPJ Parkinsons Dis. 2022 Sep 16;8(1):118. PubMed.