17 Feb 2023

In the healthy brain, α-synuclein leads a dynamic life near the synapse, where it frequently shifts its conformation as well as its association with membrane vesicles. Disruption of this active equilibrium can steer α-synuclein down the path of aggregation, leading to Parkinson’s disease. A study published February 14 in Science Signaling fingers synaptotagmin-11 as one such potential disruptor. Scientists led by Dennis Selkoe, Brigham and Women’s Hospital in Boston, report that this PD risk factor somehow coaxes α-synuclein to cling more tightly to vesicular membranes, where it loiters as disordered monomers rather than curling up into the tetramers that resist aggregation. The authors pinpoint palmitoylation of Syt11 as critical for this dangerous behavior. Without this lipid coat, Syt11 falls prey to the voracious enzymes of the endolysosomal system.

The study underscores the importance of membrane biodynamics and fatty acid modifications such as palmitoylation in maintaining proper α-synuclein homeostasis near the synapse, Selkoe said.

Aggregated clumps of α-synuclein, often tangled up with bits of cellular membrane, are among the detritus found in Lewy body inclusions. Selkoe's group previously reported that α-synuclein is most stable when folded into tetramers, while its disordered, monomeric form is prone to aggregate (Aug 2011 news). Since then, his lab has identified processes that skew this tetramerization, such as enzymes that regulate fatty acid metabolism or disease variants in the LRRK2 gene (Oct 2020 news; Sep 2022 news). Other labs have also found α-synuclein tetramers (Wang et al., 2011; Kim et al., 2018).

Protection by Palmitoylation? In neurons transduced with wild-type Syt11 (left), Syt11 formed punctate structures around the neuronal soma, dendrites, and axons. The palmitoylation-deficient version (right) was distributed similarly, but expression was reduced relative to wild-type. [Courtesy of Ho et al., Science Signaling, 2023.]

For the current study, first author Gary Ho and colleagues probed whether Syt11 might be involved. Noncoding variants near the gene have been tied to PD risk, and the protein prowls the same synaptic neighborhood as does α-synuclein (Jul 2014 conference news; Sesar et al., 2016). Studies suggest that the transmembrane synaptotagmin imbeds itself within trafficking vesicles just outside of the synapse, and can squelch dopamine release when overexpressed (Shimoju et al., 2019; Wang et al., 2018).

Before asking if and how Syt11 and α-synuclein intersect, the researchers focused on Syt11 regulation. Because other members of the synaptotagmin family are regulated by palmitoylation—the post-translational modification of cysteine residues with the long-chain fatty acid palmitate—the scientists checked if Syt11 receives similar treatment. Indeed, they found that in mouse, rat, and human brain tissue, a large fraction of Syt11 exists in a palmitoylated state. Further sleuthing mapped the greasy additions to cysteine residues 39 and 40.

To learn whether palmitoylation affects how Syt11 functions, the researchers infected induced iPSC-derived human neurons with a lentivirus carrying either the wild-type Syt11 gene or a mutated version, called Syt11-CS, in which cysteines 39 and 40 were mutated to serine. Rendering Syt11 “un-palmitoylatable” in these two spots dropped protein levels by 80 percent compared to the palmitoylated form. This was true in neurons derived from a PD patient whose α-synuclein gene was triplicated, and also in mutation-corrected neurons from the same patient. Using a series of inhibitors to determine how Syt11 is typically degraded, the researchers found that palmitoylation protected Syt11 from degradation within the endolysosomal system, rather than from the proteasome.

How did palmitate shield Syt11 from digestion? This remains unclear, but the researchers did find the modified Syt11 secluded in parts of the cell membrane that resist solubilization by the detergent digitonin. These membrane regions are poorly defined. They are rich in cholesterol, and other palmitoylated proteins also congregate there. These digitonin-insoluble regions have been implicated in protein trafficking and cell signaling (for review, see Chamberlain and Shipston, 2015). 

Because palmitoylated proteins reportedly enhance the curvature of membranes, and α-synuclein has a penchant for latching onto curved surfaces, the researchers asked if Syt11 altered α-synuclein binding to membranes. Indeed, they found that the ratio of membrane-bound to cytosolic α-synuclein doubled in human neurons overexpressing the wild-type form of Syt11. In contrast, overexpressing Syt11-CS did not affect α-synuclein’s membrane preference.

Dale Martin of the University of Waterloo in Ontario compared the curving effect to a Slinky. “The more you curve a Slinky, the more space opens up between the rungs,” he said. Similarly, when Syt11 enhances membrane curvature with its palmitate coat, more room in the membrane might open up for proteins to squeeze in. Martin added that many synaptic transmembrane proteins are palmitoylated, suggesting that the modification may play a critical role in regulating membrane biodynamics in this active compartment (Sanders et al., 2015). 

Scientists in Selkoe's group have reported that shifts in α-synuclein membrane attachment can skew its conformational equilibrium (Tripathi et al., 2022). In keeping with this idea, they found a 30 percent dip in the ratio of α-synuclein tetramers to monomers in cells overexpressing wild-type Syt11, but not in those overexpressing Syt11-CS. This glut of palmitoylated Syt11 in human neurons did not alter phosphorylation of α-synuclein at serine-129, a modification that has been tied to PD pathogenesis, but which may also regulate synaptic activity (Jan 2023 news).

At first glance, the findings might imply that inhibiting palmitoylation could stem α-synuclein aggregation. However, Ho pointed out that, like phosphorylation and other post-translational modifications, palmitoylation has different effects depending on the protein and pathways involved. In fact, Ho and Selkoe recently reported that boosting palmitoylation of MAP6, a microtubule binding protein that traffics in vesicles, dampened α-synuclein aggregation (Ho et al., 2020). A recent study reported that palmitoylation of the protein DNAJC5 instigated release of α-synuclein from cells (Wu et al., 2023). DNAJC5, a heat shock protein, has also been linked to the export of tau from neurons (Jun 2016 news). “We can’t consider palmitoylation as one thing that’s good or bad for neurons,” Ho said.

Martin agreed, comparing the palmitoylation field of today with the phosphorylation field of the 1980s and ’90s. Scientists are still at the stage of identifying enzymes that add and remove palmitate from different substrates, developing tools to measure palmitoylation, and investigating how the process is regulated in different cell types. The BrainPalmSeq database uses RNA-Seq data to track expression of palmitoylating and depalmitoylating enzymes within the mouse brain.

Another open question is how the findings relate to PD risk variants in Syt11, which land in noncoding regions near the gene. How these variants influence Syt11 expression remains unknown, but Ho plans to compare Syt11 palmitoylation and expression in brain samples from PD patients relative to controls.—Jessica Shugart

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References

News Citations

1. An α-Synuclein Twist—Native Protein a Helical Tetramer 19 Aug 2011
2. Curbing Fatty Acids Means No Parkinson’s—If You Are a Mouse 14 Oct 2020
3. LRRK2 Variants Keep α-Synuclein from Forming Tetramers 30 Sep 2022
4. Largest Meta-GWAS Yet Uncovers New Genetic Links to Parkinson’s 28 Jul 2014
5. Not All Bad: Phosphorylated α-Synuclein Modulates Neurotransmission 27 Jan 2023
6. Ushers of Propagation? More Evidence that Chaperones Evict Disease-Associated Proteins 24 Jun 2016

Paper Citations

1. Wang W, Perovic I, Chittuluru J, Kaganovich A, Nguyen LT, Liao J, Auclair JR, Johnson D, Landeru A, Simorellis AK, Ju S, Cookson MR, Asturias FJ, Agar JN, Webb BN, Kang C, Ringe D, Petsko GA, Pochapsky TC, Hoang QQ. A soluble α-synuclein construct forms a dynamic tetramer. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17797-802. Epub 2011 Oct 17 PubMed.
2. 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.
3. Sesar A, Cacheiro P, López-López M, Camiña-Tato M, Quintáns B, Monroy-Jaramillo N, Alonso-Vilatela ME, Cebrián E, Yescas-Gómez P, Ares B, Rivas MT, Castro A, Carracedo A, Sobrido MJ. Synaptotagmin XI in Parkinson's disease: New evidence from an association study in Spain and Mexico. J Neurol Sci. 2016 Mar 15;362:321-5. Epub 2016 Feb 8 PubMed.
4. Shimojo M, Madara J, Pankow S, Liu X, Yates J 3rd, Südhof TC, Maximov A. Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity. Genes Dev. 2019 Mar 1;33(5-6):365-376. Epub 2019 Feb 26 PubMed.
5. Wang C, Kang X, Zhou L, Chai Z, Wu Q, Huang R, Xu H, Hu M, Sun X, Sun S, Li J, Jiao R, Zuo P, Zheng L, Yue Z, Zhou Z. Synaptotagmin-11 is a critical mediator of parkin-linked neurotoxicity and Parkinson's disease-like pathology. Nat Commun. 2018 Jan 8;9(1):81. PubMed.
6. Chamberlain LH, Shipston MJ. The physiology of protein S-acylation. Physiol Rev. 2015 Apr;95(2):341-76. PubMed.
7. Sanders SS, Martin DD, Butland SL, Lavallée-Adam M, Calzolari D, Kay C, Yates JR 3rd, Hayden MR. Curation of the Mammalian Palmitoylome Indicates a Pivotal Role for Palmitoylation in Diseases and Disorders of the Nervous System and Cancers. PLoS Comput Biol. 2015 Aug;11(8):e1004405. Epub 2015 Aug 14 PubMed.
8. Tripathi A, Alnakhala H, Terry-Kantor E, Newman A, Liu L, Imberdis T, Fanning S, Nuber S, Ramalingam N, Selkoe D, Dettmer U. Pathogenic Mechanisms of Cytosolic and Membrane-Enriched α-Synuclein Converge on Fatty Acid Homeostasis. J Neurosci. 2022 Mar 9;42(10):2116-2130. Epub 2022 Jan 27 PubMed.
9. Ho GP, Ramalingam N, Imberdis T, Wilkie EC, Dettmer U, Selkoe DJ. Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity. Mov Disord. 2021 Feb;36(2):348-359. Epub 2020 Oct 26 PubMed.
10. Wu S, Hernandez Villegas NC, Sirkis DW, Thomas-Wright I, Wade-Martins R, Schekman R. Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers. Elife. 2023 Jan 10;12 PubMed.

External Citations

1. BrainPalmSeq database

Further Reading

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