27 Jan 2023

Phosphorylation of α-synuclein at serine 129 has long been used as a marker of synucleinopathy—p-S129syn accumulates in Lewy bodies, after all. But does this modification have a physiological function? Two recent papers suggest that neuronal activity sparks S129 phosphorylation, which, in turn, drives synaptic vesicle trafficking. One paper suggests this facilitates neurotransmitter release but the other hints it may attenuate it. “This fascinating work helps shed light on the longstanding question of a neurophysiological function for p-S129syn,” wrote Michael Schlossmacher, University of Ottawa, Canada.

In the January 16 NPJ Parkinson’s Disease, researchers led by Nagendran Ramalingam and Ulf Dettmer, Brigham and Women’s Hospital, Boston, reported that, in cultured cortical and hippocampal rat neurons, action potentials increase the amount of p-S129syn, which floods pre-synapses and increases excitatory signaling. In mice living in an enriched environment, namely in cages replete with novel objects to explore, p-S129syn filled neurons and synaptic plasticity was high. Environmental enrichment is known to boost plasticity, and learning and memory in mice (Sep 2005 news; Jankowsky et al., 2005).

In a bioRxiv preprint uploaded on December 23, last year, Subhojit Roy of the University of California, San Diego, and colleagues also reported a spike in presynaptic p-S129syn following neuronal activity. On a molecular level, p-S129 stabilized the C-terminus, allowing α-synuclein to bind synaptic vesicle proteins, they reported.

“If we are right and p-S129 is an ‘on’ switch for α-synuclein, then any past and future experiments have to be viewed through this lens—I think this is a major shift in thinking,” Roy told Alzforum. Michael Henderson of the Van Andel Institute, Michigan, agreed. “These studies are an important reminder to interpret antibody-dependent data carefully and that p-S129 staining alone is insufficient to establish the presence of Lewy pathology,” he wrote (comment below).

First author Ramalingam in Dettmer’s lab wondered if α-synuclein phosphorylation responded to neuron signaling. He used the GABA receptor antagonist picrotoxin (PTX) to dial up the firing of cultured rat cortical neurons. Six hours later, p-S129syn spiked about fourfold, while total α-synuclein did not change. On the other hand, suppressing neural activity with the sodium channel blocker tetrodotoxin (TTX) dropped p-S129syn by 25 percent. These results suggest that synaptic activity generates p-S129syn.

To see where this phosphorylation occurs, the researchers zoomed in on synapses using high-resolution confocal microscopy. They saw that p-S129syn foci overlapped with the presynaptic protein synapsin in both untreated and PTX-stimulated neurons. Synaptosomes isolated from those cells were enriched with the phosphorylated protein, hinting that p-S129syn was mostly within presynaptic vesicles.

How does neuronal activity lead to α-synuclein phosphorylation? A series of enzyme inhibition experiments confirmed that polo-like kinase 2 (Plk2) phosphorylates S129 and that protein phosphatase 2A (PP2A) dephosphorylates it, but also identified the calcium-dependent phosphatase calcineurin (CaN) as crucial for p-S129syn regulation (Inglis et al., 2009; Lee et al., 2011). The scientists believe that calcium released by action potentials activates CaN, which, in turn, increases Plk2 activity by some post-translational mechanism.

Does p-S129syn play any role in healthy neurons? Indeed, rat hippocampal neurons expressing α-synuclein with alanine at position 129, which cannot be phosphorylated, generated fewer excitatory action potentials and more inhibitory ones than did control neurons. Similarly, hippocampal slices from 1-month-old S129A mice could only muster weak short- and long-term potentiation (LTP). The authors concluded that phosphorylation of S129 stimulates synaptic firing. In vivo, environmental enrichment boosted p-S129syn in mice (see image below). Dettmer and colleagues think p-S129syn might alter plasticity by orchestrating synaptic vesicle recycling during neurotransmission.

Stimulating Synuclein. Wild-type mice living a cage with many toys (orange) had more p-S129syn (left) and enhanced long-term potentiation (right) than did animals in typical housing (purple). [Courtesy of Ramalingam et al., NPJ Parkinson’s Disease, 2023.]

Roy and colleagues found that S129 phosphorylation did just that. For their part, co-first authors Leonardo Parra-Rivas and Kayalvizhi Madhivanan overexpressed human α-synuclein in mouse hippocampal neurons. Wild-type protein tempered synaptic vesicle recycling, the phosphomimetic S129D suppressed it further, but the S129A had no effect, suggesting that S129 phosphorylation puts the brake on synaptic vesicle recycling. Though this might attenuate neurotransmitter release, Roy did not measure neural activity.

Like Dettmer and colleagues, researchers in Roy’s lab saw pre-synaptic p-S129syn rise, yet total α-synuclein remain steady, after stimulating cultured neurons. Ditto in wild-type mice. Adding TTX or a Plk2 inhibitor to cultured neurons prevented the uptick of p-S129syn. “It’s reassuring that the Roy lab saw something similar to what we did,” Ramalingam said.

Parra-Rivas and Madhivanan also exclusively detected the S129D phosphomimetic in the pre-synapses, suggesting that phosphorylation encourages α-synuclein to flock there. What was p-S129syn up to in the pre-synapses? Phosphorylation facilitated α-synuclein’s binding to two synaptic partners involved in neurotransmitter release, synapsin, and VAMP2. S129D α-synuclein bound more of both proteins than did wild-type synuclein, while the S129A mutant bound neither. In mice stimulated with the potassium channel blocker 4-aminopyridine, α-synuclein also bound more synapsin and VAMP2-than it did in control animals.

To find out how phosphorylation facilitates binding, the scientists homed in on the C-terminus of α-synuclein, which is where S129 is located and VAMP2 and synapsin bind. They modeled the structures of wild-type and S129D α-synuclein using ColabFold, publicly available software that predicts three-dimensional protein structures (Mirdita et al., 2022). While the serine left the wild-type C-terminus unstructured, the negatively charged aspartic acid snuggled with five nearby positively charged lysine residues, causing the end of the protein to curl inward (see image below). The authors think this stabilizes the VAMP2/synapsin binding region, enabling the proteins to interact.

The distribution of p-S129syn throughout the mouse brain also caught Roy’s eye. He found it only in dopaminergic neurons of the midbrain and in olfactory neurons, regions susceptible to neurodegeneration in PD. Another recent bioRXiv preprint reported a similar pattern—p-S129syn accumulating within olfactory bulb neurons in healthy mice, rats, nonhuman primates, and people (Killinger et al., 2023). The phosphoprotein interacted with presynaptic vesicle trafficking and recycling proteins.

All told, the Roy and Dettmer labs found that phosphorylation of α-synuclein drives synaptic protein-protein interactions and facilitates neurotransmitter release, extending the phosphoprotein’s known function beyond just being a sign of synucleinopathy. “These exciting results challenge a central dogma in PD and open up new opportunities to decipher this pathology through the lens of synaptic physiology,” wrote Parra-Rivas and colleagues.—Chelsea Weidman Burke

News Citations

1. Exercise Helps Mouse Elders Learn, Generate New Neurons 21 Sep 2005

Paper Citations

1. Jankowsky JL, Melnikova T, Fadale DJ, Xu GM, Slunt HH, Gonzales V, Younkin LH, Younkin SG, Borchelt DR, Savonenko AV.
   Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer’s disease.
   J Neurosci. 2005 May 25;25(21):5217-24.
   PubMed.
2. Inglis KJ, Chereau D, Brigham EF, Chiou SS, Schöbel S, Frigon NL, Yu M, Caccavello RJ, Nelson S, Motter R, Wright S, Chian D, Santiago P, Soriano F, Ramos C, Powell K, Goldstein JM, Babcock M, Yednock T, Bard F, Basi GS, Sham H, Chilcote TJ, McConlogue L, Griswold-Prenner I, Anderson JP.
   Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.
   J Biol Chem. 2009 Jan 30;284(5):2598-602.
   PubMed.
3. Lee KW, Chen W, Junn E, Im JY, Grosso H, Sonsalla PK, Feng X, Ray N, Fernandez JR, Chao Y, Masliah E, Voronkov M, Braithwaite SP, Stock JB, Mouradian MM.
   Enhanced phosphatase activity attenuates α-Synucleinopathy in a mouse model.
   J Neurosci. 2011 May 11;31(19):6963-71.
   PubMed.
4. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M.
   ColabFold: making protein folding accessible to all.
   Nat Methods. 2022 Jun;19(6):679-682. Epub 2022 May 30
   PubMed.
5. Killinger BA, Mercado G, Choi S, Tittle T, Chu Y, Brundin P, Kordower JH.
   Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis.
   bioRxiv. January 6, 2023
   bioRxiv