Not All Bad: Phosphorylated α-Synuclein Modulates Neurotransmission.

January 27, 2023

Phosphorylation of α-synuclein at serine 129 has long been used as a marker of synucleinopathy—p-S129syn eventually accumulates in Lewy bodies. But does this modification have a physical function? Two recent papers suggest that neuronal activity induces S129 phosphorylation, which, in turn, leads to synaptic vesicle trafficking. One paper suggests that it facilitates neurotransmitter release, but other cues may reduce this. “This exciting work helps shed light on the long-standing question of the neurophysiological function of p-S129syn,” wrote Michael Schlesmacher, University of Ottawa, Canada.

In NPJ Parkinson’s Disease January 16, researchers led by Nagendran Ramalingam and Alf Dettmer, Brigham and Women’s Hospital, Boston, report that, in cultured cortical and hippocampal rat neurons, action potentials increase the amount of p-S129syn. is, which pre-affects synapses. and enhances excitatory signaling. In mice living in an enriched environment, i.e. cages full of new things to explore, p-S129syn loaded neurons and synaptic plasticity were greater. Environmental enrichment is known to enhance plasticity, and learning and memory in rats (News September 2005; Jankowski et al., 2005).

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

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

First author Ramalingam in Detmer’s lab wondered whether α-synuclein phosphorylation responded to neuronal signaling. He used the GABA receptor antagonist picrotoxin (PTX) to modulate the firing of cultured rat cortical neurons. Six hours later, p-S129syn increased fourfold, while total α-synuclein remained unchanged. On the other hand, suppression of neuronal activity with the sodium channel blocker tetrodotoxin (TTX) decreased p-S129syn by 25%. 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 observed that p-S129syn foci overlapped with the presynaptic protein synapsin in both untreated and PTX-stimulated neurons. Synaptosomes isolated from these cells were enriched for phosphorylated protein, indicating 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 the important calcium-dependent phosphatase calcineurin (CaN). Stated et al., 2009; Lee et al., 2011). The scientists believe that the calcium released by the action potential activates CaN, which, in turn, increases Plk2 activity through some post-translational mechanism.

Does p-S129syn play a role in healthy neurons? Indeed, rat hippocampal neurons expressing α-synuclein with an alanine at position 129, which cannot be phosphorylated, generate fewer excitatory action potentials and more inhibitory ones than control neurons. Similarly, hippocampal slices from 1-month-old S129A mice could elicit only weak short- and long-term potentiation (LTP). The authors concluded that phosphorylation of S129 activates synaptic firing. In vivo, environmental enrichment promoted p-S129syn in mice (see figure below). Dettmer and colleagues hypothesize that p-S129syn may modulate plasticity through synaptic vesicle recycling during neurotransmission.

Activation of synuclein. Wild-type mice housed in cages have many toys (orange) with more p-S129syn (left) and increased long-term potentiation (right) than animals in normal 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 Kyalvezi Madhyavanan overexpressed human α-synuclein in mouse hippocampal neurons. The wild-type protein tapered synaptic vesicle recycling, further suppressed by the phosphomimetic S129D, but S129A had no effect, suggesting that S129 phosphorylation puts a brake on synaptic vesicle recycling. Although it may reduce neurotransmitter release, Roy did not measure neural activity.

Similar to Dettmer and colleagues, researchers in Roy’s laboratory observed an increase in pre-synaptic p-S129syn, yet stable total α-synuclein, after stimulation of cultured neurons. Similarly in wild-type mice. Addition of TTX or a Plk2 inhibitor to cultured neurons prevented the increase of p-S129syn. “It’s reassuring that RoyLab saw something similar to what we did,” Ramalingam said.

Para-Rivas and Madhivanan also specifically detected the S129D phosphomimetic pre-synaptically at synapses, suggesting that phosphorylation induces α-synuclein to localize there. What was P-S129syn already in synapses? Phosphorylation facilitates the binding of α-synuclein to two synaptic partners involved in neurotransmitter release, synapsin and VAMP2. S129D α-synuclein bound more of the two proteins than wild-type synuclein, whereas the S129A mutant did not bind. 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 learn how phosphorylation facilitates binding, the scientists focused on the C-terminus of α-synuclein, where S129 is located and VAMP2 and synapsin bond. They modeled the structures of wild-type and S129D α-synuclein using ColabFold, a 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 intercalated with five nearby positively charged lysine residues, causing the end of the protein to curl inward (Fig. see). The authors believe that this stabilizes the VAMP2/synapsin binding region, enabling the protein to interact.

The distribution of p-S129syn in the mouse brain also caught Roy’s eye. He found it only in dopaminergic neurons and olfactory neurons of the midbrain, regions that are susceptible to neurodegeneration in PD. Another recent bioRXiv preprint reported a similar pattern—p-S129syn accumulates within olfactory bulb neurons in healthy mice, rats, nonhuman primates, and humans ( Killinger et al., 2023 ). Phosphoproteins interact 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, rendering the known function of the phosphoprotein the only symptom of synucleinopathy. is extended beyond being. “These exciting findings challenge a central belief in PD and open new opportunities for understanding this pathology through the lens of synaptic physiology,” wrote Para-Rivas and colleagues.—Chelsea Weidman Burke