Phosphorylation of α-synuclein at serine 129 has long been used as a marker of synuclein disorders. After all, p-S129syn accumulates in Lewy bodies. But does this modification have a physiological function?Two of his recent papers suggest that neuronal activity triggers her S129 phosphorylation, which promotes synaptic vesicle trafficking. One paper suggests that this boosts neurotransmitter release, while others suggest that it may attenuate it. It helps shed light on long-standing questions about the neurophysiological function of ,” writes Michael Schlossmacher of the University of Ottawa, Canada.

  • Neuronal activity triggers the phosphorylation of α-synuclein at Serine 129.
  • p-S129syn binds to presynaptic vesicle proteins and enables vesicle trafficking.
  • Mice living in an enriched environment produce large amounts of p-S129syn and enhance synaptic plasticity.

In NPJ Parkinson’s disease Jan. 16, researchers led by Nagendran Ramalingam and Ulf Dettmer at Brigham and Women’s Hospital in Boston found that action potentials increased the amount of p-S129syn in cultured cortical and hippocampal rat neurons. reported that it allowed to overfill and flooded the presynapses. Increases excitatory signaling. Mice living in enriched environments, cages full of new objects to explore, had p-S129syn filling neurons and exhibiting higher synaptic plasticity. Environmental enrichment is known to enhance plasticity, and learning and memory in mice (News of September 2005; Jankowsky et al., 2005).

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

“If we’re right, and p-S129 is the ‘on’ switch for α-synuclein, then past and future experiments need to be viewed through this lens. I think it’s a big shift in mindset,” Roy told his Alzforum. Michael Henderson of the Van Andel Institute in Michigan agrees. “These studies are an important caveat for careful interpretation of antibody-dependent data and serve as a reminder that p-S129 staining alone is insufficient to establish the presence of Lewy disease.” he wrote (comment below).

Ramalingam, lead author in Dettmer’s lab, wondered whether alpha-synuclein phosphorylation responds to neuronal signaling. He used the GABA receptor antagonist picrotoxin (PTX) to modulate firing in cultured rat cortical neurons. After 6 hours, p-S129syn spiked approximately 4-fold, while total α-synuclein remained unchanged. In contrast, inhibition of neuronal activity with the sodium channel blocker tetrodotoxin (TTX) reduced p-S129syn by 25%. These results suggest that synaptic activity produces his p-S129syn.

To see where this phosphorylation occurs, the researchers used high-resolution confocal microscopy to zoom in on synapses. They confirmed that p-S129syn foci overlap with the presynaptic protein synapsin in both untreated and PTX-stimulated neurons. Synaptosomes isolated from these cells were rich in phosphorylated proteins, suggesting that p-S129syn resides mostly within presynaptic vesicles.

How does neuronal activity trigger α-synuclein phosphorylation? oxidizing, the calcium-dependent phosphatase calcineurin (CaN) was also identified as important for p-S129syn regulation (Inglis et al., 2009; Lee et al., 2011). Scientists believe that calcium released by action potentials activates her CaN, increasing the activity of her Plk2 by a post-translational mechanism.

Does p-S129syn play any role in healthy neurons? Indeed, rat hippocampal neurons expressing α-synuclein with an alanine at position 129 that cannot be phosphorylated produce fewer excitatory action potentials and more than control neurons. generated inhibitory action potentials. Similarly, hippocampal slices from her 1-month-old S129A mice were capable of only weak short- and long-term potentiation (LTP). The authors concluded that phosphorylation of S129 stimulates synaptic firing. In vivo, environmental enrichment boosted her p-S129syn in mice (see image below). Dettmer et al. propose that p-S129syn may alter plasticity by modulating the recycling of synaptic vesicles during neurotransmission.

synuclein stimulationWild-type mice (orange) living in cages with many toys have more p-S129syn (left) and enhanced long-term potentiation (right) than animals in typical captivity (purple). ). [Courtesy of Ramalingam et al., NPJ Parkinson’s Disease, 2023.]

Roy and colleagues found that S129 phosphorylation does just that. Co-first authors Leonardo Parra-Rivas and Kaylivizhi Madhivanan overexpressed human α-synuclein in mouse hippocampal neurons. The wild-type protein moderated synaptic vesicle recycling and the phosphomimetic S129D further suppressed it, whereas S129A had no effect, suggesting that phosphorylation of S129 puts a brake on synaptic vesicle recycling. there is This may dampen neurotransmitter release, but Roy did not measure neuronal activity.

Like Dettmer and colleagues, researchers in Roy’s lab observed an increase in presynaptic p-S129syn after stimulating cultured neurons, while total α-synuclein remains stable. The same is true for wild-type mice. Addition of he TTX or a Plk2 inhibitor to cultured neurons prevented the increase in p-S129syn. “It’s encouraging that Roy’s lab has seen something similar to ours,” Ramalingam said.

Parra-Rivas and Madhivanan also detected the S129D phosphomimetic exclusively at the presynaptic site, suggesting that phosphorylation promotes α-synuclein swarming. What was p-S129syn doing at the presynapse?phosphorylation facilitated α-synuclein binding to her two synaptic partners involved in neurotransmitter release, synapsin, and VAMP2. S129D α-synuclein bound more proteins than wild-type synuclein, whereas the S129A mutant bound neither. In mice stimulated with the potassium channel blocker 4-aminopyridine, α-synuclein bound more synapsins and her VAMP2 than in control animals.

To investigate how phosphorylation facilitates binding, scientists focused on the C-terminus of α-synuclein, where S129 is located and where VAMP2 and synapsin bind. They modeled the structures of wild-type and S129D α-synucleins using the publicly available software ColabFold, which predicts three-dimensional structures of proteins (Mildita et al., 2022). The serine left her wild-type C-terminus unstructured, while the negatively charged aspartic acid cuddled the nearby positively charged he five lysine residues, wrapping the protein terminus inward. (see image below). The authors believe that this stabilizes the VAMP2/synapsin binding domain and allows proteins to interact.

Opposites attract. Molecular modeling suggests that the C-terminus (purple) of α-synuclein (grey) is unstructured when S129 (green) is unphosphorylated (left). The phosphomimetic S129D (right) attracts five positively charged lysine residues (yellow) and folds into a configuration that binds VAMP2 and synapsin (not shown). [Courtesy of Parra-Rivas et al., bioRxiv, 2023.]

The distribution of p-S129syn throughout the mouse brain also caught Roy’s eye. He found it only in dopaminergic neurons in the midbrain and in olfactory neurons, a region susceptible to neurodegeneration in PD. A similar pattern was reported in another recent bioRXiv preprint of his—p-S129syn (p-S129syn) accumulates within olfactory bulb neurons of healthy mice, rats, non-human primates, and humans.Killinger et al., 2023). Phosphoproteins interacted with presynaptic vesicle trafficking and recycling proteins.

Roy and Dettmer’s lab found that phosphorylation of alpha-synuclein promotes synaptic protein-protein interactions and neurotransmitter release, suggesting that the known function of phosphoproteins is simply an indication of synucleinopathy. “These exciting results challenge the central dogma of Parkinson’s disease and open up new opportunities to decipher this pathology through the lens of synaptic physiology,” Parra-Rivas et al. is writing —Chelsea Weidman Burke

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paper citation

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    Environmental enrichment reduces cognitive deficits in mouse models of Alzheimer’s disease.
    J Neuroscience2005 May 25;25(21):5217-24.
  2. .
    Polo-like kinase 2 (PLK2) phosphorylates α-synuclein at Serine 129 in the central nervous system.
    J Biochemistry30 January 2009;284(5):2598-602.
  3. .
    Enhanced phosphatase activity attenuates α-synucleinopathy in a mouse model.
    J Neuroscience2011 May 11;31(19):6963-71.
  4. .
    ColabFold: Making protein folding accessible to everyone.
    nut method2022 Jun;19(6):679-682. Epub 2022 30 May.
  5. .
    Distribution of phosphorylated α-synuclein in the non-diseased brain suggests that olfactory bulb mitral cells are involved in the pathogenesis of synucleinopathies.
    Bio RxivJanuary 6, 2023
    Bio Rxiv