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Preformed Fibril Seeding Reshapes the Phosphorylated α-Synuclein Proximal Proteome in the Olfactory Bulb

Solji G. ChoiiD, Atousa BahramiiD, Jayda B. DuvernayiD, Tyler TittleiD, Ronald MelkiiD, Jeffrey H. KordoweriD, Bryan A. KillingeriD

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Abstract

BackgroundAggregated alpha-synuclein (syn) phosphorylated at serine 129 (PS129) accumulates in synucleinopathies, with the olfactory bulb (OB) being severely affected. Non-aggregated physiological PS129 is abundant in the mammalian OB, where it likely modulates syn-protein interactions. The impact of aggregation on the PS129 interactome in the OB remains unclear. We hypothesized that syn aggregation alters the PS129 interactome, shifting canonical synaptic partners (e.g., SNARE proteins) toward the aggregate-associated network. To test this hypothesis, we mapped PS129 interactions in the OB of PFF-injected WT and SNCAA53T/A53T (M83) mice using biotinylation by antibody recognition (BAR) and pretreated with calf-intestine alkaline phosphatase (CIAP) to distinguish physiological from aggregate-associated PS129 interactomes. Seeding and spread were assessed by immunohistochemistry and in situ seeding immunodetection assay (isSID). ResultsFollowing OB-PFF injections, CIAP-resistant aggregates and seeds were detected throughout the neuroaxis of M83 mice (e.g., OB to brainstem) but less so in WT mice. isSID seeding was concentrated near CIAP-resistant aggregates, but the overlap was only partial. BAR-PS129 identified 2,309 proteins in M83 OBs and 990 proteins in WT OBs. Of these, 357 proteins in M83 mice and 247 proteins in WT mice were associated with the CIAP-resistant, aggregate-enriched PS129 fraction. In both models, the CIAP-resistant interactome largely overlapped with the broader PS129 interactome, suggesting that seeded aggregation primarily affects existing PS129 interactions rather than directing the formation of new pathological ones. A conserved 107-protein CIAP-resistant signature shared between M83, and WT mice was enriched for axon-glia adhesion, myelin-associated, axonal/cytoskeletal, proteostatic, and synaptic vesicle-related proteins. ConclusionAggregated PS129 engages a subset of PS129 networks enriched at axon-glial interfaces. CIAP-resistant aggregates were partially associated with seed competency, indicating that CIAP resistance and seed competency are related but not equivalent. These results provide molecular details of syn seeding and spread from the OB.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (27 Jul 2026) · CC BY 4.0

Proximity proteomics in PFF-seeded mouse olfactory bulb shows aggregated phospho-S129 alpha-synuclein mostly reprioritizes existing interactions toward axon-glia, myelin and proteostasis proteins, with a conserved 107-protein signature.

Abundant physiological PS129 in the olfactory bulb obscures aggregate-specific interactions, so how aggregation changes the PS129 protein environment in vivo has been unresolved. Pairing phosphatase pretreatment with antibody-directed biotinylation separates physiological from aggregated PS129 interactomes within the same brain region in two mouse models.

Key results

  1. ShownAfter phosphatase pretreatment, aggregate-type PS129 spread widely from the olfactory bulb injection site in M83 mice but stayed mostly at the injection site in WT mice.

    M83 (n=5): CIAP-resistant PS129 appeared in downstream areas including piriform, entorhinal, hippocampal, midbrain and medulla regions; residual signal after CIAP was mainly in GCL, PA and COA. WT (n=3): seven regions, no significant PBS vs PFF differences, and residual signal mainly in the GCL. · CIAP-resistant PS129 reveals αsyn aggregate…; CIAP-resistant PS129 reveals limited αsyn…; Figure legends

  2. ShownSeed-competent species (isSID) tracked CIAP-resistant PS129 regionally in M83 mice, but the two signals overlapped only partially at the cellular level, so CIAP resistance and seeding are related but not equivalent.

    Pearson correlation between isSID and CIAP-resistant PS129: mean 0.2949 (SD 0.2304) in OB GCL and 0.4551 (SD 0.04842) in piriform area, n = 3 mice. WT showed only weak isSID signal at the injection site. · CIAP-resistant PS129 pathology coincides with…; Seeding, aggregation, and spread in OB-PFF…; Figure legends

  3. SuggestedThe aggregate-associated PS129 interactome is almost entirely contained within the broader PS129 interactome in both models, suggesting reprioritization of existing interactions rather than new ones.

    M83: 2,309 proteins (2,186 PBS, 3 PBS+CIAP, 2,248 PFF, 357 PFF+CIAP); all 357 inside the PFF interactome; no DA proteins PFF vs PBS. WT: 990 proteins (928, 8, 971, 247); about 93% shared; 5 unique to PFF+CIAP. · CIAP-resistant PS129 proximal proteome in M83…; CIAP-resistant PS129 proximal proteome in WT OBs; Axon–glia-enrichment in PS129 interactomes in…; Seeding and aggregation shifts the PS129…

  4. ShownA 107-protein CIAP-resistant signature shared by M83 and WT is enriched for axon-glia adhesion, myelin, cytoskeletal, proteostatic and synaptic vesicle proteins, with MAPT as the network hub.

    PFF+CIAP overlap between models was 22%; IS correlation among the 107 shared proteins r ≈ 0.362, R² = 0.131, p < 0.001. GFAP had top IS in M83 (27.52). MAPT betweenness 1,980 in a 95-protein STRING cluster. · Axon–glial pathways between two synucleinopathy…; PS129 at the axon-glial interface

  5. SuggestedGFAP enrichment in the M83 aggregate interactome likely reflects reactive astrocytic processes adjacent to, not overlapping, CIAP-resistant PS129 pathology.

    Pearson correlation between GFAP and CIAP-resistant PS129 in M83 OBs: mean 0.018, SD 0.034, n = 3. GFAP-positive processes surrounded Lewy body-like structures without clear signal overlap. · Axon–glial pathways between two synucleinopathy…; PS129 at the axon-glial interface; Figure legends

Methods, data and reproducibility

PFF or PBS was injected into the olfactory bulb of WT and M83 mice; sections were treated with or without CIAP, PS129 neighbors were tagged by BAR proximity biotinylation and quantified by label-free LC-MS/MS. Interactomes were ranked by an importance score combining fold change and adjusted p value, then compared across conditions and models.

  • WT C57BL/6J mice with unilateral OB injection of PFF or PBS, about 100 days survival, histology and BAR-PS129 proteomics · mouse · n = n = 5 per treatment condition
  • Homozygous M83 (SNCA A53T) mice with OB injection of PFF or PBS, histology, isSID and BAR-PS129 proteomics · mouse · n = n = 21 per treatment condition
  • M83 BAR-PS129 captures across four conditions (PBS, PBS+CIAP, PFF, PFF+CIAP) plus BAR-Neg; M83 samples run as duplicate injections · mouse · n = 15-20 pooled OBs per capture
Methods & tools
BAR (biotinylation by antibody recognition), CIAP pretreatment, isSID (in situ seeding immunodetection assay), Orbitrap Exploris 240, MaxQuant, MaxLFQ, LFQ-Analyst, g:Profiler, Cytoscape v3.10.3 with stringApp v2.2.0, CentiScaPe v2.2, R v4.4.1, GraphPad Prism 10
Reagents & environment
R v4.4.1; Cytoscape v3.10.3; stringApp v2.2.0; CentiScaPe v2.2; ImageJ v1.54g; GraphPad Prism 10; MaxQuant search against the reviewed Swiss-Prot mouse proteome (UP000000589).
Reproducibility gaps
No code URL or public repository accession for raw mass spectrometry data is printed; LFQ-Analyst outputs and importance scores are in supplementary Datasets S1–S3.
Limitations (4)
  • CIAP pretreatment may exclude intermediate, less phosphatase-resistant aggregate species, which would lead to underestimation of aggregates. · stated by the authors
  • Ectopic A53T expression from the prion promoter in M83 mice likely yields false positives of unclear relevance to human disease; WT mice develop less pathology, so conclusions rely on proteins shared across both models. · stated by the authors
  • Key spatial colocalization estimates (isSID vs CIAP-resistant PS129, GFAP vs PS129) rest on n = 3 mice, and WT regional quantification also used n=3, limiting power. · noted in this summary
  • Proximity labeling cannot separate direct binding from adjacency, and no functional perturbation tests whether the axon-glia proteins drive spread; the retrograde circuit interpretation is inferential. · noted in this summary

Read this if you study alpha-synuclein seeding, proximity proteomics or olfactory-route pathology and want protein-level evidence for axon-glia involvement in aggregated PS129.

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