Abstract

   Synucleinopathies such as Parkinson's disease (PD), dementia with Lewy
   bodies (DLB), and multiple system atrophy (MSA) are neurodegenerative
   diseases with shared clinical and pathological features. Aggregates of
   alpha-synuclein (αsyn) phosphorylated at serine 129 (PSER129) are
   hallmarks of synucleinopathies, which, for PD/DLB, are found
   predominantly in neurons, whereas in MSA, aggregates are primarily
   found in oligodendroglia. It remains unclear whether the distinct
   pathological presentations of PD/DLB and MSA are manifestations of
   unique or shared pathological processes. Using the in-situ proximity
   labeling technique of biotinylation by antibody recognition (BAR), we
   compared aggregated αsyn-interactomes (BAR-PSER129) and total
   αsyn-interactomes (BAR-MJFR1) between MSA (n = 5) and PD/DLB (n = 10)
   in forebrain and midbrain structures. Comparison between MSA and
   PD/DLB-enriched proteins revealed 79 PD/DLB-differentially abundant
   proteins and only three MSA-differentially abundant proteins (CBR1,
   CRYAB, and GFAP). Pathway enrichment analysis revealed that
   vesicle/SNARE-associated pathways dominated PD/DLB interactions,
   whereas MSA was strongly enriched for metabolic/catabolic, iron, and
   cellular oxidant detoxification pathways. A subnetwork of cytosolic
   antioxidant enzymes called peroxiredoxins drove cellular detoxification
   pathway enrichment in MSA. A network of 26 proteins, including
   neuronal-specific proteins (e.g., SYNGR3) with HSPA8 at the core, was
   shared between MSA and DLB/PD. Extracellular exosome pathways were
   universally enriched regardless of the disease or BAR target protein.
   In conclusion, synucleinopathies have divergent and convergent
   αsyn-aggregate interactions, indicating unique and shared pathogenic
   mechanisms. MSA uniquely involves oxidant detoxification processes in
   glial cells, while vesicular processes in neurons dominate PD/DLB.
   Shared interactions, specifically SYNGR3, between MSA and PD/DLB
   suggest that neuronal axons are the origin of both diseases. In
   conclusion, we provide αsyn protein interaction maps for two distinct
   synucleinopathies.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s40478-025-01958-5.

   Keywords: Neurodegeneration, Proximity proteomics, Antioxidant, Spatial
   omics, SNARE complex

Introduction

   Primary synucleinopathies are neurodegenerative diseases, including
   Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple
   system atrophy (MSA), which have common pathological hallmarks,
   specifically alpha-synuclein (αsyn) aggregates [[39]18]. The
   distribution and abundance of αsyn aggregates are highly heterogeneous
   between synucleinopathies, with copathologies (e.g., tau and
   beta-amyloid) also being common [[40]9, [41]29]. MSA is unique among
   synucleinopathies because αsyn aggregates are predominantly observed in
   oligodendroglia (glial cytoplasmic inclusions or “GCIs”, also called
   Pap–Lantos bodies) [[42]42]; in contrast, PD/DLB brain neurons exhibit
   the majority of Lewy pathology (neuronal inclusions or “NIs”) [[43]18].
   NIs are assumed to originate in neurons, but the origins of GCIs are
   less obvious because, unlike neurons, oligodendroglia do not normally,
   abundantly, express αsyn [[44]2, [45]13, [46]37]. Therefore, in the MSA
   brain, either αsyn aggregates spread from diseased neurons to
   oligodendroglia or diseased oligodendroglia ectopically express αsyn.
   There is empirical evidence to support both hypotheses [[47]48], but
   the details remain unclear.

   Research has focused on characterizing NIs in PD/DLB brains, but there
   is less understanding of the GCIs in the MSA brain, partly due to the
   lower incidence of MSA (1.6 cases per 100,000 individuals over the age
   of 40) than PD/DLB [[48]4, [49]44, [50]55, [51]62]. Recently, we
   applied an in situ proximity labeling technique called biotinylation by
   antibody recognition (BAR) to measure the αsyn interactome in the
   rodent brain and in the human PD/DLB brain and reported that vesicles,
   neurons, and neuronal synapses are strongly associated with αsyn and
   αsyn aggregates [[52]27], which is consistent with the neuronal origin
   of PD/DLB. BAR has not been applied to other synucleinopathies; in this
   study, we utilized BAR to compare the αsyn interactomes of both
   diseases to define better core molecular features, distinguishing MSA
   and PD/DLB.

Materials and methods

Tissue preparation

   Human brain tissues from individuals with a primary clinical diagnosis
   of MSA, PD, DLB, or Parkinson’s disease dementia (PDD) were obtained
   from the Rush Movement Disorders Brain Bank (see Table [53]1 for
   detailed case information). All brain samples were prepared following a
   previously described method [[54]27]. Briefly, 2-cm coronal slabs were
   fixed by immersion in a 4% paraformaldehyde solution in 0.1 M
   phosphate-buffered saline (PBS) at pH 7.4 for seven days at 4 °C. The
   fixed slabs subsequently underwent gradual equilibration in a
   cryoprotectant mixture composed of PBS, 2% dimethyl sulfoxide, and 20%
   glycerol. The brain regions of interest were cut into 40 μm coronal
   sections via a freezing stage sliding knife microtome (American
   Optical). The brain sections were stored in a cryoprotectant solution
   at − 20 °C until further processing.

Table 1.

   Characteristics of MSA and PD/DLB patients
   Sample # Sex Age at death (years) Method of fixation Hoehn and Yahr
   Scale *Disease Duration (years) Clinical diagnosis Pathological
   diagnosis
   1 M 64 Immersion fixation 7–10 days NA NA MSA MSA-C
   2 M 55 Immersion fixation 7–10 days NA NA MSA MSA-P
   3 F 75 Immersion fixation 7–10 days 4 4 MSA-P MSA-P
   4 F 65 Immersion fixation 7–10 days 5 10 MSA-P NA
   5 M 60 Immersion fixation 7–10 days 3 2 MSA-P NA
   6 M 74 Immersion fixation 7–10 days NA 9 PD Corticobasal degeneration
   with parkinsonism. Alzheimer’s
   7 F 87 Immersion fixation 7–10 days 3 2 PD LBD. Primary age related
   tauopathy
   8 M 81 Immersion fixation 7–10 days 4 3 PD PD-brainstem prominent
   9 M 84 Immersion fixation 7–10 days 4 4 PD NA
   10 M 80 Immersion fixation 7–10 days 4 6 DLB DLB diffuse subtype
   11 M 79 Immersion fixation 7–10 days 4 8 PD NA
   12 F 89 Immersion fixation 7–10 days 2 4 PD NA
   13 M 80 Immersion fixation 7–10 days 4 8 PD NA
   14 M 73 Immersion fixation 7–10 days 2 11 LBD NA
   15 M 70 Immersion fixation 7–10 days 4 5 PD/PSP NA
   [55]Open in a new tab

   ^*The duration of the disease is defined as the period from the onset
   of symptoms to death, as documented in the clinical records of the Rush
   Movement Disorder program. “NA” denotes information not available

Immunohistochemistry (IHC)

   Free-floating brain sections were washed in dilution media (“DM,” 5 mM
   Tris–HCl pH 7.6, 150 mM NaCl, 0.05% Triton X100), followed by
   heat-induced antigen retrieval (HIAR) using sodium citrate buffer
   (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at 80 °C for 30 min. The
   sections were subsequently incubated in peroxidase quenching solution
   (0.3% hydrogen peroxide, 0.1% sodium azide) containing blocking buffer
   (3% goat serum, 2% bovine serum albumin (BSA), 0.4% Triton X-100 in DM)
   for 1 h at room temperature. The tissues were then incubated overnight
   with PSER129 antibody (Abcam, "EP1536Y", ab51253, RRID: AB_869973) or
   MJFR1 antibody (Abcam, ab138501, RRID: AB_2537217) diluted 1:50,000 or
   1:20,000 in blocking buffer, respectively. The following day, the
   sections were washed in DM and incubated with a biotinylated
   anti-rabbit antibody (Vector Laboratories, BA-1000, RRID: AB_2313606)
   at a 1:200 dilution in blocking buffer for 1 h at room temperature,
   followed by rinsing in DM. The tissues were incubated with an elite
   avidin–biotin complex (ABC) reagent (Vector Laboratories, PK6100, RRID:
   AB_2336819) for 75 min at room temperature. The tissues were
   subsequently washed in DM and sodium acetate buffer (0.2 M imidazole,
   1.0 M sodium acetate buffer, pH 7.2). Sections were developed via a
   standard nickel-enhanced 3,3'-diaminobenzidine (DAB)-imidazole
   protocol, rinsed again with sodium acetate buffer and PBS (50 mM
   Tris–HCl, pH 7.2, 158 mM NaCl), and then mounted on gelatin-coated
   glass slides. Counterstaining of the tissues was performed using methyl
   green. The sections were dehydrated, cleared with xylenes, and
   coverslipped with Cytoseal 60 (Fisher Scientific). The detailed IHC
   protocol can be found at protocols.io
   ([56]https://doi.org/10.17504/protocols.io.8epv5x3mdg1b/v1).

Microscopy and imaging

   Prepared slides were imaged via an upright microscope (Olympus, BX53)
   to obtain images using a 4X objective. For 20X objective whole-section
   scans, either a Nikon A1 inverted microscope or Odyssey M imager
   (LI-COR) was utilized. The images underwent downsizing, cutting,
   autocolor balancing, and autobrightness adjustments via Adobe Photoshop
   (RRID: SCR_014199) to improve the figure and data presentation clarity.
   The edited images were imported into Adobe
   Illustrator (RRID: SCR_010279) for arrangement and figure construction.

Biotinylation by antibody recognition (BAR)

   For each case, PSER129 (BAR-PSER129), total αsyn (BAR-MJFR1), and a
   primary antibody omission negative control (BAR-NEG) were performed
   essentially as previously described [[57]27]. A notable exception was
   that the tyramide reaction was conducted in 100 mL of borate buffer
   (0.05 M sodium borate, pH 8.5) containing 14.7 μL of stock biotinylated
   tyramide (Sigma, 12.5 mg/mL dissolved in DMSO) and 10 μL of hydrogen
   peroxide (30% H[2]O[2], Sigma‒Aldrich) for 30 min at room temperature.
   After BAR labeling, the sections were washed with PBS and placed in
   crosslink reversal buffer consisting of 5% SDS, 500 mM Tris–HCl (pH
   8.0), 2 mM EDTA, 1 mM PMSF, and 200 mM NaCl. The tissues were heated to
   95 °C for 30 min and incubated at 65 °C overnight. The samples were
   centrifuged to remove insoluble debris, and the supernatant was diluted
   in TBST (150 mM NaCl, 1% Triton X-100, and 50 mM Tris–HCl, pH 7.6).
   Biotinylated proteins were captured using 40 μL of streptavidin-coated
   magnetic beads (Thermo Fisher, Catalog No. PI88817) at room temperature
   for two hours. The beads were isolated using a magnetic stand
   (Millipore), washed with excess TBST three times for 30 min each, and
   incubated overnight for 16 h at 4 °C. Following the pulldown, the
   lysates were retained for later characterization via western blotting.
   The capture proteins were eluted by heating the beads to 98 °C in 50 µL
   of sample buffer containing 1X LDS sample buffer (Invitrogen, Cat.
   B0008) and 1X sample reducing agent (Invitrogen, Cat. B0009) for
   10 min. A total of 40 µL of each sample was run on a 4–12% Bis–Tris Gel
   (Fisher Scientific, Cat. NW04127BOX) for ~ 7 min at 200 V (run until
   the sample entered the gel). The gel was incubated overnight in
   fixation buffer (50% ethanol and 10% acetic acid). The next day, the
   gel was rehydrated with ultrapure water and stained with Coomassie blue
   (Invitrogen, LC6060). The entire lane containing the captured protein
   was excised for Liquid chromatography-tandem mass spectrometry
   (LC–MS/MS) analysis.

Liquid chromatography-tandem mass spectrometry (LC–MS/MS)

   The samples were prepared and analyzed via LC‒MS/MS via optimized
   protocols as previously described [[58]28]. The gel pieces containing
   the embedded proteins were digested with trypsin according to optimized
   protocols. The resulting tryptic peptide mixture was then subjected to
   mass spectrometry.

Determination of BAR-enriched proteins

   Combining multiple search engines and quantitative software can improve
   the analysis of MS-based proteomics data sets [[59]56]. Therefore, we
   combined two common approaches, namely, Mascot/Scaffold (i.e., total
   normalized spectra, “TNS”) and Andromeda/Maxquant (LFQ). Raw files were
   independently processed and analyzed using each approach (see
   Additional files [60]12 and [61]13 for parameter details). Differential
   abundance (DA) analysis was performed using the R package DEP designed
   for proteomics analysis [[62]71]. Proteins were kept if they were
   identified in all replicates of at least one of the experimental group,
   the data were normalized via variance stabilizing transformation, and
   the QRILC method imputed missing values. Significant proteins were
   identified using protein-wise linear models and empirical Bayes
   statistics using Limma. Proteins from each analysis were combined for
   downstream pathway analysis. False discovery rate (FDR) p values were
   adjusted to account for multiple tests, and proteins with q values less
   than 0.05 were deemed significant. These values were estimated via
   Fdrtool. Proteins significantly enriched for each capture (i.e.,
   significantly enriched over BAR-NEG) were exported and analyzed by
   multiple list comparator (Molbiotools.com) to determine overlapping and
   unique proteins identified for each condition. Overlapping proteins
   were determined for all conditions (i.e., BAR capture and disease
   state, Fig. [63]3C), and individual comparisons were made within each
   capture condition (Figs. [64]4 and [65]5). Direct comparisons were also
   made between PD/DLB and MSA for each BAR capture, with any known
   background proteins (i.e., significantly enriched in BAR-NEG) deemed
   insignificant.

Fig. 3.

   [66]Fig. 3
   [67]Open in a new tab

   PSER129 and αsyn proximal proteins in the synucleinopathy brain.
   BAR-labeled proteins were identified by LC–MS/MS and quantified using
   two approaches Maxquant/andromeda label free quantification (LFQ) and
   Scaffold/mascot total normalized spectra values (TNS). Volcano plots
   comparing protein abundance (i.e., LFQ value) between capture
   (BAR-MJFR1 and BAR-PSER129) and assay background (BAR-NEG) for A MSA
   and B PD/DLB. αsyn (i.e., BAR target protein) is denoted as a star. All
   significant differentially abundant proteins appear red. LFQ results
   shown, TNS results can be found in Additional file. Names of select
   high abundance proteins are annotated. C BAR-PSER129 differentially
   abundant proteins between MSA and PD/DLB brain. Proteins previously
   found to be significantly enriched in background A, B were excluded for
   MSA with PD/DLB comparison. Both LFQ and TNS results are shown. D Venn
   diagram showing BAR enriched proteins (i.e., significant over BAR-NEG)
   for each BAR condition and disease state. Proteins from both analysis
   (LFQ and TNS) were included. E Principal component analysis (PCA) plot
   of the top 100 variable proteins (Additional file [68]1: See Figure S9
   for TNS PCA plot). F Heatmap with non-biased hierarchical clustering of
   protein abundance across all samples. LFQ shown, for TNS see Additional
   file [69]1: Figure S7 G Correlation heatmap comparing protein
   abundances between all samples. LFQ shown, for TNS see Additional file
   [70]1: Figure S8. PD/DLB, n = 10. MSA, n = 5. MJFR1 from BAR-NEG in
   both MSA and PD/DLB brains, with close grouping observed within each
   disease state (Fig. 3E). Similar to BAR-NEG, BAR-PSER129 in MSA brain
   was more related to PC2 than PC1

Fig. 4.

   [71]Fig. 4
   [72]Open in a new tab

   Pathway enrichment map for BAR-identified proteins. BAR-identified
   proteins from PD/DLB and MSA brains were analyzed by gProfiler, and
   significant (q > 0.05) GO Driver Terms were mapped using Enrichmentmap
   and annotated with Autoannotate. GO Driver terms were used to reduce
   redundancy and simplify enrichment maps. Nodes are color-coded
   according to BAR capture condition, BAR-PSER129 (Red) or BAR-MJFR1
   (Blue). Node size is proportional to the number of protein set size and
   edges denote pathway overlap size. The top-panel is MSA enrichment map,
   and bottom-panel is PD/DLB enrichment map

Fig. 5.

   [73]Fig. 5
   [74]Open in a new tab

   Protein interaction networks identified in PD and MSA brain. BAR
   identified proteins were analyzed with STRING to plot known functional
   interactions for each proximal proteome. Proteins were grouped
   according to whether they were unique to PD/DLB, MSA, or shared between
   synucleinopathies. STRING networks for A BAR-PSER129 and B BAR-MJFR1
   are shown. CentiScaPe 2.2 was used to determine the central “driver”
   node for each STRING network (i.e., highest “betweenness” score). The
   driver node was enlarged, and the first neighbors in network
   highlighted. For, BAR-PSER129 C and BAR-MJFR1 D MCL clustering was used
   to group nodes. Clusters with 6 nodes or more are depicted. Top
   enrichment (GOCC, GOMF, GOBP) are annotated onto each cluster. E
   Physical interaction network of proteins specific to MSA for all
   differential expression analysis. BAR target SNCA was included in the
   network. Nodes annotated with top enriched pathway and disconnected
   nodes not shown

Enrichment mapping

   Protein lists were input into the gProfiler web user interface.
   Enrichment analysis was conducted without ranks or weighting because we
   cannot assume that measured protein abundance is a function of distance
   to BAR target or relevance. The Gene Ontology (GO) and Reactome
   databases were queried without electronic annotations. To reduce
   redundancy and minimize the size of the enrichment map, gProfiler
   Driver terms were exclusively used to construct the final enrichment
   map. The enrichment results were exported to Cytoscape and plotted via
   Enrichmentmap V 3.4.0 with Autoannotate.

STRING

   Protein lists were imported into the STRING version 12.0 web user
   interface. The search was conducted with H. sapiens as the reference
   organism. Mapped proteins were then used to generate STRING functional
   or physical protein–protein interaction networks. The minimum required
   interaction score was set to 0.5 unless otherwise noted. STRING maps
   were then exported to Cytoscape version 3.10.2 for MCL clustering,
   pathway enrichment, network analysis (CentiScaPe 2.2), and network
   layout configuration.

Spot blotting

   Before conducting LC–MS/MS analysis, 1 µl of streptavidin bead eluent
   was applied to a methanol-activated polyvinylidene difluoride (PVDF)
   membrane and fully dried. The dried membrane was then reactivated with
   methanol, followed by washing with ultrapure water and post-fixation in
   4% paraformaldehyde for 30 min. The blots were subsequently rinsed with
   TBST (20 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) and immersed
   in blocking buffer (TBST supplemented with 5% BSA or 5% dry milk) for
   1 h at room temperature. To detect biotinylated proteins, the blots
   were incubated with prepared ABC reagent (Vector Laboratories) in
   blocking buffer for 30 min at room temperature. For αsyn detection, the
   blots were incubated overnight with the anti-αsyn antibody SYN1 (BD
   Lifesciences, #610,787, RRID: AB_398108) diluted 1:2000 in blocking
   buffer. After incubation, the blots were washed and incubated with
   anti-mouse HRP conjugate (Cell Signaling Technology, CST7076S, RRID:
   AB_10956588) diluted 1:6000 in blocking buffer. Following another round
   of washing with TBST, all membranes were imaged using enhanced
   chemiluminescence (ECL) substrates (Bio-Rad, product # 170–5060) and a
   ChemiDoc imager (Bio-Rad).

Western blotting

   10 µg of protein from the bead eluent was loaded on 4 to 12% gradient
   Bis‒Tris gels (Thermo Fisher) and run until the loading dye reached the
   bottom of the gel. Resolved proteins were blotted onto the PVDF
   membrane using wet transfer at 100 V for 1 h. Following transfer, the
   membranes were rinsed with ultrapure water (18Ω) and then fixed with 4%
   paraformaldehyde for 30 min at room temperature. Membranes were dried
   completely and reactivated with methanol. The gels were then stained
   with Revert total protein stain (LI-COR, product # 926–11,016)
   according to the manufacturer's protocol and imaged using an Odyssey M
   imager (LI-COR). Membranes were then rinsed in TBST and blocked in TBST
   containing 5% BSA or dry milk for 1 h at room temperature. Primary
   antibody incubation was performed with SYN1 (BD Lifesciences, dil.
   1:2000) or PSER129 (Abcam, dil. 1:50,000) diluted in blocking buffer
   overnight at 4 °C. The next day, the membranes were washed in TBST and
   incubated for 1 h with either anti-rabbit (dil. 1:20,000) or anti-mouse
   (dil. 1:6,000) HRP-conjugated secondary antibodies (Cell Signaling
   Technology) diluted in blocking buffer. The membranes were washed and
   developed using a chemiluminescence (ECL) substrate (Bio-Rad) with a
   ChemiDoc imager (Bio-Rad). For quantitative blots, chemiluminescent
   blots were developed via the “optimal autoexposure” method of Bio-Rad,
   which involves generating a quantitative image.

Quantitative analysis of the blots

   The mean abundance of PSER129 or αsyn for western and spot blots was
   quantified via ImageJ (version 1.54 h,
   [75]https://imagej.net/ij/download.html, RRID:SCR_003070). The data
   were then normalized to the total protein amount, or the fold
   enrichment was calculated based on negative controls. Statistical
   analysis and graphing were performed using GraphPad Prism (version
   10.2.0, [76]https://www.graphpad.com/, RRID: SCR_002798). One-way ANOVA
   with Tukey's post hoc test unless otherwise noted or two-tailed
   unpaired t-test was used to compare experimental groups.

Quantification of IHC

   Brightfield images were captured using an inverted confocal microscope
   equipped with a 20X objective (Nikon A1R). Annotation of each tissue
   section was performed within a bounding box of 2000 × 2000 pixels. All
   images underwent auto-exposure and auto-white balance adjustments using
   NIS-Elements software. RGB-based color thresholding was initially
   fine-tuned to quantify dark black pixels (i.e., PSER129 signal). This
   adjusted threshold was recorded using the Macro function in
   NIS-Elements and then uniformly to all images, irrespective of the
   disease (MSA or PD/DLB) or the region of the brain (WM or GM in the
   midbrain or forebrain) (Additional file [77]1: Figure S1 for the raw
   images and thresholding examples). The percentage area of the
   thresholded signals was subsequently exported and graphed using
   GraphPad Prism. The statistical analysis was performed using either
   One-Way or Two-way ANOVA with Tukey's multiple comparisons test unless
   otherwise noted. Matching between conditions was used for multiple
   measurements within the same case.

Results

   To perform all studies, we used 5 clinically diagnosed MSA and 10
   clinically diagnosed PD/DLB cases (See Table [78]1 for case details).
   Brain hemisections of the nigrostriatal pathway were used for all
   studies (Fig. [79]1A) because both brain regions are involved with the
   core clinical motor features of both MSA and PD/DLB, and the basal
   ganglia bears αsyn aggregates for these synucleinopathies. Tissue
   sections were positionally matched for all cases and consisted of
   forebrain regions (e.g., putamen, caudate, adjacent cortical regions)
   and the midbrain (e.g., substantia nigra).To compare MSA with PD/DLB,
   we performed in situ proximity labeling techniques BAR-MJFR1 and
   BAR-PSER129 [[80]27], which capture total αsyn and aggregated αsyn
   spatially (~ 50–100 nm radius [[81]52])-associated proteins,
   respectively (Fig. [82]1B). BAR-identified proteins were used to
   describe interactomes and compare the molecular characteristics of
   these two distinct synucleinopathies.

Fig. 1.

   [83]Fig. 1
   [84]Open in a new tab

   Summary of approach. A Depiction of specimen sampling for studies.
   Longitudinal image of the right hemisphere of the human brain with the
   approximate location of sampling sites shown (black lines). For BAR, a
   single coronal section through the Caudate Nucleus and Putamen
   (Forebrain) and a single transverse section through the midbrain were
   pooled and used. B Depiction of studies. The distribution of αsyn
   aggregates is distinct between the synucleinopathies PD/DLB and MSA.
   For PD/DLB, αsyn aggregates are observed prominently in neuronal cell
   bodies and projections, termed neuronal inclusions (NI). In contrast,
   in the MSA brain, αsyn aggregates occur prominently in glia, termed
   glial inclusions (GI). Biotinylation by antibody recognition (BAR) was
   used to identify and compare interactomes of total αsyn (BAR-MJFR1) and
   aggregated αsyn (BAR-PSER129) directly in the PD/DLB and MSA brain.
   MJFR1 antibody maps to an epitope of a.a. 118–123 of αsyn’s c-terminus
   and captures physiological monomeric forms and aggregates. PSER129
   preferentially labels αsyn aggregates, especially in the postmortem
   brain where physiological PSER129 is scarce [[85]8], and thus,
   BAR-PSER129 will capture aggregate interactions. LC–MS/MS identified
   BAR-labeled proteins and differential abundance analysis was used to
   determine interactors (i.e., proteins enriched over BAR-NEG) and
   disease-enriched proteins (PD/DLB vs. MSA). The resulting proteins were
   analyzed using a combination of approaches: overlap analysis, pathway
   enrichment mapping, and protein interaction network mapping. IHC
   staining for C PSER129 and D αsyn (i.e., MJFR1) in forebrain and
   midbrain sections. Sections were stained using nickel-DAB chromogen
   (black) and counterstained with methyl green (green). Whole-section
   scans and high-magnification images of select pathology-bearing regions
   are shown with red and blue boxes denoting the approximate area of the
   high-magnification image. Signal thresholding was applied to 20X images
   of PSER129-stained tissues, specifically in gray matter (GM, red box)
   and white matter (WM, blue box) in the forebrain and midbrain for
   quantification. Enlarged 20X images showing the application of
   thresholding E and the subsequent quantification of PSER129
   immunoreactivity across all cases F. Scale bars: C, D = 2 mm and 25 µm;
   E = 2 mm and 20 µm. MSA, n = 5; PD/DLB, n = 10.(mean ±SEM  *Tukey’s
   multiple comparisons test p-adj. < 0.05)

αSyn in the MSA and PD/DLB forebrain and midbrain

   Tissues were first characterized by IHC. Striatal containing forebrain
   and midbrain hemisections were stained for PSER129 and total αsyn
   (MJFR1). Results show widespread PSER129 pathology throughout the
   forebrain and midbrain sections (Fig. [86]1C). For PD/DLB cases,
   PSER129 pathology was dense in gray matter with substantia nigra,
   caudate, putamen, and the insular lobe was often also severely
   affected. In contrast, MSA cases had widespread PSER129 pathology in
   the white matter tracts of the midbrain and forebrain, consistent with
   known white matter involvement for MSA [[87]12, [88]64] (Fig. [89]1C).
   The morphology of PSER129-positive structures in the PD/DLB and MSA
   brains was consistent with prominent NIs or GCIs [[90]18, [91]42],
   respectively. MSA and PD/DLB exhibited αsyn (MJFR1) reactivity
   throughout the forebrain and midbrain sections, predominantly in the
   gray matter (Fig. [92]1D). GCIs were evident in the white matter
   because αsyn is of low abundance in white matter tracks of the healthy
   mammalian brain [[93]17], making αsyn bearing oligodendroglia visible,
   even at low magnification and with total αsyn staining. In contrast,
   the NIs of PD/DLB observed with PSER129 staining were obscured by a
   strong ubiquitous total αsyn signal throughout the gray matter
   (Fig. [94]1D). Despite the apparent regional specificity of PSER129
   staining for MSA and PD/DLB within some cases, a quantitative
   comparison of PSER129 between white and gray matter revealed high
   heterogeneity between cases and few overall significant differences in
   PSER129 abundance between MSA and PD/DLB (Fig. [95]1E, [96]F). For
   PSER129 abundance in PD/DLB midbrain, significant differences (Tukey’s
   post-hoc test P < 0.05) were observed between gray and white matter
   (Fig. [97]1F). Collectively, these results highlight the presentation
   of αsyn pathology and distribution of BAR targets for the cases used
   for these studies.

BAR capture in MSA and PD/DLB forebrain and midbrain tissues

   After verifying the distribution and morphology of the αsyn pathology
   consistent with PD/DLB and MSA, BAR was performed as previously
   described [[98]27] to capture proteins within proximity (predominantly
   within ~ 50–100 nm radius [[99]53]) to PSER129 (i.e., αsyn aggregates)
   and αsyn. For each case, a positionally matched forebrain and midbrain
   section were pooled (Additional file [100]1: Figure S2 average of
   81.7 ± 0.021 mg combined wet mass). Western blot (WB) analysis of
   proteins extracted from pooled forebrain and midbrain hemisections
   revealed that PSER129 and αsyn abundance was variable between cases
   (Fig. [101]2A, [102]B), which is in agreement with the results of IHC
   (Fig. [103]1F); however, PSER129 or αsyn abundance was not
   significantly different between PD/DLB and MSA (Fig. [104]2B, PSER129;
   two-tailed unpaired t test, t(13) = 1.656, p = 0.1215; αsyn two-tailed
   unpaired t test, t(13) = 0.2505, p = 0.8061). High-molecular-weight
   species (> 17 kDa) were observed on WB for PSER129 and αsyn, which may
   be SDS-resistant aggregates [[105]6] or residual formalin crosslinks
   [[106]25]. αSyn truncation/cleavage products were also observed for
   most samples, which have been widely reported in healthy and diseased
   tissues [[107]6, [108]26].

Fig. 2.

   [109]Fig. 2
   [110]Open in a new tab

   BAR capture in the PD/DLB and MSA brain. A 10 μg tissue lysate protein
   for each case were separated on 4–12% Bis–Tris gel transferred onto
   PVDF and stained for total protein (Revert Total Protein Stain,
   LI-Cor), PSER129, or αsyn. Chemiluminescence was used to detect PSER129
   and αsyn. B PSER129 and αsyn relative density values were first
   normalized to total protein (i.e., loading control) and then normalized
   to the mean intensity for each group. C Spot blots of 1 µl eluent from
   BAR captures, including BAR-NEG (Primary antibody-omission control,
   “-”), BAR-PSER129 (aggregates “P”), and BAR-MJFR1 (total αsyn “M”) for
   each synucleinopathy case. Blots were probed for either biotin (ABC
   reagent) or αsyn (BDSYN1). D BAR enrichment was calculated by dividing
   for “P” and “M” by the relative density value for “-” (i.e.,
   fold-enrichment over background) (mean ± SEM, Sidak's multiple
   comparison,**p-adj. < 0.005, n = 5–10)

   Following BAR, dot blots of BAR-captured proteins (i.e., bead eluent)
   revealed biotin and αsyn enrichment were similar between BAR captures
   for MSA and PD/DLB brains (Fig. [111]2C, [112]D). Biotin enrichment was
   significantly greater for BAR-MJFR1 than for BAR-PSER129, regardless of
   disease state (Fig. [113]2D, two-way ANOVA, F (1, 13) = 47.68,
   P < 0.0001). Biotin content was greater for BAR-MJFR1 capture in the
   MSA brain than in the PD/DLB brain (Fig. [114]2D, Sidak’s multiple
   comparison, p-adj.  = 0.0013). For PD/DLB, αsyn enrichment was
   significantly more in BAR-MJFR1 than BAR-PSER129 (Fig. [115]2D, two-way
   ANOVA F(1,13) = 13.64, P = 0.0027, Sidak’s multiple comparison, p-adj.
    = 0.0092). Together, these results demonstrate successful BAR-PSER129
   and BAR-MJFR1 enrichment for all cases in this cohort.

BAR-identified αsyn and PSER129 interactomes in the MSA and PD/DLB brain

   After filtering missing proteins, 440 (TNS method) and 209 (LFQ method)
   proteins were quantified. In the MSA brain, 38 BAR-PSER129-enriched
   proteins and 175 BAR-MJFR1-enriched proteins were significant over
   background (i.e., BAR-NEG). In the PD/DLB brain, we identified 194
   BAR-PSER129-enriched proteins and 245 BAR-MJFR1-enriched proteins. The
   BAR target αsyn (SNCA) was the most enriched protein for all captures
   when quantified by the LFQ method (Fig. [116]3A and B). For the TNS
   method, SNCA was enriched over the background but was not the most
   abundantly enriched capture protein (Additional file [117]1: Figure
   S3). BAR-NEG enrichment was observed for all the contrasts, and the
   known background biotin-binding protein propionyl-CoA carboxylase
   subunit alpha (PCCA) [[118]1] was often most abundant in BAR-NEG
   (Fig. [119]3A, [120]B). For BAR-MJRF1, a direct comparison between MSA
   and PD/DLB did not reveal any differentially abundant proteins. In
   contrast, for BAR-PSER129, direct comparisons revealed numerous
   proteins more abundant in PD/DLB, including ATP6V1B2, STX1B, and MARCKS
   (Fig. [121]3C). Three proteins (CBR1, GFAP, CRYAB) were differentially
   abundant in MSA (Fig. [122]3C). Overlap of identified proteins was
   greatest (105 proteins, 39.6% of all proteins) between groups excluding
   BAR-PSER129 in the MSA brain (Fig. [123]3D). Remarkably, despite the
   prominent GCI abundance observed in our MSA samples, not a single
   unique protein was identified for BAR-PSER129 in the MSA brain
   (Fig. [124]3D). However, BAR-MJFR1 and BAR-PSER129 identified proteins
   unique to MSA, including HSPA1B, PRDX1, SERTIN8, GSTP1, and PRDX2.
   Unique proteins were also identified for BAR-MJFR1 MSA, including
   PHGDH, TPM3, PFN1, HSPA5, ACOT7, CD47, IGHG3, UBE2N, GMFB, AQP1,
   NDUFA12, and EEF1A1. Identifying unique proteins was minimal in the MSA
   brain, and most proteins overlapped with PD/DLB (BAR-MJFR1 and
   BAR-PSER129). The principal component analysis (PCA) plot of the top
   100 BAR-captured proteins showed the separation of BAR-MJFR1 from
   BAR-NEG in both MSA and PD/DLB brains, with close grouping observed
   within each disease state (Fig. [125]3E). Like BAR-NEG, BAR-PSER129 in
   MSA brain was more related to PC2 than PC1.

   Figure [126]3F and G heatmaps show the hierarchical clustering of
   protein abundance (LFQ) and hierarchical clustering of Pearson
   correlations between all tested samples, respectively. Cases formed two
   main clusters (Fig. [127]3F), with one cluster having a strong
   “on-target” signal, containing the majority of BAR-MJFR1 captures and
   BAR-PSER129 captures in the PD/DLB brain. The second main cluster
   comprised background samples (BAR-NEG for MSA and PD/DLB) and
   BAR-PSER129 captures in MSA and PD/DLB brains showing weaker
   “on-target” signals. Regarding correlations, BAR-MJFR1 and BAR-NEG were
   closely correlated between MSA and PD/DLB, whereas BAR-PSER129 was less
   correlated between MSA and PD/DLB. (See Additional files [128]2-[129]4
   for results and 9,10 for input files).

Pathway enrichment maps for MSA and PD/DLB

   Next, we performed pathway enrichment analysis and identified hundreds
   of significantly enriched pathways (p-adj. < 0.05) for each
   experimental group (see Additional files [130]5-[131]8 for enrichment
   files). To reduce the redundancy of identified pathways, we used the
   gProfiler “go driver term” function, which first groups significant
   terms by GO relation and then uses a simple greedy search strategy to
   identify leading gene/protein sets [[132]30, [133]47]. The resulting
   top 10 driver terms are shown in Additional file [134]1 Figure S4, and
   all driver terms were used to construct the enrichment map in
   Fig. [135]4. For both MSA and PD/DLB, many clusters contained pathways
   enriched for both BAR captures, however unique pathway enrichment was
   observed for BAR-PSER129 and BAR-MJFR1. For MSA, BAR-PSER129's unique
   major clusters included “catabolic/metabolic processes,” “unfolded
   protein refolding,” “cellular oxidant detoxification,” “cell junction
   focal,” and “shock protein chaperone” clusters. For PD/DLB, all major
   clusters identified contained BAR-MJFR1-enriched pathways; therefore,
   no BAR-PSER129-specific clusters were detected. “Receptor/SNARE
   binding” was the major cluster for PD/DLB.

Protein interaction networks identified in the synucleinopathy brain

   To better understand proteins and pathways that distinguish MSA from
   PD/DLB, STRING [[136]59, [137]60] was used to plot functional and
   physical interactions between BAR-identified proteins. Each STRING
   networks' central node (i.e., driver protein) was determined using
   CentiScaPe 2.2 and highlighted in Fig. [138]5A, [139]B. Driver proteins
   (i.e., proteins with the highest “betweenness” score, a node centrality
   index) for BAR-PSER129 included MAPT (i.e., tau), SNCA, and PRDX1 for
   PD/DLB, shared, and MSA, respectively. Driver proteins for BAR-MJFR1
   included HSP90AB1, GAPDH, and PFN1 for PD/DLB, shared, and MSA,
   respectively. MCL clustering was conducted to isolate functional
   networks for each experimental group. Results showed that for PD/DLB
   BAR-PSER129 identified mostly neuron-specific networks, including
   SNARE, presynapse, and axon (Fig. [140]5C), in agreement with our
   previous observation (Fig. [141]4). Shared networks for BAR-PSER129,
   which included SNCA, involved ubiquitin ligase binding, localization,
   and follin-1-rich granule lumen. A single network of 7 interconnected
   nodes enriched for peroxidase activity, extracellular exosome, and
   response to oxidative stress was unique to BAR-PSER129-MSA. For
   BAR-MJFR1 (Fig. [142]5D), MSA specific network was also associated with
   extracellular exosome and peroxidase activity. Physical protein
   interactions for MSA-proteins were plotted in relation to BAR-target
   protein (i.e., SNCA). An MSA specific physical subnetwork emerged with
   peroxiredoxin’s (PRDX1, PRDX2, and PRDX6) as major components. SNCA was
   segregated from the MSA-specific peroxiredoxin network, with CRYAB,
   HSPA1B, and HSPA5 connected to SNCA. This BAR-identified subnetwork of
   MSA was cytosolic (e.g., GAPDH and GFAP), with the regulation of
   protein stability and response to oxidative stress being the major
   functional themes.

Discussion

   Here, we interrogated αsyn interactomes within the human brain of two
   clinically and pathologically distinct primary synucleinopathies.
   Overall, we found that total αsyn interactions (i.e., BAR-MJFR1) were
   similar between MSA and PD/DLB brains, with strong enrichment for
   presynaptic vesicle processes, which is broadly consistent with
   physiological αsyn interactions, αsyn functionality, and previous
   proximity proteomics studies [[143]10, [144]20, [145]27, [146]28,
   [147]41]. In contrast, αsyn aggregate interactions (i.e., BAR-PSER129)
   were markedly different between MSA and PD/DLB, with several
   glial-enhanced proteins (CBR1, CRYAB, and GFAP) (Human Protein Atlas
   proteinatlas.org [[148]58]) [[149]7] being more abundant in MSA αsyn
   aggregates (i.e., PD vs. MSA contrast). In addition, five proteins
   associated with MSA aggregates but also overlapped with the total αsyn
   pool, including HSPA1B, PRDX1, PRDX2, SEPTIN8, and GSTP1. Collectively
   identified MSA proteins are functionally interconnected and are
   involved with anti-oxidative mechanisms and protein stability
   (Fig. [150]5E). Multiple oxidative stress mechanisms have been
   implicated in MSA, including oxidative phosphorylation dysfunction and
   reductions in anti-oxidative machinery [[151]44]. Reduction in
   antioxidant glutathione (GSTP1 identified here) may occur in MSA
   [[152]23, [153]57], but peroxiredoxins or CBR1 (both proteins protect
   from oxidative stress) have not been directly implicated in MSA but
   have been implicated in other neurodegenerative diseases and neurotoxic
   insults [[154]21, [155]31]. Together, our findings implicate unique
   anti-oxidative mechanisms for MSA pathology. We cannot infer precisely
   how these proteins fit the MSA pathological process, whether protective
   or toxic. Interestingly, reductions in antioxidant COQ10 have been
   implicated in MSA pathogenesis [[156]3, [157]40, [158]54], albeit
   inconsistently [[159]49], and clinical efficacy trials for ubiquinol
   are currently underway [[160]38]. Notably, PFN1 was a central protein
   for MSA total synuclein; PFN1 is strongly implicated in several
   neurodegenerative diseases, including amyotrophic lateral sclerosis,
   frontal temporal dementia, and motor neuron disease [[161]51, [162]61]
   [[163]22] [[164]16].

   Our data implicates canonical αsyn pathways (i.e., vesicles/SNARE) for
   PD/DLB and anti-oxidative pathways for MSA but also demonstrates shared
   interactions between the two distinct synucleinopathies. All
   experimental conditions shared 26 proteins involved in cellular
   functions such as intracellular iron sequestering, protein refolding,
   and nucleoside diphosphate phosphorylation, representing the core
   interactome of αsyn for both diseases (Additional file [165]1: Figure
   S10). At the center of this network was HSPA8, a regulator
   chaperonin-mediated autophagy inhibitor of αsyn aggregation, which has
   been implicated in PD, DLB, MSA, and other neurodegenerative diseases
   [[166]35, [167]50]. Therefore, our data suggests a protein-refolding
   response at the center of disparate synucleinopathies. Despite the
   predominating GCIs in MSA, several neuronal vesicle proteins, including
   SYNGR3 and SV2A, were identified in the shared pathway (Additional file
   [168]1: Figure S10), supporting a neuronal origins hypothesis for MSA.
   Both proteins could originate in the few PSER129 reactive neuropil in
   our MSA cases (Fig. [169]1) or in vesicles released from neurons and
   endocytosed by proximal oligodendrocytes [[170]33, [171]39, [172]48,
   [173]70]. Indeed, extracellular exosomes (i.e., L1CAM positive
   [[174]19, [175]68]) were consistently enriched pathways in agreement
   with our previous BAR studies [[176]27], suggesting that αsyn
   aggregates in synucleinopathy brain partly resemble exosomes and
   supporting the hypothesis that αsyn/αsyn aggregates can spread via
   exosomes [[177]11, [178]15, [179]34, [180]63]. It’s unclear whether
   αsyn “exosomes” have biological importance (e.g., αsyn aggregates are
   spread by exosomes), but αsyn-containing exosomes have been isolated
   from peripheral sources [[181]5, [182]14]. Alternatively, coincidental
   exosome enrichment may occur because of αsyn aggregates' close
   association with vesicles/endosomes. Our data suggests one of two
   scenarios: αsyn aggregates spread to oligodendrocytes via presynaptic
   neuronal exosomes or, in addition to αsyn, several neuronal proteins
   are upregulated (SYNGR3 and SV2A) in diseased oligodendrocytes, but
   this is unlikely because differentiating oligodendrocytes do not
   express these genes [[183]7]. Overall, our findings support a
   hypothesis for the potential late-stage contribution of
   oligodendrocytes to the disease process of MSA (i.e., secondary to
   neuronal-derived αsyn pathology) [[184]32, [185]48].

   We identified CRYAB associated with GCI’s. CRYAB is upregulated in the
   MSA brain and directly associated with GCIs [[186]45]. Recent studies
   have identified GCI-like structures in oligodendrocytes of PD patients
   carrying αsyn gene mutations or presenting atypical clinical features
   [[187]69]. CRYAB is encoded by a subpopulation of oligodendrocytes that
   is depleted in sporadic PD [[188]36]. Functionally, CRYAB binds to the
   ends of αsyn fibrils, inhibiting aggregation and potentially playing a
   protective role. The strong association of CRYAB with GCI-related
   diseases, such as MSA, suggests that this “capping” mechanism plays a
   prominent role and may contribute to the particularly aggressive
   clinical progression observed in MSA. Additionally, MSA-derived seeds
   extracted from the human brain exhibit significantly higher
   templated-mediated seeding efficiency, which may explain the pronounced
   CRYAB upregulation and its sequestration to αsyn inclusions. These
   findings support the potential therapeutic utility of small heat shock
   proteins in managing these neurodegenerative conditions.

   The study's limitations include that most of the MSA cases here are
   MSA-Parkinsonian type (MSA-P), which is the dominant subtype of MSA in
   the western hemisphere [[189]65–[190]67], restricting the comparison of
   PD/DLB to only one subtype of MSA. Another limitation is that BAR, as a
   “spatial-omics” [[191]24] technique, has special interpretation
   considerations, including spatial resolution (~ 50–100 nm radius from
   BAR target), lack of information about the precise nature of
   protein–protein interactions (i.e., direct or indirect), and although
   cell type/cell compartment can be inferred from BAR data, it cannot be
   determined. Although not explored here, determining how BAR-identified
   proteins compare to cell-specific (e.g., oligodendrocytes or neuronal
   proteomes) or organelle-specific proteomes might help better understand
   interactions within cellular contexts. Additionally, although our
   analysis focused on MSA, the networks/proteins specific to PD/DLB
   identified here are likely valuable for follow-up analyses aimed at
   deciphering PD/DLB mechanisms.

   When interpreting BAR-PSER129 data, an important consideration should
   be made, specifically, the association of PSER129 with αsyn aggregates,
   which is not without caveats. Here, we use PSER129 as an aggregate
   marker, but physiological non-aggregated PSER129 can occur in the
   mammalian brain [[192]28, [193]43, [194]46], and thus, PSER129 is not
   an implicit marker of aggregates. However, we recently discovered that
   physiological non-aggregated PSER129 is rapidly dephosphorylated during
   post-mortem intervals [[195]8]. Correspondingly, post-mortem specimens
   used here likely contain mostly aggregated-PSER129. This conclusion is
   consistent with PSER129 staining here, which lacked distribution and GM
   abundance characteristic of the mammalian brain [[196]28].
   Nevertheless, it remains possible that some BAR-determined interactions
   are from physiological PSER129, and not αsyn aggregates.

   Proximity proteomics are powerful techniques used to study αsyn
   aggregate and non-aggregate interactomes [[197]27], but this is the
   first application of BAR comparing synucleinopathies and there
   currently aren’t standardized data analysis pipeline specifically for
   BAR. As we found before [[198]27], background captures (BAR-NEG) were a
   critical component of the study design that allowed the identification
   of BAR-antibody-specific proteins. Direct comparisons between diseases
   (e.g., MSA vs PD/DLB) could be made for each BAR capture, but
   background proteins must be removed manually, as background (BAR-NEG)
   information is lost in this approach. To increase protein
   identification and robustness of our analysis we conducted two analyses
   based on two quantitative values LFQ scores (Maxquant) and TNS
   (Scaffold). We found a correlation between methods (Additional
   file [199]1: Figure S5, S6), but LFQ was superior in that the target
   protein (αsyn) was consistently the most enriched protein, in contrast
   to TNS, where αsyn was only most abundant in the MSA-PSER129 capture
   (Additional file [200]1: Figure S3). Additional analysis might be
   fruitful in further elucidating the significance of BAR-identified
   proteins, including comparison with complementary datasets and
   incorporating analysis with different experimental assumptions
   (Additional file [201]1: Figure S11-14, Additional file [202]11).

Conclusions

   In conclusion, the results suggest that MSA involves PRDX's
   anti-oxidative processes in glial cells, while presynaptic
   vesicle/SNARE processes dominate PD/DLB. Our findings revealed a
   significant overlap between MSA and PD/DLB, suggesting shared
   pathogenic origins for these synucleinopathies. Intriguingly, we
   identified several neuronal proteins, besides SNCA and strong exosome
   enrichment, associated with GCIs, together supporting the
   neuron-to-glia spread hypothesis of MSA.

Supplementary Information

   [203]Additional file 1.^ (1.8MB, pdf)
   [204]Additional file 2.^ (89.8KB, csv)
   [205]Additional file 3.^ (187.2KB, csv)
   [206]Additional file 4.^ (24.8KB, xlsx)
   [207]Additional file 5.^ (7.3KB, txt)
   [208]Additional file 6.^ (5.4KB, txt)
   [209]Additional file 7.^ (9.8KB, txt)
   [210]Additional file 8.^ (9.8KB, txt)
   [211]Additional file 9.^ (396.2KB, xls)
   [212]Additional file 10.^ (4.4MB, xlsx)
   [213]Additional file 11.^ (12.2KB, xlsx)
   [214]Additional file 12.^ (3.8KB, txt)
   [215]Additional file 13.^ (9KB, xls)

Acknowledgements