In situ proximity labeling identifies Lewy pathology molecular interactions in the human brain

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129–labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129–enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129–identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases characterized by intracellular inclusions containing aggregated alpha-synuclein referred to as Lewy pathology (LP). Although strong evidence suggests alpha-synuclein is involved in the disease process, central questions remain regarding the molecular origins and progression of LP. A diverse range of cellular pathways have been implicated in alpha-synuclein–mediated disease processes including dysfunctional synaptic neurotransmission and vesicular trafficking, mitochondrial dysfunction, oxidative stress, neuroinflammation, calcium signaling, glycolysis, autophagy lysosomal dysfunction, and Mitogen-activated protein kinase (MAPK) signaling pathways (1). However, much of the evidence implicating these cellular processes has been derived from cell and animal models, which may or may not closely recapitulate the human disease processes. Defining the molecular details of disease process is important for progress toward the development of disease-modifying therapies.Alpha-synuclein phosphorylated at serine 129 (PSER129) is a marker of LP. PSER129 is enriched in LP with only trace quantities (<5% of total alpha-synuclein pool) of endogenous alpha-synuclein being phosphorylated (2). Evidence suggests that PSER129 may aggregate more rapidly, and PSER129 may have a role in regulating alpha-synuclein turnover (3). Evidence also suggests that PSER129 accumulates in the brain as PD progresses (4, 5) although it is not clear whether PSER129 occurs prior to, during, or following inclusion formation (6). Several antibodies have been developed to detect PSER129, including the commonly used EP1536Y antibody (Abcam), a rabbit monoclonal antibody against PSER129 that has high specificity for LP (7) and has been used extensively for immunohistochemical (IHC) labeling of LP.Determining the molecular makeup of LP in the human brain is challenging. Difficulties arise principally from the insoluble nature of LP, which precludes common biochemical methods for determining molecular interactions. In past studies, LP from the human brain has been enriched for by biochemical fractionation methods (8) and isolated by microdissection techniques (9). There are important limitations to each approach. Fractionation disturbs cellular structures and, presumably, LP composition. Microdissection exclusively captures Lewy bodies, but not other lesions (neurites, dots, etc.). Microscopy methods provide accurate data characterizing spatial and molecular makeup of LP, but these methods are limited to known molecular targets. Together, current approaches are insufficient to identify molecular details of LP in the human diseased brain.Recently, a technique referred to as biotinylation by antibody recognition (BAR) was successfully used to label insoluble cellular components within proximity to an antigen in fixed primary tissues for enrichment and downstream mass spectrometry analysis (10–13). The BAR technique (also called selective proteomic proximity labeling using tyramide, or enzyme-mediated activation of radical sources) labels a target antigen by peroxidase-catalyzed deposition of biotin moieties onto proximal molecules. BAR is particularly well suited for determining protein interactions and subcellular localization of insoluble cellular components (10). LPs are insoluble cellular inclusions, and therefore the BAR technique holds promise to study these structures. Recent demonstrations of the BAR technique, and its utility for proteomic analysis, gave us the impetus to attempt to purify LP and LP interactors from fixed primary human tissues.Here we applied a modified BAR technique to capture total alpha-synuclein (BAR-SYN1) and LP (BAR-PSER129) interactions in formalin-fixed human brain samples from individuals diagnosed with PD and DLB (SYN), as well as neurologically intact individuals (HC).