What if the antibody signal researchers use to track alpha-synuclein pathology is noisy in healthy tissue—and the cleaner method works beautifully everywhere except the human Lewy bodies it was meant to reveal? Synucleinopathies are neurodegenerative disorders characterized by intracellular aggregates called Lewy bodies.

Lewy bodies are primarily composed of alpha-synuclein, and aggregated alpha-synuclein is mostly phosphorylated at serine 129, or pS129, making pS129 a pathology marker. The problem is that commercial antibodies stain aggregates well but cross-react with other proteins in healthy brains, making physiological pS129 alpha-synuclein difficult to detect specifically.

The objective was to develop a staining procedure that detects endogenous, physiologically relevant pS129 alpha-synuclein with high specificity and low background. Because pS129 alpha-synuclein is closely tied to synucleinopathy pathology, tools that specifically detect physiological pS129 alpha-synuclein are critical.

Existing monoclonal antibodies preferentially detect phosphorylated alpha-synuclein and aggregated pS129 alpha-synuclein, but not the soluble form. Those antibodies can cross-react with other proteins carrying similar phosphorylated epitopes, including neurofilament light chain.

That cross-reactivity makes physiological pS129 alpha-synuclein staining difficult to interpret and makes its cellular localization and function difficult to study directly. Proximity ligation assay, or PLA, creates a robust signal when two primary antibodies are within an estimated range of less than forty nanometers.

When the attached oligonucleotides are close together, they can be ligated and amplified to create a fluorescent or DAB signal. For protein modifications, PLA uses one antibody against the protein and another against the modification, an approach that limits cross-reactivity and background.

Here, the system used the pS129 antibody MJF-R13 together with the total-alpha-synuclein antibody syn-1 to detect endogenous and physiological pS129 alpha-synuclein. The study used fluorescent and brightfield in situ proximity ligation assay to specifically detect pS129 alpha-synuclein in cell culture, mouse brain sections, and human brain sections.

The first specificity test used immunocytochemistry on mouse primary cortical neurons to assess whether the pS129 alpha-synuclein antibodies were monospecific. The experiments used both wild-type and alpha-synuclein knockout cultures, enabling direct comparison of antibody staining between the two genotypes in the neuronal samples.

The tested pS129 antibodies were MJF-R13, pSyn number sixty-four, and 81A, while syn-1 served as a total-alpha-synuclein reference. MJF-R13, pSyn number sixty-four, and 81A showed extensive staining in both wild-type and knockout cultures, demonstrating cross-reactivity with other antigens.

By contrast, syn-1 stained the wild-type culture only, illustrating monospecific staining in this application. The study then evaluated MJF-R13, pSyn number sixty-four, 81A, and EP1536Y by immunohistochemistry in alpha-synuclein knockout and wild-type mouse brain sections.

Across the cortex, hippocampus, and substantia nigra, staining patterns did not differ between wild-type and knockout samples. The results included cell-body staining and general background staining, while 81A showed more signal in the cortex and substantia nigra than in other areas.

None of the commercial antibodies tested specifically detected physiological levels of pS129 alpha-synuclein in immunocytochemistry or immunohistochemistry. The next goal was an assay that could detect pS129 alpha-synuclein equally well in rodent and human tissues.

The fluorescent PLA specifically stained pS129 alpha-synuclein in primary neuron and HeLa cultures and detected endogenous, physiological levels of pS129 alpha-synuclein. The brightfield PLA used DAB on fixed brain tissue, with R13 against pS129 alpha-synuclein and syn-1 against total alpha-synuclein.

The assay was compared using both primary antibodies, syn-1 alone, or R13 alone, in wild-type and alpha-synuclein knockout mouse brain sections. With both antibodies, brightfield PLA produced a robust punctate signal in the wild-type neocortex, striatum, hippocampus, and substantia nigra.

The knockout tissue produced dramatically less signal, while syn-1 alone showed very little nonspecific staining and R13 alone produced small numbers of puncta mainly in the hippocampus. Figure three tests brightfield PLA detection of phosphorylated S129 alpha-synuclein across four mouse-brain regions.

In wild-type sections, panels A through D use both R13 and syn-1, while the SNCA knockout sections in E through H and the single-antibody controls in I through P assess specificity. The regional staining patterns, together with the reduced signal in knockout tissue and controls, show why combining the antibodies is useful for detecting physiological pS129 alpha-synuclein in brain sections.

Brightfield PLA was next evaluated across additional major brain areas in the wild-type mouse brain. Punctate staining appeared throughout the stained mouse brain sections, with particularly strong staining in layer four of the prefrontal cortex.

Figure four maps physiological phosphorylated alpha-synuclein, or pS129 alpha-synuclein, across wild-type mouse brain sections using BF-PLA. The punctate staining appears across regions including the prefrontal and neocortex, striatum, hippocampus, thalamus, amygdala, substantia nigra, and cerebellum, with more localized cellular-looking labeling in areas such as cortical layer four, hippocampal CA3, thalamus, amygdala, and the cerebellar granular layer.

This matters because it shows that pS129 alpha-synuclein is detectable under normal conditions, providing a physiological reference when interpreting it as a marker of pathology. The study validated brightfield PLA in animal models because detecting soluble pS129 alpha-synuclein together with accumulated forms can show its localization, distribution, and changes.

In wild-type mice, AAV6 particles expressing green fluorescent protein, mouse alpha-synuclein, or human alpha-synuclein were injected unilaterally into the substantia nigra. Four weeks later, brightfield PLA stained pS129 alpha-synuclein in the substantia nigra and striatum, with uninjected sections used in parallel.

Mouse and human alpha-synuclein overexpression showed increased staining intensity in the substantia nigra compared with uninjected and green-fluorescent-protein control sections. Figure five presents BF-PLA staining for phosphorylated alpha-synuclein across several validation settings.

In mice, the images compare uninjected tissue with control, mouse-alpha-synuclein, and human-alpha-synuclein AAV expression, then show staining after mouse preformed-fibril injection across striatum, neocortex, prefrontal cortex, and amygdala. The lower panels extend the comparison to human frontal cortex from controls and DLB cases, placing PLA staining alongside conventional DAB pS129 staining.

The increase in staining intensity, attributed to increased alpha-synuclein expression, was found in round structures, possibly cell bodies, rather than surrounding neuronal projections or the striatum. After preformed fibrils were injected into the striatum, accumulated pS129 alpha-synuclein was detected in cell bodies in the striatum, neocortex, prefrontal cortex, and amygdala, with particularly high signals in the amygdala.

The same experiment also visualized endogenous, soluble pS129 alpha-synuclein throughout the brain. The study then tested whether brightfield PLA could detect non-aggregated and aggregated pS129 alpha-synuclein in human Lewy bodies.

In both control and dementia-with-Lewy-bodies tissue, brightfield PLA showed extensive pS129 alpha-synuclein staining in frontal cortical areas. Across additional cases and brain areas, staining intensity did not show an apparent difference between dementia-with-Lewy-bodies and control groups, and brightfield PLA did not readily detect Lewy bodies.

The 81A antibody did detect pS129 alpha-synuclein aggregates in dementia-with-Lewy-bodies tissue, while none were detected in healthy control tissue. Overall, the pS129 alpha-synuclein PLA specifically stained physiological and soluble pS129 alpha-synuclein in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal.

However, the technique was not successful in detecting Lewy bodies in human brain tissue. The R thirteen and syn-one PLA detected accumulated p S one two nine alpha-synuclein in the preformed-fibril animal model but not pathologically aggregated p S one two nine alpha-synuclein in human dementia-with-Lewy-bodies tissue.

One possible explanation is that chronically aggregated, dense Lewy bodies have a different structure or different post-translational modifications from short-term aggregates in animal models. The syn-1 antibody binds near the ninetieth amino acid, part of a motif involved in the beta-sheet structure of alpha-synuclein aggregates and including a ubiquitination site at K96.

The study suggests that these modifications may prevent syn-1 from binding aggregated alpha-synuclein in Lewy bodies while still allowing detection of endogenous and soluble human pS129 alpha-synuclein. In mouse brain, the PLA showed pS129 alpha-synuclein throughout most brain regions and denser staining in some, but not all, cell bodies.

Those somal staining patterns did not necessarily overlap with the locations of pathology seen in synucleinopathies. Human frontal cortex showed a different pattern: general staining remained, but pS129 alpha-synuclein was not localized in most cell bodies in the cases examined.

Human brain sections also showed a general staining increase compared with mouse brain sections. The PLA protocol specifically and sensitively detects endogenous, physiologically relevant pS129 alpha-synuclein in cell cultures, mouse brain, and human brain tissue.

It also shows the localization and distribution of non-aggregated pS129 alpha-synuclein in brains without pathological attributes. The method detected increased pS129 alpha-synuclein in cell cultures, alpha-synuclein-overexpressing mouse models, and preformed-fibril-injected mice, and it detected reductions caused by a kinase inhibitor.

The method could be combined with cell-organelle markers, co-expression experiments, or drug screens to investigate where and when alpha-synuclein is phosphorylated. The PLA addresses cross-reactivity by establishing a specific pS129 alpha-synuclein signal with minimal background.

It specifically detects physiological, non-aggregated pS129 alpha-synuclein in cell culture, wild-type mouse brain tissue, and human brain tissue, and it detects increased levels in cell cultures and mouse models. Soluble pS129 alpha-synuclein was readily detected in human brain tissue, but Lewy bodies were not stained in dementia-with-Lewy-bodies tissue.

Even with that limitation, the PLA can be used to investigate pS129 alpha-synuclein biology and its role in synucleinopathy pathology. This PLA method gives a more specific view of physiological and soluble pS129 alpha-synuclein across cells and brain tissue, but its failure to stain human Lewy bodies is an important biological and technical limitation.