MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity

Lewy pathology, consisting of Lewy bodies and Lewy neurites, is the pathological hallmark of synucle-inopathies such as Parkinson’s disease and dementia with Lewy bodies, but it is generally thought to represent late-stage pathological changes. In contrast, α-synuclein oligomers are regarded as early-stage pathology, likely involved in disease progression and cellular toxicity. Oligomers, however, are not de-tected by standard immunohistochemistry but require specific detection techniques such as the proxim-ity ligation assay (PLA). Here, we describe the MJF-14 PLA, a new PLA towards aggregated α-synuclein with unprecedented specificity, attained by the utilization of aggregate conformation-specific α-synu-clein antibody MJFR-14-6-4-2 (hereafter MJF-14). Signal in the assay directly correlates with α-synuclein aggregation in SH-SY5Y cells, as treatment with aggregation inhibitor ASI1D significantly lowers PLA sig-nal. In human cortical neurons, MJF-14 PLA detects pre-formed fibril-induced aggregation, especially prominent when using stealth PFFs invisible to the MJF-14 antibody. Co-labelling of MJF-14 PLA and pS129-α-synuclein immunofluorescence in post-mortem dementia with Lewy bodies cases showed that while the MJF-14 PLA reveals extensive non-inclusion pathology, it is not sensitive towards Lewy bodies. In Parkinson’s disease brain, direct comparison of PLA and IHC with the MJF-14 antibody, combined with machine learning-based quantification, showed striking α-synuclein pathology preceding the formation of conventional Lewy pathology. The majority of the PLA-revealed non-inclusion pathology was found in the neuropil, including some clearly located in the presynaptic terminals. With this work, we introduce an improved α-synuclein aggregate PLA to uncover abundant non-inclusion pathology, which deserves future validation with multiple brain bank resources and in different synucleinopathies.