Advancement in human neuroimaging based on proximity ligation assay in brain disease

Visualization of cellular connections in the brain was at the heart of the pioneering work performed by Ramon y Cajal. However, the integration of neurophysiology, histochemistry, and biochemistry became imperative for understanding the integrative communication of neurons, particularly emphasizing protein-protein interactions. The proximity ligation assay (PLA) was used to detect protein-protein interactions within nerve cells, e.g., G protein-coupled receptor heterocomplexes in the postmortem human brain [1]. PLA uses paired antibodies labelled with oligonucleotides that undergo amplification when in close proximity to generate a specific signal, detecting protein interactions/aggregates with high sensitivity (Fig. 1). Advancements in PLA methodologies have enabled the observation of A2AR-D2R heterocomplexes in the human striatum [1], contributing to strategic planning for human neuroimaging and understanding neurological and psychiatric diseases.

Fig. 1. Schematic representation of PLA.

First, a pair of well-validated primary antibodies bind to target proteins. Species-specific secondary antibodies conjugated to complementary oligonucleotides then recognize these primary antibodies. If the targets are 40 nm or less apart, the attached PLA probes (oligonucleotides) can be bridged through the hybridization of two additional connector oligonucleotides. Then a circular single-stranded DNA forms, facilitated by ligase, to create a double-stranded template for rolling circle amplification. Polymerase generates an amplified rolling circle product hybridized with labeled probes, such as fluorescents, producing a PLA signal. This method detects early protein interactions with high sensitivity, providing insights into post-translational modifications, such as oligomerization, in post-mortem human brain tissue. However, the assay’s reliability may be affected by variability in antibody quality and potential degradation of post-mortem tissue. Despite these limitations, PLA is crucial for studying protein interactions and aggregation in neuropsychiatric and neurodegenerative disorders. For example, it aids in understanding which receptors and proteins alpha-synuclein (AS) bind to in critical brain regions in PD, potentially identifying vulnerable targets for novel neuroprotective treatments. Additionally, PLA allows for the study of mutated AS and AS aggregates, as well as beta-amyloid oligomerization and Tau interactions in Alzheimer’s disease, e.g., pTau-ubiquitin interaction [2].

Prior to the introduction of PLA, limitations in protein-protein neuroimaging hindered a comprehensive understanding of protein interactions in brain disorders. However, PLA has now enabled the precise visualization and quantification of protein interactions, significantly advancing our understanding of brain pathologies. PLA approaches are increasingly applied to delineate e.g., protein-protein interactions in neuropsychiatric and neurodegenerative disorders, offering insights into disease mechanisms, treatment responses, and imaging phenotypes.

PLA has been used in human brains with Alzheimer’s disease (AD) [2] allowing for the detection, visualization and quantification of the protein complexes [2]. There was a marked alteration in the distribution of ubiquitin in brains with AD. It was associated with the usual pathologies found in tau. It was possible to validate that phosphorylated microtubule-associated protein tau aggregates, were altered by ubiquitin. Furthermore, phospho-tau-ubiquitin complexes were increased in the frontal cortex and hippocampus in AD compared to brains without. It seems possible that characterization of tau-ubiquitin action can improve our insights into AD brain pathology. It should be noticed that optimized steps for photobleaching abolished autofluorescence originating from lipofuscin and other pigments accumulated in the soma [2]. It is of interest that tau-PLA is a method to demonstrate tau multimerization with high specificity. It opens up a way for early discovery of tau pathology [3].

As to Parkinson’s disease (PD), the PLA technique has facilitated the visualization of alpha-synuclein (AS) oligomers, representing a new pathology in PD [4]. Also, based on the PLA assay, dopamine transporter/AS and synapsin-III/AS complex accumulations could be demonstrated in the caudate putamen of PD, likely having a significant role in neurodegeneration [5]. Furthermore, using PLA it was demonstrated that abundant AS oligomers accumulate in Purkinje cells in multiple system atrophy (MSA) brains [6]. AS-PLA also revealed a wide distribution of AS oligomers in MSA brains, indicating a mechanism of neuronal inclusions in MSA. Understanding AS aggregation mechanisms, identifying polymorphs, and recognizing them in previously unrecognized regions is of utmost importance.

The improved molecular imaging from ex-vivo PLA provides crucial insights into protein interactions and post-translational modifications (oligomerization), while maintaining spatial knowledge. PLA will also improve the range of molecular information obtained from human brain tissue. High-resolution image analysis combined with ex-vivo PLA provides a rigorous quantitative approach that can reveal the altered panorama of protein complexes in brain disease.

Author contributions

DOBE and KF wrote, edited, and approved the manuscript.

Funding

This work was supported by grants Hjärnfonden (F02019-0296), Karolinska Institutet Research Foundation (2022-02245), EMERGIA-2020 (39318), Plan Andaluz de Investigación, Desarrollo e Innovación 2020, Junta de Andalucía and CONSOLIDACION INVESTIGADORA (CNS2022-136008), Programa Estatal para Desarrollar, Atraer y Retener Talento, Ministerio de Ciencia, Innovación y Universidades to DOBE. KF and DOBE were also supported by Stiftelsen Olle Engkvist Byggmästare 2021.

Competing interests

The authors declare no competing interests.