Advanced multiplexing technologies with innovative Dual-Channel Dark-FRET biosensors for dynamic monitoring of alpha-synuclein pathophysiology: From cellular to in vivo models

ABSTRACT: Alpha-synuclein (aSyn) accumulation and misfolding is implicated in the pathogenesis of Parkinson's Disease (PD) and related synucleinopathies. These disorders include multiple cellular dysfunctions including impaired proteostasis (e.g., the dysregulation of the autophagy lysosomal pathway [ALP]). As preclinical disease models

become more complex to better recapitulate disease relevant pathophysiology— i.e., monoculture ➔ 2D co- culture ➔ 3D organoids (both mono- and co-cultured) ➔ in vivo — advanced multiplexing technologies that are capable of dynamic temporal and spatial monitoring of protein-protein interactions (e.g., aSyn oligomerization

and misfolding) and pathological phenotypes are required. Existing fluorescence-based biosensors are limited by their static nature and inability to differentiate signals across distinct cell types within multi-cellular environments. To address this, our proposal introduces Dark-FRET (DF) and Dual-Channel DF (DCDF) cellular

biosensors that utilize the Shadow-G/Y/R series of acceptor protein which were engineered as to have reduced quantum yield and negligible fluorescence emission, eliminating emission spillover from acceptor proteins which facilitates improved live-cell multiplexing for multiple protein-protein interaction (PPI) fluorescence assays.

This cutting-edge approach enables real-time monitoring of both aSyn folding (ShadowY-aSyn-mNeongreen) and aggregation (aSyn-mScarlet-I3/ShadowR) FRET biosensors expressed in distinct cellular populations. We expand on these capabilities by multiplexing the aSyn DCDF biosensors with our TFEB (the master regulator of

ALP), FRET and nuclear translocation biosensors. Aim 1 involves applying these biosensors to CNS-resident cell lines (neurons, microglia, astrocytes) to establish mono-, bi-, and tri-culture cellular models for dynamically tracking cell-specific aSyn interactions and ALP phenotype. Aim 2 extends DCDF technology to in vivo models

using novel C. elegans strains expressing aSyn DCDF and ALP biosensors in specific tissues. By enabling simultaneous monitoring of aSyn oligomerization and phenotypic dysfunction, this approach provides a valuable tool for investigating synucleinopathies. Its potential applications extend to 3D organoids and hiPSC models,

offering insights into diverse cell-type pathways and enabling the identification of compounds modulating aSyn oligomerization and pathological phenotypes in these advanced preclinical models. In summary, our DF/DCDF technology presents a groundbreaking approach, enabling dynamic monitoring of aSyn aggregation and pathological phenotypes across complex disease models, paving the way to address

biological questions on the supporting roles of microglia and astrocytes on neuronal health and function. Furthermore, these multiplexed biosensors represent an innovative preclinical therapeutic discovery platform that holds promise for enhancing the drug discovery process, potentially filling the therapeutic gap in

synucleinopathy treatment.