Development of molecular biosensors for the detection of alpha-synuclein aggregation in cells

Introduction: Parkinson’s Disease (PD) is the second most common neurodegenerative disorder, which affects more than 6 million people worldwide. PD is a progressive multiorgan proteinopathy, also called synucleinopathy, because of the abnormal neuronal accumulation of misfolded alpha-synuclein (αS), an intrinsically disorder protein (IDP) of 140 amino acids. αS is expressed ubiquitously in the cells and has been particularly found associated with membranes in the pre-synaptic terminal, suggesting a role in the regulation of vesicle trafficking, synaptic transmission and synaptic plasticity. Environmental toxins, genetic mutations, amplification of αS gene trigger αS misfolding and aggregation in Lewy Bodies (LBs) and Lewy Neurites (LNs), typical histopathological marker in PD. This event leads to a gradual loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the spreading of LB pathology not only in neuronal networks of the central nervous system, but also in the autonomic and peripheral nervous systems. For these reasons, PD manifest with a broad range of motor (e.g. rigidity, tremor and bradykinesia) and non-motor symptoms (e.g. anosmia, constipation, cognitive dysfunction). However, the onset of motor symptoms occurs only when about the 50% of dopaminergic neurons are lost. Therefore, identifying the early step that trigger neuronal toxicity might help in halting neurodegeneration. Misfolding, oligomerization, and fibrillization of αS are thought to be central events in the onset and progression of PD. In fact, αS oligomers acts as seeds for the nucleation dependent process of αS intracellular aggregation, and as a template for the cell-to-cell transmission of αS pathology in a prion like manner. In light of this, we developed Alpha-synuclein FRET-based Biosensors (AFBs) capable to monitor any early αS conformational changes ubiquitously in the cell or in a specific subcellular compartment such as the ER, an early site of αS aggregation, under normal and stress conditions. Methods: AFBs were designed to detect different types of αS assembly such as: - relaxed vs globular αS conformation, where both CFP and YFP are cloned respectively at the C-terminal and N-terminal of the same molecule of WT human αS (intramolecular AFB); - parallel multimeric structures based on N-N and C-C termini interactions between molecules, where both fluorophores (CFP and YFP) are cloned separately at the C-terminal of WT human αS (intermolecular AFBs CC); - antiparallel multimers based on C-N termini interactions, where CFP and YFP are cloned separately and respectively at the C-terminal and N-terminal of WT human αS (intermolecular AFBs CN). AFBs were tested in two different cell lines: SH-SY5Y human neuroblastoma cell line and an inducible cell line stably expressing αS in the ER (iSH-SY5Y). Two different FRET methodologies were used to analyses AFBs behaviour: Sensitized Emission (SE) in SH-SY5Y living cells and Acceptor Photobleaching (AP) in iSH-SY5Y fixed cells. Results: interesting outcomes arose by the analysis of AFBs in iSH-SY5Y cells with AP. AFBs provided a radical different behaviour in the ability of detecting conformational differences in αS structure. Intramolecular AFB revealed a closed conformation of monomeric αS under normal conditions, whereas intermolecular AFBs highlighted a multimerization of αS in the ER, which occurred in an antiparallel orientation of αS monomers in normal condition and with a parallel orientation under the effect of leupeptin, an inhibitor of αS degradation pathway. Discussion and Conclusions: these findings suggested that AFBs represent a valuable strategy to detect conformational changes in αS structures in physiological condition and under the effect of stress stimuli. To the best of our knowledge they are the first fluorescent probes capable of detecting different type of αS multimers in the ER, an early site of pathological αS aggregation. Therefore, AFBs represent an interesting investigative approach on αS multimerization and, in the future, might be employed as a powerful tool for the screening of therapeutic agents which aim to halt αS pathological aggregation from the early phase and to prevent the irreversible progression of PD.