Lighting Up Alpha-synuclein Oligomers.

Based on diverse evidence for its role in the pathogenesis of Parkinson's disease (PD), alpha-synuclein is probably the most extensively studied protein in the field. A mutation in the gene encoding alpha-synuclein (SNCA) represents the first identified cause of familial PD (Polymeropoulos et al., 1997). Subsequently alpha-synuclein was found to be a main component of Lewy bodies, a pathological hallmark of sporadic PD (Spillantini et al., 1997). Additionally, elevated expression of alpha-synuclein due to gene multiplications or nucleotide polymorphisms can cause PD or significantly increase the risk to develop the disease. While the crucial role of alpha-synuclein is thus established, it is still not fully understood how alpha-synuclein pathology connects to neurodegeneration, and why specific neuronal subtypes such as the dopaminergic neurons of the substantia nigra are preferentially affected in PD. Sufficiently answering these most critical questions will likely facilitate the development of urgently-required disease modifying therapy. The in vivo application of bimolecular fluorescence complementation (BiFC) of alpha-synuclein in the mouse presented by Cai et al. in this issue of EBioMedicine (Cai et al., 2018) presents a valuable tool to tackle these questions. The authors used viral vectors with venus yellow fluorescent protein (venusYFP) fragmented into N and C termini, then fused with human wildtype alpha-synuclein to form non-fluorescent fusion proteins V1S and SV2, respectively. Recombinant adeno-associated virus (AAV) was chosen to mediate the overexpression of alpha-synuclein constructs in the substantia nigra of mice. When alpha-synuclein associates into oligomers, the complementary N- and C-terminal halves of venusYFP combine, thereby reconstituting a full, fluorescent complex. The authors report progressive appearance of venusYFP fluorescence, indicating formed alpha-synuclein oligomers in nigral dopaminergic neurons. Eight weeks following transduction, affected neurons showed alterations in morphology which spread, likely via anterograde transport, to terminals in the striatum. Some fluorescent signal remained after proteinase K treatment, suggesting formation of insoluble alpha-synuclein aggregates. The authors replicated previous observations in AAV-mediated alpha-synuclein overexpression models such as dopamine loss, inflammatory response and altered motor behavior. These initial findings should motivate further in vivo studies on the spread and transmission of alpha-synuclein species visualized via BiFC. Why is the direct spatial and temporal tracking of alpha-synuclein oligomerization desired? Physiologically, alpha-synuclein, as indicated by its name, concentrates presynaptically and in the nucleus. The protein was suggested to form alpha-helically folded tetramers, but there is also compelling evidence for a monomeric state in mammalian cells (Theillet et al., 2016). Probably triggered by factors such as higher expression, disturbance in metabolism, or interaction with other agents, alpha-synuclein becomes prone to oligomerization and ultimately forms the amyloid fibril with a cross beta-sheet quaternary protein structure which constitutes Lewy bodies. During this process, alpha-synuclein forms multiple kinds of species, or strains, which seem to differ in their capacity to cause acute cell death and to spread in a prion-like fashion (Luk et al., 2012; Peelaerts et al., 2015). In fact, alpha-synuclein pathology was detected in the peripheral nervous system of PD patients, and there is evidence for transport across nerves to central neurons (Del Tredici and Braak, 2012). Studying such complexity requires tracking of the protein across all formed species. Visualizing the early alpha-synuclein oligomerization, independent of whether those small oligomers represent the most toxic species, would aid detection of initiation and propagation of alpha-synuclein pathology and its potential therapeutic inhibition (Richter et al., 2017). Here lies a major challenge, because immunohistological techniques only detect a subset of aggregation species, and early stage aggregates often dissolve in protein assays. Researchers are therefore following different strategies such as ligation assays, fluorescent labeling of alpha-synuclein prior to inoculation and novel imaging techniques to visualize protein structure with high resolution (Roberts et al., 2015; Rodriguez et al., 2015). With BiFC, oligomerization of the locally overexpressed protein becomes trackable and potential transmission of these fluorescent oligomers to other cells can be monitored in vivo. One disadvantage of fluorescent labels is the potential interference with the protein function as well as with the aggregation it is supposed to monitor. Another disadvantage is the requirement of AVV as a shuttle, thereby inducing secondary effects such as local inflammation or mechanically-induced cell death, which can artificially contribute to pathology. Therefore, these studies require a number of control groups as provided by Cai et al. Regardless of their specific limitations, each additional tool to track alpha-synuclein pathology provides researchers with the opportunity to visualize an aspect of pathogenesis, which in combination will hopefully provide a convincing picture of how alpha-synuclein contributes to neurodegeneration. Importantly, such tools can be applied to other synucleinopathies, such as multiple system atrophy, where aggregation curiously affects mainly oligodendroglia. For this purpose, Cai et al. suggest further validation of their virus approach for mediating overexpression in oligodendrocytes. In summary, the findings of Cai et al. contribute a valuable tool to piece together the puzzle of alpha-synuclein pathology and thus PD pathogenesis.

Disclosure

The author declared no conflicts of interest.