In some ways, Alzheimer disease research is about a decade ahead of research on Parkinson’s. As PD research expands and catches up, observers of both fields can’t help but notice how the latter appears to follow rather surprisingly in the tracks of the former. For example, one idea that is gaining currency in PD research holds that it is a conspiracy of oligomeric/protofibrillar species of the presynaptic protein α-synuclein that harms neurons, more so than do mature fibrils or the pathologic hallmark, i.e., the Lewy bodies. At the 37th annual conference of the Society for Neuroscience last month in San Diego, California, two presentations illustrated how this line of research is evolving in the wings of similar work on AD.
The hypothesis that monomeric α-synuclein passes through a series of aggregation states starting with a native monomer and ending with insoluble fibrils, and that the intermediate states may well be the worst, goes back to Peter Lansbury at Boston’s Brigham and Women’s Hospital (Goldberg and Lansbury, 2000; Volles and Lansbury, 2003). The number of labs working to test this hypothesis is steadily growing. In San Diego, Martin Ingelsson of Uppsala University, Sweden, presented early data of a new line of investigation that aims to characterize the fibrillization propensity of the α-synuclein mutations known to be at the root of early-onset Parkinson’s. Previously, the larger Uppsala group led by Lars Lannfelt has been studying protofibril formation by the arctic Aβ mutation in familial early-onset AD. As in AD, the heat is on in PD research to find the most toxic α-synuclein species. Unlike in AD, however, the work is still in vitro and no candidates have as yet been isolated from either genetic or sporadic PD brain (for comparison, see ARF related news story).
In PD, the A30P and A53T mutations in α-synuclein are known to cause early-onset disease. More recently, a third mutation, E46K, proved to cause early-onset dementia with Lewy bodies. DLB is an understudied double whammy of a bad disease, which robs people of their cognition and movement. In addition, rare α-synuclein duplications cause early-onset PD, whereas triplications cause early-onset DLB. With regard to the aggregation propensity of these mutant proteins, earlier work by others has shown that the A30P mutation slows aggregation down, whereas A53T speeds it up. In San Diego, Ingelsson reported how he, Joakim Bergström, and their colleagues compared side by side the speed with which the three mutant forms aggregate. This experiment confirmed the previous finding, and discovered further that the E46K mutation causing DLB was the most aggressive of all.
Next, the Swedish scientists combined size exclusion chromatography with high-performance liquid chromatography (SEC-HPLC) to characterize the intermediate species that form over time when wild-type α-synuclein is incubated in vitro and left to associate with itself. At the beginning, Ingelsson reported, the researchers saw only monomer. After about a day, smaller species than the monomer began appearing. These initial species could represent truncated monomer, invoking the speculation that they might act as seeds for aggregation, Ingelsson said. (Some scientists also suspect truncated species of tau of seeding aggregation.) At a later time point, aggregation proceeded to oligomeric forms larger than 600 kDa. Putting a precise molecular weight on these oligomers is difficult because their apparent weight on the chromatogram may not reflect their true size, Ingelsson said. But the Swedes are confident that one major species they are seeing are large protofibrils comprising about 50-100 monomers each. They do not see low-N oligomers in this system. When added to HEK cells in initial tests of toxicity, only the protofibrils managed to kill significant numbers of the cells; neither the monomer nor the fully formed fibrils did. (Tests in primary neurons are underway, Ingelsson said.)
Clearly, aggregation in vivo is more complicated because α-synuclein interacts with a multitude of factors, some of which are likely attempts to try to prevent damage to the cell. AD research has shown one such line of protection to come from the chaperone HSP70 and its partner CHIP, which mark the Aβ peptide for degradation if it cannot be properly folded (Kumar et al., 2007). In this regard, too, α-synuclein is following in the footsteps of Aβ in that its misfolding and aggregation appear to be subject to rescue attempts and disposal by CHIP, at least in vitro. Researchers at Massachusetts General Hospital had reported that CHIP can degrade α-synuclein (Shin et al., 2005). In San Diego, Julie Tetzlaff, a postdoctoral fellow in Brad Hyman and Pam McLean’s laboratory there, followed up by asking whether this happened to monomers or oligomers. To do that, Tetzlaff used a new cellular imaging trick, where she split green fluorescent protein and fused each non-fluorescent half to one α-synuclein molecule. When the α-synuclein monomers interact, dimers fluoresce. Using this bimolecular fluorescence complementation (BiFC) assay in transfected cells, Tetzlaff observed that CHIP reduced the fluorescence. A series of further experiments suggested that CHIP recognizes toxic oligomeric forms of α-synuclein and mediates their degradation. The present data are from human neuroglioma cells; work with dopaminergic cell lines is ongoing, Tetzlaff said. Beyond these two research groups, others as well have picked up on parallels in the aggregation of these two proteins. The focus lies on exploiting the similarities not only for mechanistic studies but also toward biomarker development in Parkinson disease.—Gabrielle Strobel.
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