Trehalose On The Aggregation Of Alpha Synuclein

Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by protein amyloidogenesis or fibrillogenesis in human brain. PD, a common neurodegenerative movement disorder, is featured by loss of dopaminergic neurons in the substantia nigra, accompanied by formation of Lewy bodies. The key component of those inclusions is fibrillar a-synuclein (AS), a 140-residue protein which is largely unfolded in its physiological conformation. Recently, it was shown that the aggregation of AS into fibrils is a critical step in the pathogenesis of PD, and that the precursor of AS fibrils, namely soluble-sheet oligomeric species, has been suggested to be a toxic conformation in PD. However, there is still much controversy in this field and the molecular mechanisms underlying the AS aggregation remain unknown. Therefore, better understanding the mechanism of AS aggregation and developing novel strategies to prevent its neurotoxicity are being one of the most important topics attracting neuroscientists interests and efforts in PD research. Any compound which is able to slow down and/or prevent aggregation of AS might be promising to lead to a novel therapy for PD. Trehalose was recently found to be effective in the intervening the protein aggregation in various neurodegenerative disorders, including Alzheimer's disease, Huntingdon's and prion diseases, which are all associated with the formation of amyloid deposits in the fibrils with a characteristic cross-structure. The striking similarity in the aggregates of AS to that of beta-amyloid enabled us to hypothesize that the trehalose might be able to disrupt AS aggregation and contribute to PD therapy. Thus, we used recombinant human A53T mutant AS, which assembles into the filaments more readily than its wild type, to investigate the impacts of trehalose on the aggregation of AS A53T. An Investigation into the Effect of Trehalose on the Aggregation of Alpha-SynucleinAim:To investigate the impacts of trehalose on the aggregation of Alpha-Synuclein.Methods:2.0 mg/ml A53T Mutant Alpha-Synuclein (AS A53T) was co-incubated without or with 10 mM or 100 mM trehalose in 20 mM phosphate buffer (pH 7.5) at 37℃for 0 to 7 days. The incubated solution was extracted at every given time to assess the AS A53T aggregation by means of CD spectroscopy, ThT fluorescence assay as well as AFM image.Results:(1) In the absence of trehalose,2.0 mg/ml AS A53T was highly unfolded in the solution with the most negative ellipticity CD spectroscopy at 198 nm at the beginning. As the incubation time increased, the value of negative ellipticity at 198 nm decreased gradually along with one more negative peak at 219 nm increasing. ThT shows that the ThT fluorescence intensity increased during the incubation from day 0 to day 4, and reached saturation after being incubated longer than 5 days. AFM shows that when the incubation time increased from 2 to 4 days, those oligomers continued to prolong and thicken, and some fibrils started to be bifurcated like a "crotch". Further incubation for 6 days, the bifurcated fibrils entwisted and cross-linked into the large fibrils, and eventually turned into fibrillar plaques when incubation for 7 days. (2) In the solution of 2.0 mg/ml AS A53T in the presence of 10 mM trehalose, the negative peak at 219 nm slightly increased during the incubation from day 0 to day 3 as the initial conformer of random coil attenuated simultaneously. During the incubation from day 4 to day 7, it was surprisingly found that the peak at 198 nm went more negatively while the negative peak at 219 nm decreased simultaneously. It indicates that in the 2.0 mg/ml AS A53T solution with 10 mM trehalose presence, the ThT fluorescence intensity increased during the incubation from day 0 to day 3 and then decrease afterward. AFM shows that in the 2.0 mg/ml AS A53T solution with 10 mM trehalose, there was no significant difference in the morphologies of AS A53T protein between those two samples during the incubation from day 0 to day 3. However, it was amazingly found that during 4 and 5 days of the incubation the formed protofibrils or fibrils of AS A53T disassembled into doublestrand rosaries along with some cake-like aggregates. Further incubation to day 7, the doublestrand rosaries were dissolved eventually into the small aggregates. (3) In 2.0 mg/ml AS A53T solution in the presence of 100 mM trehalose, there was a typical evolution of the CD spectra during the whole period of incubation from day 0 to day 7, which is similar to the trend observed in incubation of AS A53T without trehalose. ThT fluorescence increased before day 3 but went slower than that without trehalose. However, the ThT fluorescence continued to increase from day 4 to day 6, and reached the saturation afterward. The saturated fluorescence intensity in the presence of 100 mM trehalose was higher than that without trehalose. AFM shows that in 2.0 mg/ml AS A53T solution with 100 mM trehalose, there was no formed large fibrillar aggregates of AS A53T except for some small oligomers and protofilaments during the incubation from day 0 to day 7.Conclusion1. AS A53T in absence of trehalose assembles into oligomers firstly. Moreover, in the late period of incubation time, the AS A53T continued to assemble into the entwisted or cross-linked large fibrils even the fibrillar plaques.2.10 mM trehalose was not able to inhibit the assembling of the random coil AS A53T monomers into protofibrils or fibrils at the early time, in the late period it could disassemble the formed protofibrils or fibrils into the small oligomers.3.100 mM trehalose could slow down the formation of oligomers, stabilize the formed oligomers or protofibrils in the aqueous solution, and even prevent the formation of AS A53T fibrils.