Kinesin‐1 inhibits the aggregation of amyloid‐β peptide as detected by fluorescence cross‐correlation spectroscopy

Abstract

Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-b (Ab) generated by the proteolytic processing of amyloid-b precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Ab generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Ab with kinesin-1 at the singlemolecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Ab over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

How fluorescent tags modify oligomer size distributions of the Alzheimer-peptide Aβ(1-40)

“…This corresponds to literature finding which report on the use of Atto647N the formation of high MW oligomers, with e.g. a 70-fold increase of the diffusion time (27) or a time-independent, 10-fold increase in the diffusion time (48). The latter value agrees well with our finding of N r el of 10.9.…”

“…Thus, investigation methods which can work at much lower concentrations, as optical techniques based on absorption and/or fluorescence detection, have been most widely employed to monitor in real-time the kinetics of the early aggregation processes in solution (20). In the last two decades techniques as fluorescence absorbance (22), photobleaching (19,23), self-fluorescent-quenching (20), fluorescence correlation spectroscopy (FCS) (24,25), fluorescence cross-correlation spectroscopy (FCCS) (26), dual-color fluorescence cross-correlation spectroscopy (dc-FCCS) (27), Förster resonance energy transfer combined with fluorescence correlation spectroscopy (FRET-FCS) (26), confocal two-color coincidence detection (cTCCD) (28), but also fluorescence imaging of labeled peptides (19,(29)(30)(31) or binding of Thioflavine T (ThT) (32) or Congo Red (33), have been widely applied for the study of such systems at very low concentrations. Despite the great sensitiveness of such optical techniques, one draw-back about using fluorescence is to be found in the inherent, yet necessary, modification of the original peptide system due to the attached fluorophore.…”

The effects of fluorescent labels on Aβ42 aggregation detected by fluorescence correlation spectroscopy

“…And then the hydrodynamic radii of Aβ 42/BP , Aβ 42/RB , and Aβ 42/RITC increased to their maximal values at 1 hour with that of 34.5, 22.4, and 11.9 nm, respectively, after which the hydrodynamic radii of Aβ 42/BP decreased slightly at the next detection time (the R H at 1 hour is significantly different from those at other detection times as calculated by one‐way analysis of variance with LSD post hoc test, P < .05), while that of Aβ 42/RB and Aβ 42/RITC did not have big changes. These data of Aβ 42/BP resemble our previous results and the reported literature, and the slight decrease may have been caused by reorganization of the oligomers. However, the hydrodynamic radii of Aβ 42/5‐SFX and Aβ 42/5(6)‐FITC did not show obvious changes between 0 minute and 4 hours.…”

“…A previously described method was used to prepare Aβ 42 . A purified synthetic Aβ 42 peptide (0.5 mg, purity ≥98%; Sangon, Shanghai, China) was dissolved in 0.5 mL of Hexafluoro‐2‐propanol (HFIP, Sigma, St. Louis, Missouri) and incubated for 24 hours at room temperature with gentle shaking.…”

How Fluorescent Tags Modify Oligomer Size Distributions of the Alzheimer Peptide

“…Thus, investigation methods that can work at much lower concentrations, such as optical techniques based on absorption and/or fluorescence detection, have been most widely employed to monitor in real time the kinetics of the early aggregation processes in solution ( 20 ). In the last two decades, techniques such as fluorescence absorbance ( 22 ), photobleaching ( 19 , 23 ), self-fluorescent-quenching ( 20 ), fluorescence correlation spectroscopy (FCS) ( 24 , 25 , 26 , 27 ), fluorescence cross-correlation spectroscopy ( 28 , 29 ), Förster resonance energy transfer combined with FCS ( 28 ), and confocal two-color coincidence detection ( 30 ) but also fluorescence imaging of labeled peptides ( 19 , 31 , 32 , 33 ) or binding of Thioflavine T (ThT) ( 34 ) or Congo Red ( 35 ) have been widely applied. Despite the great sensitiveness of such optical techniques, one drawback of using fluorescence is to be found in the inherent, yet necessary, modification of the original peptide system because of the attached fluorophore.…”