Most studies on Aβ aggregation take place in test tubes. Does the peptide clump up differently inside a nerve cell? Researchers led by Gabriele Kaminski Schierle, University of Cambridge, U.K., have developed a combination of fluorescence-based microscopy techniques to find out. Outlined May 22 in Chemistry and Biology, their methods visualize Aβ aggregates while they form inside acidic compartments of living cells. “Tracing the kinetics and dynamics of Aβ aggregation in a natural context provides insights that you cannot get from in-vitro or traditional cell biology experiments,” said first author Elin Esbjörner. Her methods may help evaluate potential therapies that prevent the build-up.
Esbjörner and colleagues used a technique called fluorescence lifetime imaging microscopy (FLIM) to study the speed with which Aβ fibrils formed. They labeled either Aβ40 or Aβ42 with fluorescent dye and doused SH-SY5Y cells with it. The cells endocytosed the peptide and concentrated it in acidic organelles, likely late endosomes and lysosomes. FLIM measures the average time the dye stays in its excited state before emitting a photon; this time shortens when Aβ aggregates, bringing dye molecules closer together. For Aβ40, the researchers found a six-hour lag time between when the peptides were added to cells and the start of its aggregation. In contrast, Aβ42 began to aggregate right away. That confirms a finding from many previous studies that Aβ42 aggregates much faster than Aβ40, implying that monomers interact differently to drive fibril formation.
To examine the size and shape of the fibrils that formed, the group used a superresolution fluorescence microscopy technique called direct stochastic optical reconstruction microscopy (dSTORM). This method combines thousands of images of the same sample into one. For each image, only a few different fluorescent dye molecules—each decorating a single amyloid monomer—emitted a photon, which makes it possible to map the placement of single molecules. A computer then puts all the images together to reconstruct an image of the sample (see Kaminski et al., 2011). In this way, the researchers found that Aβ40 fibrils in cells formed spherical structures that reached an average of 160 nm. Aβ42 fibrils assumed both spherical and elongated shapes, reaching around 225 nm (see image below right).
The results largely corroborate previous findings from in-vivo experiments, namely that Aβ42 aggregates more readily than Aβ40, the authors wrote (see Gouras et al., 2000). However, they report a difference in lag times of aggregation in living cells compared to in vitro. In vitro, both Aβ40 and Aβ42 show a lag phase, while in live cells only Aβ40 does, suggesting that the intraneuronal environment selectively enhances Aβ42 aggregation. Being able to measure the kinetics of amyloid formation will help scientists study genetic factors and pharmacological compounds that might slow or block it, Esbjörner told Alzforum.
Gunnar Gouras, Lund University, Sweden, praised the work and agreed that screening for potential drugs to block aggregation could be an exciting use of this technology. This is the first time researchers have observed the kinetics of amyloid formation inside the physiologically relevant environment of the cell, he said. “It’s a different world inside these low-pH vesicles,” he said. Currently available in-vivo imaging with multiphoton microscopy or positron emission tomography with amyloid tracers only captures the end products of this process, he pointed out. “This is a big step forward for the field of protein aggregation,” he said.—Gwyneth Dickey Zakaib
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