André Delambre Symposium on ALS 2009, Québec: STOMPing on Inclusions May Reveal their True Identity

The nervous systems of people with neurodegenerative disease are often pocked with tiny inclusions, which are packed with a variety of proteins, many still unidentified. Many of those proteins may be involved in disease pathology, and figuring out what they are is the first step to discovering how they might relate to disease. Researchers at the University of Toronto are working on a new method to collect proteins from small inclusions for identification by mass spectrometry. Kevin Hadley presented their progress in a poster at the Andre-Delambre Foundation Symposium on amyotrophic lateral sclerosis, held 25-26 September in Quebec City. They call their technique STOMP: Spatially Targeted Optical Microproteomics.

Mass spectrometry requires less than a nanogram of protein, but with inclusions measuring less than 10 microns across, acquiring even a nanogram of material is challenging, Hadley said. Current techniques are not good enough to collect and identify the contents of these tiny aggregates. One option, laser microdissection, lacks the fine resolution needed to capture them. Alternatively, researchers can homogenize tissues and collect insoluble material. However, this brute-force approach may miss some inclusion components and collect other, non-inclusion but insoluble material. By simply grinding up tissue and collecting the insoluble fraction in a centrifuge, we are probably losing a lot of interesting stuff, Hadley said.

Along with principal investigator Avijit Chakrabartty, also at the University of Toronto, and Rishi Rakhit, now at Stanford University in Palo Alto, California, Hadley is working on a method to isolate and identify small inclusions with resolution and specificity that supersedes microdissecting out the protein globs or simply collecting insoluble bits. The technique relies on the strong and specific interaction between the vitamin biotin and the protein streptavidin to pull out proteins from inclusions.

First, Hadley soaks tissue samples in a photoactivatable biotin analog. This compound crosslinks biotin to proteins only when exposed to ultraviolet light. If Hadley were to wash the sample at this stage, all proteins would remain unlabeled. The key to the technique is to only biotin-tag proteins in inclusions.

To do that, the researchers must first find the inclusions. They rely on immunofluorescence to pick out aggregates under the microscope—so an antibody must be available for at least one inclusion component. Hadley uses a confocal microscope to obtain multiple images of the tissue along the vertical axis, resulting in a three-dimensional record of where all the inclusions are.

Next, he targets an ultraviolet laser only at the inclusions, leaving the surrounding tissue unlit. This ensures that only proteins in the aggregates will be biotin-tagged. The tedious way to do this would be to manually define each area, then zap it with the laser. Since this is too time-consuming, the researchers developed a computer program to do it for them. With the picture of the inclusions as input, the program figures out where to aim the laser to light up only the labeled areas.

Then, it is time to turn on the UV and link inclusion proteins to biotin. A standard laser lacks the resolution to finely define tiny aggregates, so Hadley relies on two-photon excitation, which allows two red photons to stand in for a single UV photon. UV radiation, the kind needed to activate the biotin tagging, contains twice as much energy as red light. But very occasionally, with a red laser, two red photons will hit the same spot at the same time, and the double hit packs a punch similar to a single UV photon. It may be one of those things that is forbidden in physics, but works in real life, Hadley quipped. Because the doubling of the red photons is a rare event, it is much more likely to occur in the very center of the laser beam than out on the edges. Therefore, it hits the sample with a much finer resolution, and allows Hadley to label the tiniest aggregates without blasting the adjacent tissue.

Thus far, the researchers have achieved the fine resolution they need to tag inclusions. To test the system, Hadley showed that he could hit a six-micron-diameter fluorescent bead dead center with the laser. The next step is to try it out in real tissue samples. First, the researchers will validate the system in tissue samples from a mouse model of Alzheimer disease. They know exactly what the inclusions should contain: Aβ.

If the techniques works, proteins in the inclusions will be biotin-tagged, but peptides in the surrounding tissue will not. Hadley will homogenize the tissue and apply it to a column of streptavidin-coated beads. The streptavidin will grab the biotin-tagged proteins and let the rest wash through. Then, Hadley can collect the proteins that remained in the column, run them out on a gel, and send the bands in for mass spectrometry to identify them. With the Alzheimer model tissue, they are looking to see Aβ in their results, but Aβ inclusions contain a wide variety of other proteins as well, some potentially unknown. If we find something else, that would be interesting, too, Hadley said.

Once they have verified their approach, the researchers will move on to tissue from people with ALS. They can expect to see well-known aggregate components such as SOD1 and TDP-43—but they may see something else that gives new clues to the disease. And since aggregates are common in many neurodegenerative diseases, the technique has far-reaching potential.

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