That was slow. “We spent a lot of time, probably in the first few years, basically just working out the immunopanning and culturing the astrocytes,” Caldwell recalls. One challenge was to ensure the media contained few proteins to begin with – these would have interfered with their measurements. The scientists also had to ensure that culturing the astrocytes in a petri dish did not alter their behavior relative to how it behaved in the brain.
After noting that the cultured cells behaved normally and retained their ability to direct neuronal development, the scientists examined the proteins they made and the genes they expressed. Then they compared these to normal cells. In all three models of the disorder, they found 88 proteins and about 11 genes that were upregulated — meaning their amount or expression increased.
Both Caldwell and Allen were surprised that the two were often out of sync. While one might think that an increase in the expression of a gene would correlate with an increase in its associated protein, this wasn’t exactly the case. In all three disorders there was little overlap between the most overexpressed genes and the most overproduced proteins. “I think it really underscores, especially in different diseases, that you really need to look at proteins,” says Allen, rather than just focusing on gene expression.
Baldwin, who was not involved in the study, agrees, noting that this lack of overlap is a “conspicuous” finding. “What sequencing can’t capture, what proteomics can, is all the regulation that occurs when the protein is produced,” she says. Sequencing tells you which gene transcripts are available, she adds, but “doesn’t necessarily tell you which ones are turned into protein or at what rate they’re turned into protein.”
Allen’s team focused on a few specific proteins that popped up in all three models of the disorder. One is called Igfbp2, which inhibits the gene pathway for insulin-like growth factor (IGF) — a hormone that normally helps brain development. “The idea was that too much of this inhibitor was being produced by the astrocytes,” says Allen. So the lab tried to suppress it. They gave living mice with Rett syndrome an antibody that blocked Igfbp2 and found that their neurons grew more normally.
Another protein that was overproduced in all three animal models is called Bmp6. It is thought to regulate astrocyte maturation. Again, the team tested what happened when they turned off the protein. First, they placed mouse neurons in a dish and then added the proteins secreted by astrocytes from mice with Fragile X. The neurons were unable to grow many neurite tendrils. But when the scientists tried again, this time using the sludge of Fragile X astrocytes treated with a Bmp6 inhibitor, those tendrils grew. Shutting down the production of the Bmp6 protein appeared to lead to more normal neuron development.
And as it turns out, the two proteins could be linked — showing up of Bmp6 can also lead to showing up of Igfbp2, Allen says, “and that leads to some of these deficits.”