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NEUROSCIENCE
Knocking Down Cell Cycle Protein Picks Up Axon GrowthFootball would not be the same without special teams--neither would we. Even in cells of the simplest organisms, specialized multi-enzyme complexes have evolved to tackle such complicated tasks as DNA synthesis. But it is perhaps in eukaryotes where the concept is taken to its highest degree of sophistication. For example, to guarantee that cell division goes off without a hitch, special protein teams must be rolled on and off the cellular playing field with split-second timing. It is curious then, that among all this dedicated machinery, we occasionally find in mammalian cells, as in football, a player sitting on more than one special team.
Take the anaphase promoting complex (APC). This is a refined ubiquitin ligase system that tags the mitotic cyclins, among other proteins, for destruction and ensures a smooth transition through mitosis. Yet the complex is robustly expressed in neurons, which are postmitotic and never divide. So what is it doing there? "Something we would not have predicted," said HMS assistant professor of pathology Azad Bonni, who reports in the Jan. 8 Science Express that APC may play a crucial role in regulating the growth of axons. Cell Cycle to Cell DeathIn 2002, work from Bonni's lab showed that the protein kinase CyclinB/Cdc2 can phosphorylate the pro-apoptotic protein BAD, freeing it from innocuous protein complexes and plunging neurons into a suicidal spiral. This work dovetailed nicely with reports from other labs suggesting that in postmitotic neurons, activated cell cycle proteins play a role in programmed cell death rather than the cell cycle itself. The finding also suggested that APC's role in neurons may be to control apoptosis.To test this idea, Bonni and postdoctoral fellows Yoshiyuki Konishi and Judith Stegmuller decided to target cdh1, one of two known proteins that ensure APC passes ubiquitin on to the correct substrates. Using an RNAi strategy, Konishi succeeded in substantially reducing cdh1 in primary cultures of cerebellar granule neurons, but surprisingly, they found that this neither kicked off cellular proliferation nor induced cell death. What the authors did notice, however, was that under the microscope the cells looked morphologically different.
The data suggested that axonal growth can be regulated by APC. To test this hypothesis, Konishi transfected neurons with a dominant-negative form of the APC complex, one with a mutation in the RING finger motif of the ligase subunit. The transfected cells also grew axons twice as long as those found in untreated neurons, confirming that APC plays a direct role in tempering axonal growth. Overcoming InhibitionPrimary neurons are relatively easy to work with, but are not necessarily the best model to mimic the complex cellular milieu that surrounds granule neurons in vivo. Would inhibiting APC also promote axonal lengthening in a more natural environment? To test this, Stegmuller joined the project and developed a tissue assay in which granule neurons were overlaid on a slice of cerebellum. Though the RNAi treatment did not have as dramatic an effect as on cells grown in primary cultures, axons grew 40 percent longer than in untreated cells. But in a further surprise, axons stretched in substantially different directions.The cerebellar cortex is composed of layers of specialized cells with the external granular/molecular layer being outermost, followed by the Purkinje layer, the inner granular layer, and the white matter. In the tissue slice assay, axons from overlaid granule neurons generally extend along the external/molecular layer mimicking the normal pattern in vivo, but Stegmuller found that axons from RNAi-treated neurons extended in a more random fashion, crossing layers almost 60 percent of the time and sometimes penetrating as deeply as the white matter. Using an in vivo RNAi electroporation method recently developed by HMS's Connie Cepko (see Research Briefs, Focus, Dec. 12, 2003), Konishi was then able to confirm that cdh1 knockdown has the same effect in the cerebellar cortex in rat pups. In these animals, axons from RNAi-targeted neurons broke with the flow. Instead of following the normal parallel extension of most axons, they "defasiculated," heading off in random directions. The results indicate that even in vivo, inhibiting cdh1 can have a dramatic effect on axonal growth and patterning. "We are not sure yet if these axons are functional," noted Bonni, "but they look normal and have growth cones. It will be crucial," he said, "to determine if cdh1 knockdown can improve the ability of axons to regenerate after injury." He has reason to be hopeful, since inhibiting APC/cdh1 appears to send especially strong growth signals to axons. Bonni explained that brain myelin is one of the strongest inhibitors of axonal growth known, so his team was encouraged to see, in the tissue slice experiments, that axons from RNAi-treated neurons penetrated into the white matter layer, which is laden with myelin. In fact, the same neurons had little problem growing on a myelin bed, and even extended their axons further than untreated cells grown on an inert polyornithine layer. --Tom Fagan |
Available evidence suggested that the anaphase promoting complex (APC) was key to preventing programmed cell death in neurons, so (from left) Azad Bonni, Yoshiyuki Konishi, and Judith Stegmuller were surprised to find that the complex actually regulates axon growth. When the complex is ablated (above), axons grow more than twice as long as in control neurons (top). (Images courtesy of Science; photo by Jeff Cleary)