1999 Highlights

from the 1999 Annual Report HIGHLIGHTS OF THE YEAR The research and education programs at the Laboratory remain strong and vibrant. The recruitment of the first students in the Watson School of Biological Sciences and the com- pletion of their intensive core courses highlighted progress this year. The DNA Learning Center introduced new, Web-based educational tools, and the meetings and courses programs at the main campus and the Banbury Center continued to provide a valuable service to the scientific community. Significantly, the Laboratory Press saw the continued development of its exciting textbook-publishing program as part of the Laboratory’s goal to promote innovative teaching programs for graduate students and senior undergraduate students. These educational programs occur in the surrounding of a strong research environment, with research and education braided together in a common enterprise. Research Neuroscience Roberto Malinow and Karel Svoboda have continued to push the limits of brain imaging by observing the changes that occur in individual neurons as they respond to electrical stimuli. Combining their expertise in electrophysi- ology and high-resolution imaging of the brain, they study the cellular events that underlie “synaptic plasticity”—the ability of brain neurons to reorganize their connections in response to experience, such as during learning and consolidation of memories. In particular, Roberto and Karel are exploring the mechanisms involved in long-term potentiation (LTP), the strengthening of Roberto Malinow synaptic connections between nerve cells in response to trained stimuli. Their studies focus on a region of the brain involved in learning and memory called the hip- pocampus, a region required for a form of memory called declarative memory, or the con- scious memory of learned events. This year, Roberto and Karel studied the distribution of AMPA receptors within dendrites, a nerve cell property they suspect contributes to LTP. AMPA and other receptors mediate the effects of glutamate, a key neuro- transmitter that is one chemical which crosses the synapse or gap between neurons. They first engineered hippocampal neurons to express a fluores- cent version of the AMPA receptor so they could track its movement within the processes called dendrites that branch from the neuronal cell. They used a high-resolution optical-maging system called two-photon laser-scanning microscopy, both before and after electrical stimulation. Before stimulation, AMPA receptors were distributed evenly along the branch-like shafts of dendrites, but were largely restricted from the dendritic spines that project from shafts and are where the synapses lie. After stimulation, however, AMPA receptors moved rapidly into the spines. Synapses are typically located at the tips of spines, but without functional AMPA recep- Karel Svoboda tors, such synapses are inactive, or “silent.” Therefore, the movement of AMPA receptors into dendritic spines may contribute to LTP, at least in part, by converting silent synapses to 5 active synapses. Moreover, evidence from Roberto’s lab suggests that the constant replen- ishment of AMPA receptors at synapses may be involved in maintaining synapses in an active state over long periods of time. It is an intriguing possibility that this form of synaptic plasticity may underlie the development of the brain’s neuronal network and function of neurons in an adult brain. Roberto and Karel’s studies provide some of the first real-time, high-resolution images of the events that are likely to power the restructuring of vast neural networks within an important site for learning and memory in the brain. Yi Zhong and his colleagues are also studying the neurological basis of learning and memory, but in the fruit fly Drosophila. They have recently focused their efforts on explaining the puzzling observation that a human disease called neurofibromatosis causes both cancer and learning defects. Neurofibromatosis is one of the most common inherited genetic disor- ders affecting humans. The disease is characterized by discoloration and tumors of the skin and is caused by mutations in the NF1 gene. NF1 encodes a protein that inhibits the activity of the Ras protooncogene prod- uct, another protein that is altered in many human tumors. Thus, when the NF1 gene is mutated, the resulting uncontrolled activity of Ras can lead to cancer. But how mutations in NF1 lead to cancer and learning defects was unknown. From previous work, Yi knew that an enzyme called adenylyl cyclase was Yi Zhong required for learning in Drosophila. He also knew that adenylyl cyclase activity was stimulated by the Drosophila NF1 protein (human and Drosophila NF1 are remark- ably similar, so much so that human NF1 can substitute for its Drosophila counterpart in transgeneic flies). Yi and his colleagues first established that Drosophila—like humans and mice—requires NF1 for learning (in this case, learning to avoid a particular odor that was paired with a small electrical shock). It was first necessary to determine whether the defects in adult flies lacking NF1 were an indirect result of developmental abnormalities due to the loss of NF1 activity as the brain devel- ops. But experiments demonstrated that the learning defect in flies lacking NF1 could be cor- rected during adulthood by a burst of NF1 from an externally controlled gene. Then, in a series of elegant experiments, Yi showed that the effects of NF1 on learning are mediated by the enzyme protein kinase A, an enzyme that lies downstream from NF1 and adenylyl cyclase in the regulatory pathway controlling learning. Since protein kinase A is a well-characterized enzyme, the studies suggest that by activating it, the learning deficit could be reversed. Yi’s studies reveal the role of NF1 in a new mechanism that contributes to normal learning and memory in Drosophila. Because this pathway is conserved from fruit flies to humans, there is a distinct possibility that pharmacological intervention in the adenylyl cyclase pathway might reverse the NF1 defect in humans. Virus-induced Pathogenicity: The Case of HIV The genomes of human and simian immunodeficiency viruses, HIV and SIV, encode several auxiliary proteins that modulate the function of infected cells and optimize virus propagation in part by enabling infected cells to evade the host’s antiviral immune response. Determining how these auxiliary proteins work is therefore important for understanding the pathogenesis of AIDS and how an effective vaccine might be formulated. 6 Jacek Skowronski and his colleagues are studying how one of these auxiliary proteins, Nef, promotes HIV infection. The Nef proteins of HIV and SIV are similar, and each fulfills mul- tiple roles in the course of viral infection. The goal of the experiments in Jacek’s laboratory is to determine how Nef interacts with protein sorting and signal transduction machinery in T cells and macrophages, two immune system cell types that are infected by HIV. Jacek and his colleagues have found that Nef is required for the “down-regulation” or removal of particular host-cell proteins from the cell surface (CD4 and class I MHC proteins). Moreover, a collaboration with scientists at Erlangen University (Germany) and Harvard University has led to the identification of certain mutations in Nef that selectively disrupt its ability to down-regulate CD4, thereby greatly decreasing viral replication during the acute phase of viral infection. One future challenge for the Skowronski group is to determine precisely how down-regulation of CD4 and class I MHC by Nef promotes viral propagation. One possibility is that by removing CD4 and MHC I from the cell surface, Nef enables HIV-infected cells to evade the body’s immune response. Alternatively, Nef might be required to optimize host-cell survival so that the virus has a chance to reproduce before it kills the cell. Cancer Research: Regulation of Cell Death The genetically programmed death of cells is a normal part of growth and development. In addition, programmed cell death, or “apoptosis,” safeguards organisms from cancer by triggering the self-destruction of precancerous cells. A hallmark of many types of cancer is a defect in apoptosis that allows precancerous cells to survive and proliferate, and eventually form a tumor. Yuri Lazebnik and his colleagues are exploring the possibility that restoring the ability of cancer cells to self-destruct via apoptosis might be an effective therapeutic strategy. They study a family of enzymes called caspases that act in a proteolytic cascade to execute programmed cell death. Among several known caspases, caspases 2, 8, and 9 are thought to be “initiator” caspases. In response to cellular self-destruct signals, these caspases trigger a chain reaction in which a series of “effector” caspases are converted from an inactive to an active form. Once activated, effector caspases cleave key cellular proteins, and cell death fol- lows due to destruction of DNA, disruption of nuclear structure, and disintegration of cells into small vesicles. Previous work from Yuri’s lab suggested that a caspase cascade leading to self-destruction remains functional in cancer cells, but it is uncoupled from the signal that triggers its activation. To study this critical link between a cell death signal and the caspase-mediated cell death machinery, Yuri and his colleagues purified a cellular factor that could trigger caspase- mediated apoptosis. They found that a known protein called APAF-1 (apoptosis activation factor-1) plus caspase-9 are key components in triggering apoptosis in tumor cells. Another lab had shown that APAF-1 stimulated the biochemical activity of caspase-9, but precisely how APAF-1 worked on caspase-9 remained a puzzle. Effector caspases are synthesized as inactive precursors that are activated upon cleav- age by other “upstream” caspases. For example, caspase-3 (an effector caspase) is cleaved and activated by caspase-9 (an initiator caspase), but how the initiator caspase is cleaved and activated was unclear.

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