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Molecular Mechanisms Regulating Neuronal Phenotype
Summary: Gail Mandel is interested in revealing molecular mechanisms underlying global regulation of gene expression and function in the nervous system.
One of the major goals in neuroscience is to understand how the functions of individual neurons lead to higher order brain functions such as learning and memory. Many years ago, we demonstrated that REST, a transcription factor first identified in our lab, controls expression of genes that encode hallmarks of the neuronal phenotype. These genes encode excitability proteins such as certain ion channels and receptors.
In our most recent experiments, we discovered, surprisingly, that the REST-binding site is more complex than previously appreciated, and that target genes contain many different variations of the originally identified site. The result is that a much larger repertoire of genes is potentially under REST control, including large gene families that control our sense of smell. Genes regulating cell migration also contain REST-binding sites, suggesting a mechanism to explain the finding (see below) that mutations in REST are associated with motility defects in certain cancers. This new work provides further evidence that REST is indeed a master regulator of the neuronal phenotype. A similar mechanism for specifying identity of other tissues has not yet been identified, suggesting that nervous system development is unique.
New Roles for REST?
The REST gene is expressed widely in the animal, in nonneural, terminally differentiated cells and in presumptive nervous tissue that is not yet neuronal in nature. During development of the nervous system, REST expression decreases dramatically as neurons undergo terminal differentiation. If REST levels are not allowed to decrease, there is no conversion of progenitor cells to fully functional differentiated neurons. In contrast to the situation in neuronal cells, in nonneural tissues and neural progenitor cells, REST levels remain high throughout the lifetime of the animal. Historically, it was assumed that the role of REST in nonneuronal cells was primarily to block aberrant expression of neuronal proteins that would be deleterious to these cell types, but this hypothesis had never received experimental support. In collaboration with the Stephen Elledge's lab (HHMI, Brigham and Women's Hospital), we showed that REST functions as a tumor suppressor in epithelial cells such as mammary cells or colonic epithelium.
Although the mechanism by which REST suppresses tumors is not known, it is likely to involve suppression of sets of genes containing the consensus REST-binding sites. Interestingly, paraneoplastic diseases, as well as some forms of prostate cancer, are characterized by the aberrant expression of neuronal genes. In the case of a mouse model for prostate cancer, more than 90 percent of the neuronal genes aberrantly expressed are REST target genes. We are interested in determining whether REST plays an active role in these neoplasms. (Grants from the National Institutes of Health provided support for aspects of this work.)
REST Regulates RNAs as well as Protein-Coding Genes
One current impediment for curing diseases such as brain cancers is a lack of understanding of the complex interactions among the factors responsible for normal cell function. We developed a new strategy for identifying the complete set of genes regulated by any transcription factor. We have used this strategy successfully to study the network of genes regulated by REST. Many new and unexpected findings have resulted from this approach, including the observation that some important regulators of neuronal function are not proteins but, rather, small regulatory mRNA molecules, called microRNAs or miRNAs, that somehow interfere with protein synthesis. MicroRNAs are present in plants, worms, and humans, and bind to the mRNAs in the cell that produce proteins. As a result of this binding, the proteins are not made or are degraded.
We have identified one specific miRNA that is expressed only in mature neurons. Like classical neuronal-specific genes, this miRNA is repressed outside of the nervous system because of the presence of REST. The miRNA is absent in neural precursor cells, but when REST goes missing at late stages of neuronal differentiation, the miRNA is expressed. In the mature neuron, the miRNA has an unusual function in that it begins to degrade mRNAs that are normally found only in nonneuronal cells, the reverse function of REST.
Our studies have led to a new model for how cells preserve their cellular identity: in both neuronal and nonneuronal cells, REST represses genes that are not wanted. Moreover, miRNAs not only block nonneuronal proteins but also induce expression of neuronal genes that help promote the neuronal phenotype. This occurs because the miRNAs regulate repressors of neuronal genes. When the repressor mRNAs are blocked, neuronal gene targets are up-regulated. If our working model is correct (see figure), we have identified a feedback loop that reinforces and amplifies the neuronal phenotype in a terminally differentiated neuron and perhaps helps block interconversion of neurons to other types of cells. (Grants from the National Institutes of Health provided support for aspects of this work.)
Mechanisms Controlling Synapse Formation
We have initiated a new line of studies aimed at elucidating post-transcriptional mechanisms underlying plasticity at vertebrate synapses. In addition to misexpressing proteins in mammalian neurons and then assessing the consequences on synaptic activity, we are identifying animals with aberrant behavior and then working backward to find the culprit gene. For both approaches, we have been exploiting the advantages offered by zebrafish. In particular, there are many existing mutant lines of zebrafish that exhibit behavioral traits due likely to alterations in synaptic function. Our strategy is to use electrophysiological and behavioral analyses to screen these mutant fish for defects in synaptic communication. The defects could be either in the presynaptic nerve or in the postsynaptic nerve or muscle cell. In collaboration with Paul Brehm (Vollum Institute), we have also initiated a new genetic screen for identifying mutants in locomotory behaviors. The screen entailed the design of specialized chambers for picking out fish with mutant swimming behaviors from hundreds of fish at a time. With these screens, we have identified new proteins—or ascribed new functions to old proteins—that are culprits in human disorders, including myasthenia gravis, episodic infantile apnea, Brody disease, and certain myotonias.
Last updated: June 18, 2007
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