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Scientific Report 2005


Neurobiology




Chairman’s Overview


Members of the Department of Neurobiology continue to focus their efforts on primary cellular processes of development, with emphasis on development of the nervous system in vertebrates. Our earlier work on cell adhesion molecules and the emergence of new technologies prompted us to examine the control of fundamental processes of gene expression in eukaryotic cells. Much of this effort has emphasized the regulation of translation of mRNA into protein, including basic mechanisms of translation and the specific regulation of translation at synapses that occur as the result of particular patterns of synaptic activity. We have also been studying the nature and differentiation of neural stem cells and the basic factors that regulate gene transcription. These various activities are connected by overlapping fundamental principles as well as by the application of common technologies.

Translation in eukaryotes is initiated via 2 mechanisms, cap dependent and internal ribosome entry site (IRES) dependent, which differ in how ribosomes are recruited to the mRNA. In the first mechanism, ribosomes are recruited at the cap structure, a modified nucleotide found at the 5′ ends of mRNAs. In the second mechanism, which has been the focus of many of our studies, ribosomes are recruited by IRES elements contained within the mRNA. Differential use of these 2 mechanisms appears to be important in processes such as synaptic plasticity in the brain.

Vince Mauro and his colleagues found that short mRNA sequences can function as IRESs and suggested that some of these sequences affect translation by base pairing to the RNA component of ribosomes, rRNA. Detailed analysis of one of these IRES elements showed that it does form base pairs with rRNA. This pairing was accomplished by altering either the mRNA or the rRNA sequences in a yeast experimental system. Both approaches showed that an intact complementary match was required for activity. These findings are the first conclusive evidence that base pairing is a prominent mechanism in eukaryotic translation. Dr. Mauro and his group have also developed a novel positive-feedback vector-selection system to identify IRES elements from libraries of random nucleotide sequences and have used these individual elements to generate powerful translational enhancers that have applications in biotechnology and gene therapy.

Consolidation of the mechanisms that underlie learning and memory involves an elaborate set of molecular mechanisms that alter the shape and function of the synapse. Some of these changes require protein synthesis, and recent work has revealed a new set of events that link the activity of the synapse to changes in synaptic strength. Apparently, granules containing mRNAs and parts of the translation machinery can be transported to the vicinity of dendritic spines. Synaptic activity can trigger local translation by the elements in the granules, providing specific synaptic proteins at sites of activity.

Peter Vanderklish, Bruce Cunningham, and their colleagues are analyzing factors that regulate local mRNA translation and are determining how the mechanisms that lead to changes in dendritic spines are altered in fragile X syndrome, the most common inherited form of mental retardation. In the mouse model for this syndrome, mice lack the gene for the fragile X mental retardation protein. In these mice, the spines that support the synapses are abnormally long and thin. This abnormality may arise from defects in the regulation of protein synthesis at the synapse. Fragile X mental retardation protein can suppress translation in dendrites, and Dr. Vanderklish and his group have now found that large granules that transport mRNAs and translation machinery to the synapse are reduced in mice that lack this protein.

Working with Dr. Mauro, Dr. Vanderklish also found that the RNA-binding protein RBM3 is present in a subset of dendritic granules and that overexpression of RBM3 can enhance protein synthesis as much as 3-fold. In seeking a mechanism for this increase, these researchers found that RBM3 is associated with the large ribosomal subunit. More critically, overexpression of RBM3 significantly reduced the amount of a microRNA. Because microRNA can influence protein synthesis, this alteration could be a major mechanism by which RBM3 regulates protein expression.

In addition to translational mechanisms, transcriptional control is a major factor in regulating gene expression. Homeodomain transcription factors are critical regulators of gene expression in development. In previous work, we focused our studies on cell adhesion molecules as targets for these transcription factors, which influence the ability of the adhesion molecules to act as regulators of morphogenesis. For more detailed mechanistic studies, Robyn Meech and her colleagues have focused on the homeodomain protein Barx2, which affects a number of developmental processes, particularly in muscle and cartilage development. Moreover, they found that Barx2 affects estrogen-dependent growth and responses to estrogen in breast cancer cell lines.

Overexpression of Barx2 causes the invasion of estrogen receptor–positive breast cancer cells into the extracellular matrix. In cells that lack estrogen receptors, Barx2 expression is lost. These findings and others suggest that coordinate expression of Barx2 and the estrogen receptor α protein occurs. In addition, Dr. Meech has found that Barx2 acts in concert with other factors critical for development, including the homeodomain protein Sox9, the muscle differentiation factor Myo D, and the estrogen receptor.

The vertebrate nervous system is derived from multipotent stem or progenitor cells in the neural tube that divide and differentiate into mature neurons and glial cells. Cell adhesion is a pivotal process in differentiation, and our previous studies indicated that the neural cell adhesion molecule promoted the formation of mature neuronal networks from cultures of neural progenitor cells. In recent studies, Kathryn Crossin and her colleagues have explored the role of energy metabolism in the differentiation of stem cells. Compared with progenitor cells, mature neurons expressed high levels of reactive oxygen species, primarily from mitochondrial metabolism. Dr. Crossin has shown that this property can be used with fluorescence-activated cell sorting to isolate highly enriched fractions of both multipotent stem cells and neurons. Understanding the role of reactive oxygen species in neuronal differentiation and maturation may provide new means of intervention in development, neurodegeneration, and aging.

The goal of all of these activities is to understand the molecular and cellular events that define and regulate the development of the nervous system. Our efforts have remained focused on the fundamental processes of morphogenesis and neuronal function as well as on a number of related diseases. This strategy is based on the belief that understanding even a single primary process of development can provide the necessary framework for defining key mechanisms that underlie a variety of diseases.

 

Gerald M. Edelman, M.D., Ph.D.
Chairman and Professor


Faculty