The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Fundamental Processes in Neural Development

G.M. Edelman, S. Aschrafi, A. Atkins, S. Chappell, J. Dresios, O. Harismendy, D.C.Y. Koh, G.W. Rogers, T. Stevens, M. Tsatmali, W. Zhou

We continue to focus on primary cellular processes and their influence on the development of the vertebrate nervous system. 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 during synaptic plasticity. Equally important have been studies of the nature and differentiation of neural stem cells and the basic factors that regulate gene transcription.

In examining the mRNAs expressed in response to neuronal adhesion, Vince Mauro noted that many of them had sequences that matched or were complementary to ribosomal RNA. He and his colleagues, Stephen Chappell, Wei Zhou, George Rogers, Jr., John Dresios, and Dora Koh, are working on several mRNAs to characterize a mechanism of translation enhancement that uses base pairing of mRNAs to ribosomal mRNA sequences. Dr. Zhou developed an important positive feedback vector to identify translational enhancers. In collaboration with Drs. Zhou and Chappell, Dr. Dresios used a related system in yeast to show definitively that base pairing was the underlying mechanism by which a 9-nucelotide sequence from the mouse Gtx homeodomain mRNA enhanced translation. This study was the first to confirm such a mechanism in eukaryotic translation, similar to the Shine-Dalgarno sequence in prokaryotes.

Drs. Rogers and Koh investigated a mechanism that regulates the translation efficiency of the BACE1 mRNA, which encodes the enzyme β-secretase. This enzyme is overexpressed in Alzheimer’s disease without a corresponding increase in BACE1 mRNA levels, indicating that the translation efficiency of this mRNA is increased. Earlier studies suggested that the translation of this mRNA is regulated by factors that affect the accessibility of the initiation codon. Dr. Koh is testing this notion by adapting a method that was used to probe RNA secondary structures in bacteria.

Translation of dendritic mRNAs is involved in many functions of the nervous system. For example, several forms of changes in efficacy occur in a brain region called the hippocampus, and translation of dendritically localized mRNAs is required for each change to persist beyond a few hours. Peter Vanderklish has been studying how translation is regulated by receptor-mediated signaling that occurs during the induction of changes in synaptic efficacy.

In collaborative studies, Bruce Cunningham, Armaz Aschrafi, Dr. Dresios, Ann Atkins, and Dr. Vanderklish have examined 2 RNA-binding proteins important in synaptic function: RNA-binding motif protein 3 (Rbm3) and the fragile X mental retardation protein (FMRP). The protein Rbm3 is associated with a subset of large granules containing specific proteins, mRNAs, and much of the translation machinery. Rbm3 binds ribosomes and alters microRNA levels to enhance translation globally. FMRP also occurs in large mRNA granules, where it acts as a translational suppressor. Loss of FMRP results in enhanced translation of granule mRNAs in response to metabotropic glutamate receptor signaling.

Transcription of genes into messenger RNAs is a highly regulated process that is essential for normal development and when altered can result in disease states. Transcription requires several factors that maintain the DNA in an appropriate conformation and activate the ability of DNA polymerase to synthesize mRNA. Robyn Meech and colleagues are studying 2 such transcription factors: Barx2 and the neural restrictive silencer factor (NRSF).

Barx2, discovered in our laboratory, is critical for the development of muscle and cartilage, and Tracy Stevens showed that it regulates the expression of the gene for estrogen receptor α in breast cancer cells. She showed that Barx2 regulates the expression of 2 estrogen receptor α mRNAs that differentially affect gene expression and cell growth. In addition, overexpression of Barx2 promoted anchorage-independent cell growth and induced cellular invasion. These mechanisms coordinately regulate cell growth, invasion, and gene expression that are critical in normal development of the mammary gland and in progression of breast cancers.

NRSF is a transcription factor that represses the expression of neuron-specific genes in nonneuronal cells. Oliver Harismendy is developing a novel chromatin immunoprecipitation-based technology for genome-wide analysis of transcription factor binding sites. This method, serial analysis of genomic sites, should increase the efficiency of the identification of active targets of transcription factors such as NRSF in various cells and phases of development. When used in conjunction with bioinformatics, the method can provide unique and essential information about the use of DNA-binding sites in particular contexts.

Understanding the factors that stimulate the conversion of stem cells to neurons and that sustain neuronal survival are critical issues for understanding neuronal development. Kathryn Crossin and her colleagues have shown that newborn neurons have higher levels of reactive oxygen species (ROS) than their progenitors do, a surprising finding because high levels of ROS are usually a reflection of dying cells. More recently, Marina Tsatmali pursued the hypothesis that elevated ROS levels play an important role in neuronal differentiation. She perturbed the expression of ROS and found that this manipulation dramatically alters neuronal morphology, although not neuronal fate. She also examined the physiologic properties of neurons in brain slices with high or low levels of ROS and showed that the cells with high levels of ROS have normal physiologic properties in situ.

The aim of all of these activities is the study of the molecular and cellular events that define and regulate the development of the nervous system. We are focusing on fundamental processes rather than on specific diseases. This strategy is based on the notion that understanding even a single primary process can provide the necessary framework for defining the mechanisms that underlie not just one but a variety of diseases.

Publications

Aschrafi, A., Cunningham, B.A., Edelman, G.M, Vanderklish, P.W. The fragile X mental retardation protein and group I metabotropic glutamate receptors regulate levels of mRNA granules in brain. Proc. Natl. Acad. Sci. U. S. A. 102:2180, 2005.

Atkins, A.R., Gallin, W.J., Owens, G.C., Edelman, G.M., Cunningham, B.A. Neural cell adhesion molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by intramolecular domain-domain interactions. J. Biol. Chem. 279:49633, 2004.

Dresios, J., Aschrafi, A., Owens, G.C., Vanderklish, P.W., Edelman, G.M., Mauro, V.P. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis. Proc. Natl. Acad. Sci. U. S. A. 102:1865, 2005.

Meech, R., Edelman, D.B., Jones, F.S., Makarenkova, H.P. The homeobox transcription factor Barx2 regulates chondrogenesis during limb development. Development 132:2135, 2005.

Tsatmali, M., Walcott, E.C., Crossin, K.L. Newborn neurons acquire high levels of reactive oxygen species and increased mitochondrial proteins upon differentiation from progenitors. Brain Res. 1040:137, 2005.

Vanderklish, P.W., Edelman, G.M. Differential translation and fragile X syndrome. Genes Brain Behav. 4:360, 2005.

Zhou, W., Edelman, G.M., Mauro, V.P. A positive feedback vector for identification of nucleotide sequences that enhance translation. Proc. Natl. Acad. Sci. U. S. A. 102:6273, 2005.