Scientific Report 2005
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 receptorpositive
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.