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RESEARCH PROGRAM -
Lab of Dr. Michael Brenner Background Current
studies of the CNS are assigning an increasing number of activities to
astrocytes. Such activities now include contributing to the development and
maintenance of neurons and oligodendrocytes, establishment of the blood-brain
barrier, recycling of glutamate, potassium homeostasis, and modifying neuronal
activity. Recent findings that astrocytes produce and/or have receptors for a
large array of neurotransmitters, neuropeptides, cytokines, and growth factors
have further stimulated speculation concerning the roles of astrocytes. Nearly all
of the suggested activities for astrocytes are based on observed correlations,
and many of these have been made on cultured cells, whose properties may differ
from those in vivo. As an alternative approach to understanding astrocyte
function, our group is studying their cell-specific transcription and the role
of the GFAP protein. GFAP was selected for study because its gene is expressed
fairly strongly, and almost exclusively, in astrocytes. Expression of the gene
is also turned on about the same time as astrocytes mature, and its activity
increases dramatically following almost any CNS injury. Thus, study of GFAP
transcription should yield insights into mechanisms governing development,
reaction to injury, and cell specificity. The interesting regulation of the GFAP
gene, and the fact that astrocytes have elaborated their own specific
intermediate filament protein, predict an important role for GFAP in these
cells. We have discovered two such roles for the protein.
We have found that the absence of
GFAP renders mice hypersensitive to traumatic spinal cord injury, revealing a
novel role for GFAP in structural support. We have also discovered that
mutations within the coding sequence of the GFAP gene are responsible for many
cases of Alexander disease, a rare but often fatal neurodegenerative disorder of
humans. Methods A wide
repertoire of molecular biological techniques is used in our studies. These
include screening of gene libraries, subcloning, DNA sequencing, Southern,
northern and western blotting, synthesis of reporter genes and transgenes,
site-directed mutagenesis, in vitro transcription and translation, primer
extension, riboprobe protection, culture of cell lines and primary cells,
transient and stable transfections, DNA footprinting, gel mobility shift assays,
polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR), and
fluorescent double-label immunocytochemistry. Summary
of Results We have
isolated cDNA and genomic clones for the human GFAP gene, and used these to
determine the GFAP mRNA and protein start sites (1), to analyze the structure of
its basal promoter (2,3), and to identify cis-acting regions responsible for its
cell-specific expression (4). In these latter studies cell transfection
experiments showed that a 2 kb 5'-flanking segment of the GFAP gene produced
astrocyte-specific expression of a linked reporter gene. Deletion analyses then
identified two subregions that were necessary and sufficient for this activity
in transfected cells. One of these regions is located about 100 bp upstream of
the RNA start site, while the other is located about 1500 bp upstream. A 124 bp
subsection of the upstream region was found to be particularly important for
transcriptional activity. Site-directed mutagenesis of contiguous blocks
throughout this region revealed the presence of multiple sites that contribute
to transcriptional activity (5). Subsequent
analyses have been performed almost exclusively in transgenic mice, which
produce more reliable results than cell transfection.
An initial study demonstrated that the 2 kb 5'-flanking segment of the
human GFAP gene directs expression of a b-galactosidase
(lacZ) reporter gene in astrocytes throughout the brain of transgenic mice, and
also shows the upregulation of GFAP expression following injury that is typical
of reactive gliosis (6). However, under certain circumstances this promoter is
not completely astrocyte specific, but may express in neurons as well (7).
Interestingly, we found that when a promoter composed of the two critical
subregions of this segment is used, expression is largely limited to the cortex
and hippocampus (8). This result reveals an unexpected regional heterogeneity
among astrocytes, and suggests that astrocytes in different areas of the brain
use different regulatory regions of the GFAP gene To
investigate the function of the GFAP protein, we have analyzed both mice that
have had their GFAP gene disrupted, and mice that over-express the protein. The
GFAP null mice are hypersensitive to traumatic spinal cord injury (9), revealing
a previously unrecognized role of GFAP and astrocytes in providing mechanical
support to the spinal cord. Mice that strongly express the human GFAP gene die
young, and display hypertrophic astrocytes containing Rosenthal fibers—protein
inclusion bodies that are associated with a number of human neurological
diseases, and are the hallmark of Alexander disease (10). Prompted by this
latter observation, we tested whether Alexander disease might be caused by
mutations in the GFAP gene. Sequencing of DNA obtained from Alexander disease
patients has indeed shown that coding mutations are associated with many cases
of the disease (11,12). These findings may provide important insights into other
protein aggregate diseases, such as amyotrophic lateral sclerosis
(Lou Gehrig’s disease), Parkinson’s disease and various muscle and liver
diseases. Finally, in addition to our own studies, we have filled requests from
over one hundred other laboratories for the GFAP promoter for use in analyzing
astrocyte function, producing disease models, and gene therapy. Continuing
Research Analysis
of GFAP transcription remains a central task of the laboratory. We are pursuing
the factors responsible for the restriction of expression to astrocytes, that
control brain region-dependent expression, and that mediate the increased
synthesis of GFAP following injury. We have narrowed our search for DNA
sequences that contribute to astrocyte-specificity and to the response to injury
to segments as small as 50 bp, and are now working to identify the precise
sequences required. We
will then use this information to isolate and characterize the mediating
transcription factors to parse out the signaling pathways involved. We are also
working to increase the utility of the GFAP expression system by developing
cassettes that are more dependably astrocyte-specific, direct expression to
particular subregions of the brain, are more compact and have higher expression
levels. In continuing studies of Alexander disease we are
investigating the mechanism by which GFAP coding mutations produce the disorder.
Mutation-specific antibodies are being developed to permit comparison of
the properties of the mutant and wild type proteins in both mouse models and
human patients.
Proteomics/mass spec is being used to identify the proteins present in
the aggregates, with the expectation that this will provide clues to their
formation and to their biological effects. References Cited 1. Brenner, M., Lampel, K., Nakatani, Y., Mill, J., Banner,
C., Mearow, K., Dohadwala, M., Lipsky, R., and Freese, E. (1990)
Characterization of human cDNA and genomic clones for glial fibrillary acidic
protein. Mol. Brain Res. 7: 277-286. 2. Nakatani, Y., Brenner, M., and Freese, E. (1990) An RNA
polymerase II promoter containing sequences upstream and downstream from the RNA
startpoint that direct initiation of transcription from the same site. Proc.
Natl. Acad. Sci. USA 87: 4289-4293. 3. Nakatani, Y., Horikoshi, M., Brenner, M., Yamamoto, T.,
Besnard, F., Roeder, R.G., and Freese, E. (1990) A downstream initiator is
essential for efficient binding of TFIID to the TATA box. Nature 348:
86-88. 4. Besnard, F., Brenner, M., Nakatani, Y., Chao, R., Purohit,
H.J., and Freese, E. (1991) Multiple interacting sites regulate astrocyte-specific
transcription of the human gene for glial fibrillary acidic protein. J. Biol.
Chem. 266: 18877-18883. 5. Masood, K., Besnard, F., Su, Y., and Brenner, M. (1993)
Analysis of a segment of the human GFAP gene that directs astrocyte-specific
transcription. J. Neurochem. 61: 160-166. 6. Brenner, M., Kisseberth, W.C., Su, Y., Besnard, F., and Messing, A. (1994) GFAP promoter directs astrocyte-specific expression in transgenic mice. J. Neurosci. 14, 1030-1037. 7.
Su, M., Hu, H., Lee, Y.,
d'Azzo, A., Messing, A. and Brenner, M. (2004).
Expression specificity of GFAP transgenes.
Neurochem. Res. 29:2075-2093. 8.
Lee, Y., Su, M.,
Messing, A. and Brenner, M. (2006). Brain-region
dependent expression of a GFAP regulatory sequence in transgenic mice.
Glia (in press). 9. Nawashiro, H., Messing, A., Azzam, N., and Brenner, M.
(1998) Mice lacking GFAP are hypersensitive to traumatic cerebrospinal injury.
NeuroReport 9: 1691-1696. 10. Messing, M., Galbreath, E.J., Head, M.W., Goldman, J. E., and
Brenner, M. (1998) Fatal encephalopathy with astrocyte inclusions in GFAP
transgenic mice. Amer. J. Pathol. 152: 391-398. 11. Brenner, M., Johnson, A.B., Boespflug-Tanguy, O., Rodriguez, D.,
Goldman, J.E. and Messing, A. (2001). Mutations
in GFAP, encoding glial fibrillary
acidic protein, are associated with Alexander disease. Nature Genetics 27:
117-120. 12. Li, R., Johnson, A.B., Salomons, G.S., Goldman, J.E., Naidu, S., Quinlan, R., Cree, B., Ruyle, S.Z., Banwell, B., D’Hooghe, M., Siebert, J.R., Rolf, C.M., Cox, H., Reddy, A., Gutiérrez-Solana, L.G., Collins, A., Weller, R.O., Jakobs, C., Messing, A., Van der Knaap, M.S., and Brenner, M. (2005). GFAP mutations in infantile, juvenile and adult forms of Alexander disease. Annals Neurol. 57(3):310-326. (For a complete list of publications, see Curriculum Vitae.) |
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