Michael Brenner, Ph.D.
Primary Department Affiliation: Neurobiology
Primary Research Area: Neurodevelopment and Developmental Disabilities
Neurodegeneration and Neurodegenerative Disorders
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.
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), fluorescent double-label immunocytochemistry, protein purification and mass spectrometry.
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), and to identify cis-acting regions responsible for its cell-specific expression (3). 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 (4).
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 (5). However, under certain circumstances this promoter is not completely astrocyte specific, but may express in neurons as well (6). 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, and astrocyte specificity is compromised (7). These results reveal an unexpected regional heterogeneity among astrocytes, and suggest that astrocytes in different areas of the brain use different regulatory regions of the GFAP gene. Reinsertion into this truncated promoter of regions that had been removed are localizing the DNA elements required for astrocyte and regional specificity (8).
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 (11-13), establishing Alexander disease as the first genetic disorder of astrocytes. 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 three hundred other laboratories for the GFAP promoter for use in analyzing astrocyte function, producing disease models, and gene therapy.
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.
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., 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Lee, Y., Su, M., Messing, A. and Brenner, M. (2006). Astrocyte heterogeneity revealed by expression of a GFAP-lacZ transgene. Glia 53:677-687.
8. Lee, Y., Su, M., Messing, A., Su, M. and Brenner, M. (2008) GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56: 481-493.
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 associated with Alexander disease. Nature Genetics (in press).
12. Brenner, M., Goldman, J.E., Quinlan, R.A. and Messing, A. (2009). Alexander disease: a genetic disorder of astrocytes. In Parpura, V. and Haydon, P., Astrocytes in (patho)physiology of the nervous system, Springer, pp. 591-648.
13. Messing, A., Li, R., Naidu, S., Taylor, P., Silverman, L., Flint, D., van der Knaap, M., and Brenner, M. Archetypal and new families with Alexander disease and novel mutations in GFAP. Arch. Neurol., in press.
Michael Brenner received his Ph.D. in Biochemistry from the University of California, Berkeley. He served on the faculty of Harvard College and Temple University Medical School, and was a Research Scientist at the National Institutes of Health before joining UAB in 1998. He is presently Professor of Neurobiology with a joint appointment in the Department of Physical Medicine & Rehabilitation.