Coordinate regulation of a protective phenotype in astrocytes

During ageing astrocytes become 'reactive', displaying a characteristic series of phenotypic changes. The reactive phenotype, usually defined by a thickenening of intermediate filaments immunoreactive for glial fibrillary acidic protein (GFAP) and a change of shape is part of a complex, and incompletely described response termed gliosis. While some neuroprotective pathways may be induced, there is also a loss of some important functions including glutamate uptake. The interplay between the different intracellular pathways in astrocytes that determine induction of protective pathways and induction of shape changes are largely unknown. It may be possible to drive protective pathways, without an accompanying gliosis. The PhD student will use molecular and pharmacological strategies to identify the pathways which are able to drive astrocytes into a 'protective' phenotype (which we define here biochemically by glutamate uptake, glutathione release, BDNF and GDNF release), and define the relationship between those changes and 'reactivity' (shape, GFAP). A multitude of studies, including some from our own laboratories, have identified a number of pathways important in co-ordinating protective functions of astrocytes. For this project, the student will focus on four key pathways which signal to a transcriptional response and have been implicated strongly in the reactive phenotype, and induction of beneficial functions of astrocyte function, namely Calcineurin/NFAT [1], SHH/Gli1 [2], antioxidants/ARE [3] and TNF/kappaB [4]. The student will use mouse primary astrocytes, but also stem-cell derived mouse astrocytes [5] for the in vitro studies. The student will transfect cells with luciferase reporter constructs to measure, in 96 well plate format, NFAT, ARE, Gli1 and kB pathway activities. First the student will use a variety of stimuli that we, or others, have used in our laboratories that mimic biochemical aspects of the ageing process in vitro, namely oxygen-glucose deprivation, oxidative stress and growth factor deprivation (serum withdrawal). The student will measure the effect of these stimuli on luciferase activity then, having established suitable parameters he/she will probe the interdependence of these pathways using a combination of knockout technologies (e.g. siRNAs against the transcription factors) and pharmacological agents to interfere with the pathways leading to transcriptional activation. This work will establish the hierarchy and interactions of these four pathways, and the key intracellular pathways involved in their regulation. We anticipate to focus on the role of p38MAPK-dependent pathways, and Rho GTPase dependent processes to co-ordinate responses, but will also identify whether other protein kinases and pathways which are of interest as drug targets for treatment of age-related diseases are involved. For this part of the project, we anticipate that the student will benefit from the use of compound libraries and screening strategies that will be carried out during placement in the Pfizer Neuroscience Research Unit Laboratories in Groton, CT. Finally, the relationship between the key signalling events, and phenotype will be carried out using a range of techniques including immunocytochemistry and ELISA to monitor changes in GFAP levels, biochemical assays to measure glutathione release, cell-based ELISA to monitor glutamate transporter levels, and ELISA to meaure GDNF and BDNF synthesis and release. In particular we hope to have dissected the pathways sufficiently to find agents which can drive increases in GDNF, BDNF, glutamate uptake and glutathione without altering the cytoskeleton or cell shape. 1. Norris CM, et al 2005 J Neurosci. 25:4649-58.. 2. Atkinson PJ, et al. 2009 J Neurochem. 108:1539-49 3. Bahia PK et al 2008. J Neurochem. 106:2194-204. 4. Dvoriantchikova G et al. 2009 Eur J Neurosci. 30:175-85. 5. Conti L et al. 2005 PLoS Biol. 3:e283.