Molecular characterisation of EGFR in neural cells

Epidermal growth factor receptor (EGFR) mutant mice develop epithelial phenotypes and a neurodegenerative disease affecting astrocytes and neurons exclusively in the frontal cortex and olfactory bulbs. Besides controlling the normal development of epithelial cells and astrocytes, EGFR overexpression has been detected in many human carcinomas and glioblastomas, which are tumors of epithelial and glial origin respectively. Therefore, the study of the physiological signaling pathways of the EGFR is of fundamental importance for understanding how aberrant EGFR signaling can lead to tumor formation and progression. Within this grant proposal we would like to investigate the role of EGFR during neural cell development and characterize the molecular mechanisms leading to the development of the cortical degeneration observed in EGFR mutant mice. Currently it is not clear whether the massive cortical degeneration is caused by an intrinsic defect of the neurons, a defect of the astrocytes, which can not support the survival of neurons or a combination of both. Recent work from our laboratory shows that lack of EGFR affects specifically astrocytes isolated from the cortex, but not astrocytes isolated from the midbrain. Mutant cortical astrocytes are not able to support neuronal survival, whereas mutant midbrain astrocytes can keep neurons alive in an in vitro co-culture system. These results suggest that cortical astrocytes do not produce sufficient survival signals for neurons and provide an explanation why in the absence of EGFR the neurodegeneration occurs mainly in the cortex. To verify this hypothesis in vivo, we plan to characterize the phenotype of mice harbouring conditional deletions of the EGFR in different neural cells. In parallel, we will perform rescue experiments by generating transgenic mice expressing the EGFR in a Cre- inducible manner in astrocytes, neurons and neural precursor of EGFR deficient mice. Primary cells isolated from these mutant mice will be characterized biochemically to elucidate the molecular mechanisms, which lead to the phenotypes observed in vivo.

Epidermal growth factor receptor (EGFR) mutant mice develop epithelial phenotypes and a neurodegenerative disease affecting astrocytes and neurons exclusively in the frontal cortex and olfactory bulbs. The goal of this research proposal was to characterize the cellular and molecular mechanisms responsible for the cortical degeneration observed in EGFR mutant mice. Work from our laboratory has shown that lack of EGFR affects specifically astrocytes isolated from the cortex, but not astrocytes isolated from the midbrain. Mutant cortical astrocytes are not able to support neuronal survival, whereas mutant midbrain astrocytes can keep neurons alive in an in vitro co-culture system. These results suggest that cortical astrocytes do not provide sufficient survival signals for neurons and provide an explanation why in the absence of EGFR the neurodegeneration occurs mainly in the cortex. To investigate the contribution of astrocytes, neurons and oligodendrocytes to the development of the neurodegeneration in vivo, we employed mice carrying conditional EGFR alleles (EGFR f/f ) which were bred to GFAP-Cre and Nes-Cre transgenic mice to delete the EGFR only in astrocytes (EGFR astro ) or in all neural cells (EGFR np), respectively. EGFRastro and EGFRnp mice are viable but smaller than their control littermates. Similar to EGFR knock-out mice ectopic neurons could be found in the hippocampus of both EGFRastro and EGFRnp mice. Surprisingly only a small percentage of EGFRnp mice developed the cortical neurodegeneration observed in EGFR knock-out mice and the majority of mice remained disease-free. We could show that the absence of the neurodegeneration can most likely be explained by the stability of the EGFR protein, which was still present in the brain of EGFRnp mice at 1 months of age, despite the complete Cre-mediated deletion of the EGFR at the genomic level. Complete absence of the EGFR protein could be detected only 3 months after genomic EGFR deletion suggesting that the EGFR is a very stable protein in vivo under physiological ligand concentration. Indeed, we could show in primary astrocytes from EGFRnp mice that at low ligand concentration the EGFR is recycled to the cell surface whereas at high EGF concentrations the EGFR is degraded. This explains why the majority of EGFRastro and EGFRnp mice do not develop the neurodegeneration. To investigate if EGFR is required in adult brain, we injured the brains by applying stab wounds to EGFRastro and EGFRnp brains and could show that adult wound healing is normal in the absence of EGFR. Moreover, EGFRastro and EGFRnp brains did not show increased susceptibility to Prion-induced neurodegeneration. Interestingly, we found that mice lacking EGFR in the brain are more susceptible to kainate-induced seizures. We discovered that glutamate transporters are expressed at reduced levels in EGFR mutant brains and astrocytes. These findings may provide a molecular explanation for the increased susceptibility to kainate as it is known that glutamate transporters are required to quench the excess glutamate released after kainate receptor stimulation.