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Role of a regulator of the P13K - Akt pathway and two FOXO target genes in the developing nervous system and childhood brain tumours
Dr Jonathan Ham and Dr Thomas Jacques
Hypothesis: The aim of this PhD project is to study the role of the Pik3ip1, Mxi1 and Fbxo32 genes in the developing nervous system and in childhood brain tumours. We hypothesize that each gene regulates specific aspects of apoptosis (programmed cell death) in developing neurons. We also hypothesize that PIK3IP1, Mxi1 and Fbxo32 act as tumour suppressor proteins in the nervous system and that their expression is lost or reduced in paediatric brain tumours.
Aims and methods: The phosphoinositide 3-kinase (PI3K) - Akt signalling pathway has important functions during normal nervous system development (Ref. 1). However, hyperactivation of this pathway contributes to the development and maintenance of a variety of cancers including brain tumours (2). The binding of growth factors to tyrosine kinase receptors activates Class 1a PI3Ks, which in turn activate the protein kinase Akt (1,2). Akt promotes cell growth and inhibits apoptosis. It can inhibit cell death by phosphorylating and thereby inactivating the proapoptotic BH3-only protein Bad and the transcription factor FOXO3a (1,2)
Developing sympathetic neurons have been an important model for studying the mechanisms by which cell death and survival are regulated in the mammalian nervous system (1). These cells require nerve growth factor (NGF) for survival and die by apoptosis in its absence in a transcription-dependent manner. The binding of NGF to its receptor TrkA on the surface of sympathetic neurons promotes neuronal growth and survival and activates the PI3K-Akt pathway (1). When sympathetic neurons are deprived of NGF, PI3K and Akt activity rapidly decrease. This leads to an increase in the activity of FOXO3a, which induces the transcription of proapoptotic genes, such as bim, which encodes a BH3-only protein (3,4). In a recent Affymetrix exon array study with sympathetic neurons we identified many new genes induced after NGF withdrawal, which include Pik3ip1, Mxi1 and Fbxo32 (5). Pik3ip1 encodes an inhibitor of PI3K; Mxi1 codes for a transcriptional repressor and Fbxo32 an E3 ubiquitin ligase, and both genes are directly activated by FOXO transcription factors (5).
Aim 1: Investigation of the function of PIK3IP1, Mxi1 and Fbxo32 in sympathetic neurons
In immunoblotting and immunostaining experiments, the student will study the pattern of expression of the three proteins in sympathetic neurons dying by apoptosis after NGF withdrawal in vitro. The student will then determine the role of the three proteins in NGF withdrawal-induced death using the single cell microinjection technique or recombinant adenoviruses to overexpress and to knockdown each protein in sympathetic neurons (3,4). The student will also investigate the downstream mechanism of action of each protein.
Aim 2: Investigation of the pattern of expression of PIK3IP1, Mxi1 and Fbxo32 during normal nervous system development
The student will investigate the pattern of expression of PIK3IP1, Mxi1 and Fbxo32 in the normal cerebral cortex, cerebellum and superior cervical ganglia during postnatal development of the rat (P1 – adult), in particular during and after the periods of naturally occurring developmental neuronal death. Tissue sections will be prepared and immunohistochemistry performed using PIK3IP1, Mxi1 and Fbxo32 antibodies and an anti-active caspase-3 antibody to identify cells dying by apoptosis.
Aim 3: Characterization of the pattern of expression of PIK3IP1, Mxi1 and Fbxo32 in human brain tumour samples
To determine whether the expression of PIK3IP1, Mxi1 or Fbxo32 is reduced in specific types of childhood brain tumour the student will perform immunostaining of paraffin-embedded human brain tumour sections to detect the expression of the genes of interest at the protein level. Tissue arrays of more than 100 samples per tumour type will be constructed in the GOSH Histopathology Laboratory for the following types of paediatric brain tumour: medulloblastomas; ependymomas; pilocytic astrocytomas / low grade gliomas; glioblastomas; low grade glio-neuronal tumours.
1. Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Current Opinion in Neurobiology 10, 381-391.
2. Yuan, T.L. and Cantley, L.C. (2008) PI3K pathway alterations in cancer: variations on a theme. Oncogene 27, 5497-5510.
3. Whitfield, J., Neame, S.J., Paquet, L., Bernard, O. and Ham, J. (2001) Dominant negative c-Jun promotes neuronal survival by reducing Bim expression and inhibiting mitochondrial cytochrome c release. Neuron 29, 629-643.
4. Gilley, J., Coffer, P. and Ham, J. (2003) FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. Journal of Cell Biology 162, 613-622.
5. Kristiansen, M., Menghi, F., Hughes, R., Hubank, M. and Ham, J. (2011) Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death. BMC Genomics 12: 551.