Inhibition of a novel cell death pathway to reduce oxygen deprivation-induced neural damage

Supervisors: Dr Patrizia Ferretti and Professor Charles Marson

Hypothesis: We have evidence that activation of PAD3, a calcium-dependent enzyme that modifies protein structure and function by reducing its positive charges through a process called citrullination, is associated with early neural injury response and extensive tissue damage [1, 2]. We have also preliminary evidence that citrullination increases following hypoxia/ischaemia in an animal model [2]. Therefore we wish to test the following hypotheses:

  • PAD3 activation is involved in oxygen and glucose deprivation (OGD) expression of and human neural cell death
  • hypoxia-induced cell death can be antagonized by blocking PAD3 expression/activity in a human in vitro model and in a perinatal in vivo chick model
  • the new generation PAD inhibitor recently synthesized and partially characterized by the applicants [3] will be more specific and effective in reducing neural damage than a general PAD inhibitor currently available

Aims and methods: The aim of this study is to investigate further the role of PAD(s) in the damaged nervous system, and test the hypothesis that targeting PAD enzymes may have a broad therapeutic potential in neural pathologies. To this purpose, we will characterize a novel putative PAD inhibitor recently synthesized by the co-applicant using animal (perinatal hypoxia and traumatic injury) and human (in vitro neural cell hypoxia/ischaemia) models, and develop new more specific inhibitors that could be of therapeutic value. The study will increase our understanding of mechanisms underlying neural damage and will contribute to the development of novel drugs that will reduce it. A reduction in neural tissue damage will be pivotal to improving functional recovery in children who have suffered mechanical or hypoxic damage.

The student will use a broad range of techniques to investigate PAD role(s) in in vitro and in vivo neural injury models:

1. In vitro human model: Human neural cell lines we have recently generated, primary human embryonic neural cultures and a human neuroblastoma cell line will be used to study PAD3 role in response to hypoxic damage and the effect of a new PAD inhibitor on cell death and PAD activity. The student will develop a 3 dimensional in vitro model of human hypoxia/ischaemia reperfusion based on the well-established oxygen and glucose deprivation (OGD) method where reoxygenation after hypoxia results in increased intracellular Ca++ and cell death [4, 5].
2. In vivo animal model: The chick perinatal hypoxia model and the spinal cord crush injury model [1, 3] will be used to test in vivo the ability of the new PAD inhibitor and related compounds to inhibit citrullination and cell death and reduce cavity size (indicator of the extent of tissue damage), as compared to the currently available inhibitor and to inhibition of PAD3 using siRNA.

Altogether, the student will gain experience in several areas, such as cell culture, neural stem cell biology, experimental embryology, microsurgery, protein and RNA analysis (e.g. immunocytochemistry, q-PCR), enzymatic assays, manipulation of gene expression (siRNA), detection of apoptotic cells, imaging techniques (fluorescence, confocal and time-lapse microscopy). The student will also receive some training in chemical synthesis and drug development

References:
1) Lange, S., Gogel, S., Leung, K.Y., Vernay, B., Nicholas, A.P., Causey, C.P., Thompson, P.R., Greene, N.D., Ferretti, P., 2011. Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability. Dev Biol 355, 205-214.
2) Lange, Ferretti, P., Lange, S., U, K. P., Raivich, G., Deimination in the developing CNS - role in its response to traumatic and hypoxic injury. In: A. P. Nicholas, S. Bhattacharya, Eds.), Protein Deimination in Human Health and Disease, Vol. In press. Springer, New York, 2013. In press.
3) Ferretti, P., U, K. P., Vagaska, B., Matthew, C., Merchant, R., Marson, C., 2013b. Discovery of a potent, synthetic, drug-like and reversible inhibitor of peptidylarginine deiminase Med Chem Comm Accepted pending minor revision.
4) Bai, Y., Meng, Z., Cui, M., Zhang, X., Chen, F., Xiao, J., Shen, L., Zhang, Y., 2009. An Ang1-Tie2-PI3K axis in neural progenitor cells initiates survival responses against oxygen and glucose deprivation. Neuroscience 160, 371-381.
5) Liu, Y., Kintner, D.B., Begum, G., Algharabli, J., Cengiz, P., Shull, G.E., Liu, X.J., Sun, D., 2010. Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation. J Neurochem 114, 1436-1446.