Neuronal Cell Death Post Trauma
Preventing neuronal cell death following brain trauma
Chief Investigator: Professor Seong-Seng Tan
Lead Organisation: Howard Florey Institute
TAC Neurotrauma Funding: $1,662,375
Project Dates: 1 November 2006 – 16 November 2009
Background:
Traumatic brain injury is a serious medical and social problem worldwide, with the highest number of sufferers falling in the younger and middle age groups. There is at present no cure available, and once initiated, the process of neuron death occurs inexorably until neurons in the traumatised region and surrounding brain areas are reduced in number. The consequences are permanent mental and physical disability, and in extreme cases, fatal. We have discovered that the brain harbours endogenous mechanisms for protecting neurons from death following injury. One of these consists of over-expression of Ndfip1, a protein that is normally present at low levels, but is massively upregulated following trauma. Our research is predicated on the strategy of increasing Ndfip1 expression in all vulnerable neurons in the traumatised region, thereby protecting these neurons from death during the critical period of stress (hours to days).
Methods:
Ndfip1 was expressed in cultured cells to allow sufficient protein to be extracted and purified. This was used to immunize rodents and rabbits to generate antibodies (polyclonal and monoclonal) for further study. Ndfip1 was over-expressed in cultured neurons from cell lines and from embryonic mouse and human embryos (from medical termination of pregnancies) to test its ability to protect neurons from stressful stimuli. To discover the targets of Ndfip1, proteins that bind to Ndfip1 were screened and sequenced. Validation of binding was carried out using co-immunoprecipitation followed by protein sequencing. To analyse the ability of Ndfip1 to protect neurons from metal poisoning, flow cytometry was used to measure the proportions of surviving versus dying neurons. To create mouse models for studying Ndfip1 protection, we removed the Ndfip1 gene by homologous recombination in embryonic stem cells, followed by creation of mouse chimeras that resulted in mice lacking Ndfip1.
Results:
We have demonstrated beyond any doubt that Ndfip1 is a valid intrinsic mechanism present in the brain for protecting neurons from death following injury. Normally, Ndfip1 is only protective in a small number of neurons following injury, due to an unknown reason. However, since all neurons express low levels of Ndfip1, they are in principle capable of upscaling Ndfip1 to protective levels during stress. We confirmed this using cell culture experiments, using neurons obtained from mouse embryo and human embryo brains. We also identified the targets of Ndfip1 binding, these include DMT1, a metal transporter that normally allows the passage of metal ions into neurons. According to this scenario, Ndfip1 during stress restricts metal entry by binding to DMT1, followed by its degradation. We also discovered a second mechanism that might account for neuron protection, namely that Ndfip1 is carried is small vesicles known as exosomes, and during injury, more exosomes are carried out and export harmful proteins.
Conclusions:
We have identified Ndfip1 as a robust intrinsic mechanism present in a minority of neurons to protect it against death during injury. Our experiments have shown that it is possible to amplify this response, extending the protection to a greater number of neurons following stress in vitro. We have identified a number of mechanisms that oversee this protection, and future strategies will be aimed at discovering drugs that can increase Ndfip1 in neurons, raising the protective mechanisms.
Publications:
SANG Q, KIM M, KUMAR S, BYE N, MORGANTI-KOSSMAN MC, GUNNERSEN J, FULLER S, HOWITT J, HYDE L, BEISSBARTH T, SCOTT HS, SILKE J, and TAN S-S. Nedd4-WW domain-binding protein 5 (Ndfip1) is associated with neuronal survival following acute cortical brain injury. J. Neurosci. 2006;26:7234-7244
GUNNERSEN JM, KIM MH, FULLER SJ, DE SILVA M, BRITTO JM, HAMMOND VE, DAVIES PJ, PETROU S, FABER ES, SAH P, TAN S-S. Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons. Neuron. 2007;56(4):621-39
CHIU C, REID CA, TAN HO, DAVIES PJ, SINGLE FN, KOUKOULAS I, BERKOVIV SF, TAN S-S, SPRENGEL R, JONES MV, PETROU S. Developmental impact of a familial GABAA receptor epilepsy mutation. Annals Neurol. 2008;64:284-293
FOOT NJ, DALTON HE, SHEARWIN-WHYATT L, DORSTYN L, TAN S-S, YANG B, and KUMAR S. Regulation of the divalent metal ion transporter DMT1 and iron homeostatis by a ubiquitin-dependent mechanism involving Ndfips and WWP2. Blood 2008;112:4268-4275
PUTZ U, HOWITT J, LACKOVIC J, FOOT N, KUMAR S, SILKE J, and TAN S-S. Nedd4-family interacting protein 1 (Ndfip1) is required for the exosomal secretion of Nedd4-family proteins. J. Biol. Chem. 2008;283:32621-32627
TAN S-S, TRUONG H, BINDER M, CATE H, KILPATRICK T, KALLONIATIS M, HAMMOND V. Oligodendrocyte positioning in cerebral cortex is independent of projection neuron layering. Glia. 2008;57:1024-1030
HOWITT J, PUTZ U, LACKOVIC J, DOAN A, DORSTYN L, CHENG H, YANG B, CHAN-LING T, SILKE J, KUMAR S, and TAN, S-S. Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons. Proc. Natl. Acad. Sci. USA. 2009;106:15489-15494
Presentations:
TAN S-S. Molecular mechanisms of neuroprotection by Ndfip1 following brain Injury. 31st Annual Meeting of the Japan Neuroscience Society; 2008 July, Tokyo, Japan.
TAN S-S. Ndfip1 neuroprotection following brain injury. Australian Neuroscience Society Annual Meeting; 2008 January 27-30, Hobart, Australia.
TAN S-S. Traumatic Brain Injury: Understanding the mechanisms of neuroprotection before finding a cure. Federation of Asian and Oceanic Neurosciences (FAONS) Meeting; 2009 May 17-19, Bangkok, Thailand
Additional Grants:
NHMRC Project Grant App ID: 566621, 2009 – 2011
Chief Investigators Seong-Seng Tan, Jason Howitt and Ulrich Putz
Title: Neuroprotection by Ndfip1 in brain injury – identifying targets and understanding mechanisms.
$271,000 (2009); $266,000 (2010); $266,000 (2011)