Proton sensing G protein coupled receptors and TRPC channels - novel players in neuronal proliferation and development

In order for our human body to function properly, it has to offer a stable environment to its building blocks, the cells. Cells work best in a tightly controlled environment in which factors such as nutrient supply and pH are kept more or less constant. Any change in environmental settings may have detrimental effects on cell functioning and can even cause cell death. One of the parameters that is kept constant is the extracellular proton concentration that determines the tissue pH. A too low (acidic) extracellular pH (equivalent to a high extracellular proton concentration) has been shown to cause cell death. One such example for acidosis-induced cell death is cell loss following a stroke (ischemia). Surprisingly, however, under certain conditions a low external pH promotes rather than compromises cell survival. This astonishing observation has been made in cancerous tissue: here, extracellular acidosis is not only tolerated by cancer cells, but it is essential for cancer progression and formation of metastases. An equally intriguing finding is that the detrimental effects of ischemia (in large part due to acidosis of the ischemic tissue) is less dramatic and more readily reversible in younger compared with old patients. All this suggests that the ability of cells to respond to a low extracellular pH with enhanced survival rather than death may be cell-type specific and can to some extent depend on the developmental stage of the tissue involved. Cancers mimic early developmental stages of tissues in that both cancer cells and developing cells continually multiply to build up tissue mass. We have recently made the important observation that developing brain cells, just like cancer cells, respond to external acidification with increased cell survival rather than cell death. This surprise finding has led us to propose the following project: to investigate how a fall in extracellular pH promotes survival of normal, developing brain cells. This study is the first to address the effects of extracellular acidosis on developing tissue and will open up an entirely new field of research. Understanding the cellular mechanism triggered by a fall in external pH that induce cell survival in normal cells will provide important insights into how cancerous cells may take advantage of these pathways and use them to enhance survival of transformed as opposed to unftransformed cells. Ultimately, it may provide us with novel therapeutic strategies to combat debilitating diseases such as cancer, which affect 1 in 3 people.

The main objective of this study is to investigate how a fall in extracellular pH can promote cell survival of granule precursor cells, which are neuronal precursor cells of the cerebellum, through activation of the proton sensing G protein coupled receptor OGR1 and canonical Transient Receptor Potential (TRPC) channels. Key methods to be employed throughout the project are the patch-clamp technique (whole cell voltage clamp recordings), fluorescence Ca2+ imaging and cell survival and proliferation assays. 1) We will establish that activation of the proton sensing G protein coupled receptor OGR1 is crucial for the acidosis-mediated enhanced survival of granule precursor cells using wildtype and OGR1 knock-out mice as well as specific (ant)agonists of the OGR1 receptor in primary mouse granule cell cultures and brain slices of the cerebellum (cells in their native environment). 2) We will investigate the physiological consequences of OGR1 activation. We have previously shown that OGR1 can link to a family of receptor operated channels, the TRPC channels. We will determine which TRPC subunits are activated in granule precursor cells following stimulation of OGR1 by protons using RNA knock-down approaches. We will also study how knock-down of these subunits interferes with acidosis-mediated enhanced granule cell survival. 3) Finally, we will try to identify genes expressed in response to a fall in extracellular pH using quantitative PCR approaches. The effect of TRPC knock-down on gene expression will be investigated to assess a contribution of TRPC channels to gene expression. Taken together, these experiments will provide the first mechanistic explanation as to how extracellular acidification can promote cell survival and will have important implications for our understanding of normal precursor cell proliferation as well as tumour progression in an acidic environment.

Extracellular acidosis is generally thought to induce cell death. However, in solid cancers acidosis of the interstitial fluid promotes cancer progression and metastasis. We now find that the ability of cells to respond to external acidosis with enhanced cell survival is not limited to transformed cells but can also be observed in normal proliferating precursor cells during development, which may point towards a common mechanism used by proliferating cells to respond to increases in external proton concentrations with cell survival rather than cell death. Our hypothesis that extracellular acidosis promotes survival of proliferating neuronal precursor cells through activation of G protein coupled proton sensing receptors and subsequent activation of TRPC channels is a first step to identify signalling cascades involved in proton-mediated cell survival, and our research may have wide impact both for researchers as well as the wider community. 1. Who will benefit from this research? a) Our research will be of immediate interest to people working in the fields of proton sensing G protein coupled receptors, TRPC channels, neuronal development and cancer research. b) Furthermore, this proposal has the potential to indirectly benefit people in the wider public, in particular cancer sufferers. One in three people will at some stage of their life be affected by cancer, and radiation therapy (despite all its detrimental side-effects, particularly in younger patients) is still one of the methods of choice for treating this devastating disease. Finding alternative treatments that target as many different cancers as possible is therefore of huge general interest. If we understand how normal precursor cells, which resemble transformed cells in that they proliferate, can respond to external acidosis with enhanced cell survival, then this may open up novel therapeutic strategies for targeting the acidosis-mediated progression of solid cancers. 2. How will they benefit from this research? a) Findings resulting from this research proposal will significantly enhance our understanding of proton-mediated signalling cascades and will therefore open up new pathways of investigation for other researchers interested in any of the scientific fields mentioned above. b) We collaborate with Dr. Klaus Seuwen from Novartis, and it would be an obvious next step to look into the development of specific drugs targeting proton-mediated signalling pathways. The ultimate aim would be to develop drugs that can be used to suppress acidosis-mediated signalling in cancer tissue, thereby leaving transformed cells vulnerable to the detrimental effects of external acidosis. Hence, in the long term our research may benefit cancer sufferers because it may lead to the identification of novel targets for cancer treatment. 3. What will be done to ensure that they benefit from this research? All our findings will be published in peer-reviewed journals and hence be in the public domain. Other researchers will likely pick up on our ideas and get involved. Hence, the general knowledge of proton-induced survival pathways will increase, and this will increase the likelihood of pharmaceutical companies being interested in the research and trying to develop drugs targeting different aspects of the signalling cascades.