Dr Julia Gerasimenko
My primary research interest is directed but not limited to study of mechanisms and causes of acute pancreatitis. 80% cases of acute pancreatitis are associated with either bile reflux or alcohol abuse. The toxic effects of alcohol are principally mediated by fatty acid ethyl esters (FAEE), which are produced in the pancreas when oxygen levels in the organ are low. FAEEs induce an excessive increase in the calcium ion concentration inside pancreatic acinar cells, which in turn leads to premature activation of digestive enzymes. One of the hallmark responses of acute pancreatitis is a premature, intracellular activation of trypsinogen and its conversion from zymogen to active trypsin. The main result is massive necrotic cell death and inflammation of the pancreas.
The plan of my research is based on two recent key findings:
[A] Physiological Ca2+-liberating internal messengers release Ca2+ not only from the endoplasmic reticulum, but also from acid pools in the zymogen granule area.
Conclusions from two-photon permeabilized cell experiments measuring calcium store (Gerasimenko et al J Cell Sci 2006 33, Faculty of 1000 Biology Section: (http://www.f1000biology.com/article/16410548)
[B] Bile acids, alcohol and alcohol metabolites release calcium from the endoplasmic reticulum and the acid pools. We shall investigate the specific mechanisms underlying toxic Ca2+ release from the various pools as well as the subsequent vacuole formation and pathological digestive enzyme activation that causes the disease. Specific interventions in the pathological signalling cascade will be tested to pave the way for eventual rational treatments investigation of the involvement of alcohol and its metabolites in the induction of pancreatitis.
Calcium store changes from granular (blue) and basal (red) areas of permeabilized pancreatic acinar cell loaded with Fluo-5N AM. The bile acid TLC-S releases Ca2 selectively from acid store when ER Ca2+ pump is inhibited by thapsigargin (TG) (Gerasimenko et al JBC 2006).
As the result of successful collaboration with Dr. K. Mikoshiba’s laboratory in RIKEN Brain Science Institute, Tokyo, Japan, we found that pancreatic protease activation by alcohol metabolite mainly depends on Ca2+ release via acid store IP3 receptors (Gerasimenko J. et al, PNAS, 2009). Currently there is no specific pharmacological treatment for pancreatitis. However, now our research has identified the critical proteins responsible for the excessive calcium release which is where the problem begins with the possibility to search for specific chemical compounds for the treatment of acute pancreatitis.
I am investigating the action of nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+ releasing messenger and its role in the induction of pathological processes of exocrine pancreas. Our findings (Gerasimenko J, et al., JCS, 2006) show that the NAADP-sensitive Ca2+ pool is located in the endoplasmic reticulum and in acidic organelles, which are represented by secretory granules, endosomes and lysosomes. So far there was much uncertainty on the action of NAADP in mammalian systems. We shall investigate further NAADP-elicited Ca2+ release from different organelles in permeabilized pancreatic acinar cells and pancreatic cell cultures using transfection and knockouts techniques including recently reported link to TPC2 channels. I am also developing optical applications involving two-photon microscopy for the tasks outlined above and in particular two-photon permeabilization technique that allow us to study small intracellular Ca2+ stores in different organelles.
A-D. Example of permeabilization of the cell by high intensity two-photon excitation light (735 nm)
A. A pancreatic acinar cell doublet loaded with Fluo-5N AM before permeabilization. Blue dot shows the position of two-photon light application.
B. Same cell doublet after permeabilization and perfusion with Texas Red dextran (3000 MW). Only the lower cell has been permeabilized and is therefore bright due to diffusion of Texas Red dextran into the cytoplasm.
C. Same cell doublet after washing out of Texas Red dextran. Note reduced fluorescence of Fluo-5N in the lower permeabilized cell.
D. Transmitted light picture of the doublet (after permeabilization) shown in A-C.
E. We have demonstrated that NAADP –like IP3 and cADPR – releases Ca2+ not only from the ER, but also from an acid, bafilomycin-sensitive pool in the secretory granule area when ER Ca2+ pump is inhibited by thapsigargin (TG). Calcium store changes from granular (blue) and basal (red) areas. (Gerasimenko et al J Cell Sci 2006)
F. Ca2+ signalling components and Ca2+-mediated interactions in the apical secretory pole of pancreatic acinar cells. Summary of conclusions from experiments on two-photon permeabilized cells and from patch clamp whole cell current recording studies (Gerasimenko et al J Cell Sci 2006; Menteyne et al Curr Biol 2006)
Current Sources of funding
- Medical Research Council
- Wellcome Trust
Collaboration with other labs
- Prof. Alexei Tepikin, Liverpool University, UK
- Prof. Steven Pandol, University of California, Los Angeles, USA
- Prof. Anna Gukovskaya, University of California, Los Angeles, USA
- Dr. Katsuhiko Mikoshiba, RIKEN Brain Science Institute, Tokyo, Japan
Contributions to books
- Oleg Gerasimenko and Julia Gerasimenko, Two-photon permeabilization and calcium measurements in cellular organelles, Chapter 12 in METHODS IN MOLECULAR BIOLOGY SERIES (Series Editor J. Walker) LIVE CELL IMAGING (Volume Editor D.B. Papkovsky).
- O. Gerasimenko and J. Gerasimenko, Measuring calcium in the nuclear envelope and nucleoplasm, Chapter 7 in Calcium signalling, Second Edition, Oxford University Press, 2001: A Practical Approach, Edited by A. Tepikin, pp. 125-135
- J. Gerasimenko. Measuring Ca 2+ in Endosomes of Intact Cells, Chapter 11 in Measuring Calcium and Calmodulin Inside and Outside Cells, Springer Lab Manual, Edited by O.H. Petersen, pp.231-247