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RESEARCH PROFILES
In the funny papers, the universal sign of stress is steam coming out of one’s ears. In real life, the signals are less clear, and this makes measuring stress a difficult prospect for researchers. Analysis of stress in living subjects typically relies on measurements of corticosterone levels in blood samples or functional MRI of blood oxygen levels. But Yoshio Umezawa, Sung Bae Kim, and Takeaki Ozawa at the University of Tokyo, the Institute for Molecular Science, and the Japan Science and Technology Agency (all in Japan) have developed a noninvasive imaging method for measuring physiological stress. In the October 15 issue of Analytical Chemistry (pp 6588– 6593), Umezawa and colleagues report the construction of a pair of genetically encoded indicators that, once transfected into cultured cells and implanted in mice, enable in vivo imaging of endogenous corticosterone as an index of stress intensity. Physiological responses to stress, such as gluconeogenesis, deposition of liver glycogen, and anti-inflammatory responses, occur following the transport of the glucocorticoid receptor (GR) from the cytosol to the nucleus of live cells, where corticosterone-bound GR activates the transcription of specific target genes. This is the initial step for regulating the magnitude and specificity of stress-related gene expressions in response to physical and emotional stress. Umezawa and colleagues have found a way to exploit this GR translocation to detect stress. “The principle of the approach is an extension of our earlier work using reconstitution [of split enhanced green fluorescent protein] to identify mitochondrial proteins,” says Umezawa (Nat. Biotechnol. 2003, 21, 287–293). In the current work, the researchers use a split Renilla luciferase (R Luc) protein that is reconstituted by protein splicing after nuclear translocation. The approach is particularly effective because when R Luc is split into two fragments, it has no bioluminescence. Umezawa and 410 A
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Bioluminescence intensity
Visualizing stress
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A cooled-CCD pseudocolor image of corticosterone in mice. Cells expressing both the Cterminal RLuc–DnaE–GR and the N-terminal RLuc–DnaE indicators were injected at site 4.
colleagues, therefore, constructed two complementary DNA indicators. One is composed of the N-terminal half of a Synechocystis species DnaE intein connected to the N-terminal R L uc fragment, which localizes to the nucleus. The other is composed of the C-terminal halves of R L uc and DnaE joined to the GR, which localizes to the cytosol. When corticosterones are present, the RLuc–DnaE–GR indicator translocates to the nucleus, where the two halves of RLuc are joined by protein splicing. The addition of the R Luc substrate coelenterazine results in a luminescent product, which can be visualized using a cooled CCD. In developing this technique, Umezawa and colleagues first used transfected NIH 3T3 cells to confirm that correct localization, expression, and protein splicing of their indicators occurred in vitro. Next, transfected cells containing no indicators, just one indicator, or both indicators were injected into live mice at four separate locations. The injected mice were forced to swim for 5 min, then injected with the RLuc substrate coelenterazine. CCD images taken 2 h after the swimming period showed only background luminescence in all sites, except
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where transfected cells containing both indicators had been injected. In those sites, mice that had been forced to swim had 32.7% higher luminescence intensities compared with nonswimming mice. Blood corticosterone levels in swimming mice were 7.1 those in nonswimming mice, as determined by ELISA. After Umezawa and colleagues successfully imaged mice subjected to the emotional stress of forced swimming, they tested their system with a metabolic stress. For this, they again injected the mice with cells transfected with indicators and stimulated them with 2-deoxyD-glucose (glucoprivation). Metabolically stressed mice again showed higher luminescence intensities (20%) than non-stressed mice; this indicates that the group’s method can detect both interoceptive (metabolic) and exteroceptive (emotional) types of stress. The new method, Umezawa says, may “provide a wide variety of applications for pharmacological or toxicological purposes, such as testing various endo- and exogenous risk factors and identifying specific molecular events responsible for diseases in living subjects.” Further down the line, Umezawa thinks this research might also “facilitate the development of transgenic animals that express the indicators in [their] tissues or organs with controllable promoters.” The technique holds potential for use in more specific regions in vivo. Last year, Umezawa and colleagues used a similar technique to demonstrate that particular chemical compounds were capable of crossing the blood–brain barrier and inhibiting the translocation of androgen receptor to the nuclei of transfected cells implanted in the brains of mice (Proc. Natl. Acad. Sci., U.S.A. 2004, 101, 11,542–11,547). Umezawa says that more recently, they have “developed similar genetically encoded bioluminescent probes for illuminating protein nuclear transport induced by phosphorylation or by proteolysis.” a —Amy Hodson Thompson
