Molecular stress response in the CNS of mice after systemic exposure to interferon-α, ionizing radiation and ketamine
Introduction
Neuropsychiatric diseases are often related to stress and stress modifies the onset and progression of neuropsychiatric diseases (Garcia-Bueno et al., 2008). Current knowledge of stress-induced response in the brain includes regulation of hypothalamic paraventricular nucleus, hypothalamic–pituitary–adrenal (HPA) axis activity and oxidative/nitrosative, neuroinflammatory changes (Cerqueira et al., 2008). However, the underlying mechanism(s) of stress response in brain are not well understood, nor is it known whether diverse stressors act through common “gatekeeper” molecules or pathways.
IFN-α, an anti-viral cytokine (Licinio et al., 1998), is currently used for the treatment of certain viral illnesses, cancers, chronic hepatitis C and other conditions (Wichers et al., 2005, Capuron et al., 2007). There is growing clinical evidence that IFN-α treatment of patients with chronic hepatitis or cancer can cause neuropsychiatric complications (Kraus et al., 2005, Wichers et al., 2005, Capuron et al., 2007). Debien et al. (2001) showed that more than 30% of patients treated by IFN-α presented various psychiatric disorders including depression, anxiety, intense and fluctuating personality disorders, manic or psychotic symptoms, and suicidal tendencies. Others have described declines in concentration, attention, memory and episodic dizzy spells and headaches (Kraus et al., 2005). Yet, the target cells of systemic IFN-α treatment on the neurological and psychiatric effects remain undefined.
There are also growing concerns regarding the cognitive sequelae of whole-brain irradiation for cancer patients (Shi et al., 2006). Gamma-radiation can induce cell death in fetal rat brain (Borovitskaya et al., 1996). Achanta et al. (2007) reported that whole-brain irradiation induced gene expression changes in rodent hippocampus involving pathways important for learning, memory and apoptosis. A study conducted in our laboratory (Yin et al., 2003) demonstrated that whole-body radiation treatment at 10 cGy and 2 Gy modulated the transcript expression of genes in adult mouse brain. The radiation exposure modulated genes related to stress response, cell-cycle control and DNA synthesis/repair. Further bioinformatics analyses demonstrated that low-dose radiation (at 10 cGy whole body) affected nine neural signaling pathways, which are also affected in human aging brain and Alzheimer's disease patients (Lowe et al., 2009).
Clinical treatment with glutamate N-methyl-d-aspartic acid (NMDA) receptor antagonists such as ketamine, have been found to induce a broad range of cognitive adverse effects and symptoms that resemble various aspects of neuropsychiatric diseases (Krystal et al., 1994). We have previously demonstrated that troponin T1 (Tnnt 1) was dramatically induced in the brain of adult mice 30 min after treatment with a single intraperitoneal (i.p.) injection of 80 mg/kg ketamine (Lowe et al., 2007). Through bioinformatics analysis, Tnnt 1 gene was found to be directly regulated by FoxO1, a transcription factor involved in multiple metabolic pathways including glycolysis, lipogenic and sterol synthetic pathways (Zhang et al., 2006) and central energy homeostasis (Kim et al., 2006). Tnnt 1 is also indirectly regulated by dexamethasone, a known stress marker and a key hormone in energy homeostasis (Wu et al., 2004). Khodarev et al. (2001) showed that modulation of cytoskeleton genes including troponin T1 was induced by irradiation at dose of 1 Gy, 3 Gy or 10 Gy in a human malignant gliomas cell line. In addition, PAK3, a GTPase-dependent kinase involved in multiple signaling pathways including axonal guidance signaling, directly interacts with the troponin T complex (Buscemi et al., 2002). These lines of evidence indicated that the function of Tnnt 1 is associated with diverse cellular processes beyond its role in regulating striated muscle contraction.
In vertebrates, troponin T complex includes three troponin T (Tnnt) genes that have evolved for the regulation of striated muscle contraction: slow skeleton TnT (sTnT; Tnnt 1), cardiac TnT (cTnT; Tnnt 2), and fast skeleton TnT (fTnT; Tnnt 3) (Wang et al., 2001). However, troponin T is also expressed in non-muscle tissues including prostate cancer cells (Ashida et al., 2004), pancreatic cancer cell lines (Basso et al., 2004), a variety of mammalian cell lines (fibroblasts, foreskin fibroblasts, PtK1 cells, CHO cells and HeLa cells) (Lim et al., 1986), embryonic and neonatal rat brain stems (De Vitry et al., 1994) and in the brain (Fine et al., 1973, Mahendran and Berl, 1977, Lim et al., 1986, Lowe et al., 2007). Thus, it has been speculated that troponin T may function in cellular processes other than muscle contraction (Lim et al., 1986). However, its role and function in non-muscle tissues remains unresolved.
We hypothesized that induced Tnnt 1 expression in the brain may be associated with early molecular responses to induced CNS stress. To test this hypothesis, we investigated the effects of systemic exposure to two stressors, IFN-α and ionizing radiation, on Tnnt 1 expression in mouse brain tissues. The aims of this study were to determine whether (a) the expression of Tnnt 1 was induced in CNS of mice by IFN-α or ionizing radiation, both of which are known to induce neuropsychiatric symptoms; (b) there were similar expression patterns compared to those induced by ketamine and (c) there were tissue and cellular-specific expression patterns. Our results showed for the first time, that the all three exposures induced the CNS expression of Tnnt 1 and suggest that Tnnt 1 may have a role as a common molecular biomarker of CNS stress.
Section snippets
Animals and treatments
B6C3F1 male mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). The use of animals in the study was approved by the Lawrence Livermore National Laboratory IACUC.
Results
Two groups of adult male mice were treated with either single i.p. injection of IFN-α or whole-body radiation. Various brain sections of mice were tested for the expression of Tnnt 1 using RNA in situ hybridization at 4 h post-treatment. Expression of Tnnt 1 was significantly induced in various CNS regions of mice treated with IFN-α and in mice treated with radiation when compared with concurrent sham exposed controls.
Systemic IFN-α treatment and whole-body radiation induced the expression of Tnnt 1 in adult mouse brain
Using RNA in situ hybridization, we demonstrated that the expression of Tnnt 1 was consistently induced in pyramidal neurons of cerebral cortex and hippocampus, but not in glial cells after IFN-α, whole-body radiation, as well as ketamine treatment. IFN-α apparently had nearly no effect on Purkinje cells of cerebellum, while radiation treatment had similar effects in both regions of Ammon's horn and DG of hippocampus, in contrast to IFN-α and ketamine. Using fluorescence immunohistochemistry
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Ms. Sylvia Ahn for the technical assistance of RNA in situ hybridization; Dr. Eric Yin (Ph.D.) for mouse radiation treatment and brain tissues collection after radiation.
This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Berkeley National Laboratory under contract DE-AC02-05CH11231 and Lawrence Livermore National Laboratory under contract W-7405-ENG-48. Funded in part by DOE Low Dose Research Program grant (SCW0391) to
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