Elevated Expression of HSP72 in the Prefrontal Cortex and Hippocampus of Rats Subjected to Chronic Mild Stress and Treated with Imipramine

Abstract

The HSP70 and HSP90 family members belong to molecular chaperones that exhibit protective functions during the cellular response to stressful agents. We investigated whether the exposure of rats to chronic mild stress (CMS), a validated model of depression, affects the expression of HSP70 and HSP90 in the prefrontal cortex (PFC), hippocampus (HIP) and thalamus (Thal). Male Wistar rats were exposed to CMS for 3 or 8 weeks. The antidepressant imipramine (IMI, 10 mg/kg, i.p., daily) was introduced in the last five weeks of the long-term CMS procedure. Depressive-like behavior was verified by the sucrose consumption test. The expression of mRNA and protein was quantified by real-time PCR and Western blot, respectively. In the 8-week CMS model, stress alone elevated HSP72 and HSP90B mRNA expression in the HIP. HSP72 mRNA was increased in the PFC and HIP of rats not responding to IMI treatment vs. IMI responders. The CMS exposure increased HSP72 protein expression in the cytosolic fraction of the PFC and HIP, and this effect was diminished by IMI treatment. Our results suggest that elevated levels of HSP72 may serve as an important indicator of neuronal stress reactions accompanying depression pathology and could be a potential target for antidepressant strategy.

Keywords: HSP70; HSP90; chronic mild stress; constitutive HSC70; constitutive HSP90B; depression; heat shock protein; imipramine; inducible HSP72; inducible HSP90A.

Figure 1

Schematic showing the experimental groups and consumption of 1% sucrose solution in rats exposed to CMS (stress) for (A) 3 weeks and (B) 8 weeks and simultaneously treated with IMI for the last 5 weeks (IMI, 10 mg/kg, ip). Vertical lines represent the standard error of the mean, n = 6; *** p < 0.001 relative to the sham or sham/sal group; ++ p < 0.01 relative to the stress-reactive group; ## p < 0.01 relative to the stress/IMI resp group; reactive—group of animals responding to CMS in the behavioral test.

Figure 2

Levels of (A) mRNA and (B) protein expression of HSP72 in PFC of rats subjected to the CMS (stress) for 8 weeks and simultaneously treated with IMI (10 mg/kg, ip) for the last 5 weeks. Results were determined by real-time PCR and Western blot methods. Rats were sacrificed 24 h after the last administration of IMI or saline (sal). Results are expressed as a percentage of sham/sal group ± standard error of mean (SEM); n = 6; (C) representative blots; * p < 0.05 relative to sham/sal, # p < 0.05 relative to stress/IMI resp group; stress/IMI resp—group of CMS rats that responded to IMI treatment (reversed anhedonia in sucrose test); stress/IMI nonresp—group of CMS rats that did not respond to IMI.

Figure 3

Levels of (A) mRNA and (B) protein expression of HSP72 in HIP of rats subjected to the CMS (stress) for 8 weeks and simultaneously treated with IMI (10 mg/kg, ip) for the last 5 weeks. Results were determined by real-time PCR and Western blot. Rats were sacrificed 24 h after the last administration of IMI or saline (sal). Results are expressed as a percentage of sham/sal group ± standard error of mean (SEM); n = 6; (C) representative blots; * p < 0.05 relative to sham/sal, # p < 0.05 relative to stress/IMI resp group; stress/IMI resp—group of CMS rats that responded to IMI treatment (reversed anhedonia in sucrose test); stress/IMI nonresp—group of CMS rats that did not respond to IMI.

Figure 4

Levels of (A) mRNA and (B) protein of HSP90B in HIP of rats subjected to the CMS (stress) for 8 weeks and simultaneously treated with IMI (10 mg/kg, ip) for the last 5 weeks. Results determined by real-time PCR and Western blot. Rats were sacrificed 24 h after the last administration of IMI or saline (sal). Results are expressed as a percentage of sham/sal group ± standard error of mean (SEM), n = 6; (C) representative blots; * p < 0.05 relative to sham/sal; + p < 0.05 relative to stress/sal group; stress/IMI resp—group of CMS rats that responded to IMI treatment (reversed anhedonia in sucrose test); stress/IMI nonresp—group of CMS rats that did not respond to IMI.

Figure 5

Representative double immunofluorescence staining for HSP72 or HSP90B and NeuN. Arrows indicate HSP72-positive or HSP90B-positive cells colocalized with neurons in the (A–F) PFC and (G–L) HIP areas. Scale bar 25 μm.

Figure 6

The HSP72 as a potential target of antidepressant treatment. Signaling molecules involved in the mechanism of anhedonia are shown in the rectangles. Directions of changes within the cerebral prefrontal cortex (PFC) and/or the hippocampus (HIP) of animals with anhedonia phenotype are illustrated by arrows. HSP72 working directly and/or by cooperation with HSP90 in the GR chaperone complex is the upstream regulator of apoptosis and inflammatory processes, as described in the Section 3.2.3. Therefore, modulation of HSP72 activity may normalize the apoptotic and inflammation pathways affected by depression (indicated by the dotted arrows). The HSP72 mRNA expression is increased in the PFC and HIP of imipramine-resistant CMS rats as revealed by the current study. Bcl-2, B-cell lymphoma 2 apoptosis regulator; Casp-9, Caspase 9; Casp-3, Caspase 3; Casp-12, Caspase 12; Il-1β, Interleukin 1β; Il-6, Interleukin 6; GR, glucocorticoid receptor; HSP72, Heat Shock Protein 72; HSP90, Heat Shock Protein 90 [15,47,73,74,79,80,81].