Molecular Mechanisms of IRE1α-ASK1 Pathway Reactions to Unfolded Protein Response in DRN Neurons of Post-Traumatic Stress Disorder Rats

The goal of this study was to further elucidate the molecular mechanisms of post-traumatic stress disorder (PTSD) pathogenesis and to provide experimental evidence for new drug targets for effective PTSD treatment. Expression changes of IRE1α, ASK1, and other downstream molecules of the IRE1α-ASK1 endoplasmic reticulum stress (ERS) signaling pathway were investigated. JNK, P38, CHOP, Bcl-2, and Bax were analyzed at both protein and mRNA levels of dorsal raphe nucleus (DRN) neurons of PTSD rats. The rat PTSD model was established via the single-prolonged stress (SPS) method. Animals were randomly divided into five groups: a normal control group, a 1-day SPS group, a 4-days SPS group, a 7-day SPS group, and a 14-day SPS group. Spatial memory and learning ability of rats were evaluated subsequent to SPS using the Morris water maze test. Changes of IRE1α expression in the control and SPS groups were detected via immunohistochemistry (IHC). Protein and mRNA expressions of IRE1α, ASK1, JNK, P38, CHOP, Bcl-2, and Bax in the control and SPS groups were detected via Western blot and RT-PCR, respectively. The Morris water maze test revealed significantly longer average escape latencies in all SPS groups compared to the control group. In the spatial probe test, the percentage of time spent in the target quadrant was significantly lower in the SPS groups compared to control. IHC revealed increased positive expression of IRE1α subsequent to SPS challenge, reaching maximal levels on days four and seven (P < 0.01), while significantly decreasing on day 14 (P < 0.01). Western blot and RT-PCR revealed that protein and mRNA expressions of IRE1α, ASK1, JNK, CHOP, and P38 were significantly increased compared to control, peaking on days one, four, and seven post-SPS before returning to previous levels. Compared to control, expressions of Bcl-2 and Bax presented an initial increasing tendency followed by a decrease. A peak of Bcl-2 expression appeared early on day one following SPS, then decreased to a steady level. Bax expression in the SPS groups remained constant during early stages after SPS (days one to three) compared to control; however, expression significantly increased on day four and maintained a high level. In summary, 1) SPS challenge significantly activated the IRE1α-ASK1-JNK and IRE1α-ASK1-P38 apoptosis-signaling pathways in DRN neurons of PTSD rats. This resulted in a cascade of downstream reactions and ultimately apoptosis of DRN neurons. 2) Increased expression of apoptosis-associated molecules Bcl-2 and Bax in DRN neurons following SPS challenge was revealed as a central mechanism, inducing apoptosis of DRN neurons in PTSD rats.