MiR-34c And CRFR1 Regulate Susceptibility To Posttraumatic Stress Disorder Induced By Early Adolescent Traumatic Stress

1. IntroductionPost-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experiences. The dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is the key factor in etiology of PTSD because activation of the HPA axis is the most important endocrine component of the stress response and its abnormal activities result in neurotransmitter dysfunction and behavioral disorders. Most studies suggested that excessive corticotrophin releasing factor (CRF) activity or greater responsiveness of corticotrophin releasing hormone (CRH) to stressors was involved in the pathophysiology of PTSD. Therefore, as the key upstream factors in HPA axis activation and regulation, CRF and CRF receptors play a significant role in HPA axis dysfunction involved in anxiety, depression and PTSD pathogenesis. It has been hypothesized that antagonism of corticotrophin-releasing factor receptor type 1 (CRFR1) may provide an effective pharmacological treatment for stress-related mental disorders.Several studies have found that exposure to early life stress is a risk factor for developing PTSD. Adolescence is an important and special period of neurobiological and behavioral development, in which period-prominent developmental transitions are observed in the limbic brain regions and prefrontal cortex. It was suggested that modulation of CRF receptors’expression and function in early life may be a persistent and important factor in neuronal dysfunction throughout the lifetime. MiR-34c, one of the prominent stress-related miRNAs, was found to be strongly up-regulated by exposure to stress, resulting in reduced symptoms of anxiety in the mice. Interestingly, CRFRlmRNA is one of the targets of miR-34c. A limited number of studies, however, have explored the influence of traumatic stress in early adolescence on HPA function, central CRFR1 and miR-34c expression, and their relative vulnerability to PTSD in adulthood. As a potential non-pharmacological therapeutic approach, regular exercise has considerable beneficial effects on brain function and plasticity, as observed in many studies. Our study focused on the effects of early adolescent traumatic stress on anxiety-like and depression-like behaviors, functional state of HPA axis, central CRFR1 and miR-34c expression, to reveal the effects of early life stress on PTSD susceptibility and the effects of regular exercise on PTSD-like symptoms.2. Objectives(1) To investigate the long-term effects of adolescent traumatic stress on anxiety-like behavior, depression-like behavior, memory damage, dysregulation of HPA axis function and central CRFR1 expression in adulthood.(2) To compare the effects of regular treadmill exercise and administration of CP-154,526 on anxiolytic effect, antidepressant effect, memory functional improvement and HPA axis function, as well as central CRFR1 receptor expression.(3) To investigate the short-term and long-term effects of adolescent stress on anxiety like behaviors, CRFR1 and CRFR1mRNA expression, as well as miR-34c expression in both adolescence and adulthood.(4) To observe the effect of CP-154,526 on anxiety like behaviors, CRFR1 and CRFR1mRNA expression, as well as miR-34c expression in both adolescence and adulthood.3. Materials and methods(1) Experiment grouping designExperiment 1:A total of 64 male Wistar rats (21 days old) were randomly divided into four groups (n=16 in each group):the control group (CON), the stress group (S), the stress and exercise group (S+E), and the stress and antagonist group (S+A). With the exception of the control group, the rats in other groups received the repeated inescapable electric foot shock.Experiment 2:A total of 72 male Wistar rats (21 days old were randomly divided into three groups (n=24 in each group):the control group (CON), the stress group (S) and the stress and antagonist group (S+A). After animal modeling and drug administration,12 rats randomly chosen from each group were sacrificed after behavioral tests. The rest of the animals were raised to adulthood and were sacrificed after behavioral tests.(2) Animal model of PTSDWith the exception of the control group, the rats in other groups received the repeated inescapable electric foot shock. The animals received 0.5 mA of electric foot shock for six consecutive days. Electric foot shock continued for 6 s, repeated 20 times with a random interval in a 30-min period in each trial, with two trials per day. The interval between the two trials was not less than 4 h.(3) Antagonist administration CRFR1 antagonistCP-154,526 was administered intraperitoneally. The rats in S+A group were treated with 3.2 mg/kg/day of CP-154,526 (in vehicle) for 14 consecutive days starting on the day after the foot shock procedure. Rats in other groups were treated with 80% polyethylene glycol 400 (vehicle) to balance the systematic error.(4) Treadmill exercise protocolThe rats in the S+E group ran on a motorized treadmill for five days/week, for six weeks, starting one day after the last electric foot shock.(5)DEX/CRHtestTo examine the functional state of HPA axis, rats were subjected to the DEX/CRH test after behavioral tests. CORT was detected in 10 μl samples with an Iodine [125I] Corticosterone Radioimmunoassay Kit.(6) Behavioral testBehavioral tests were taken two weeks or six weeks later after foot shock. Behavioral tests including Open field (OF) test, Elevated plus maze (EPM), Y maze test and Morris water maze.(7) Immunohistochemistry assay for CRFR1 expressionAfter behavioral test, rats were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Tissue sections of mPFC, hypothalamus, hippocampus and amygdala were cut coronally from paraffin blocks at 5-um thickness to detect CRFR1 expression by immunohistochemical staining.(8) Western blotting analysis for CRFR1 expressionHypothalamus, mPFC, hippocampus and amygdala tissues were homogenized in cold lysis buffer. The samples were centrifuged at 14000 rpm for 30 min at 4℃, then the supernatant was collected and protein content was assayed colorimetrically.20μg total proteins were loaded onto a 12%SDS-PAGE gel, electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies at 4℃ overnight following incubation of secondary antibodies for 1h. The membranes were developed using an enhanced ECL detection system. The intensity of bands was determined using the Image J 14.0 software.(9) TaqMan microRNA assay for CRFR1 mRNA and miR-34C50-100mg tissues was mixed with 1 ml Trizol (Invitrogen) to extract total RNA from frozen specimens following the manufacturer’s guide.1mL of RNA was used to measure the expression of CRFRlmRNA or miR-34c by quantitative RT-PCR (qRT-PCR) with the TaqMan miRNA reverse transcription kit. Finally, and relative expression levels were calculated as 2^-[(Ct of CRFR1mRNA)-(Ct of GAPDH)] or 2^-[(Ct of miR-34c)-(Ct of U6)].3. Results(1) Early adolescent traumatic stress induced long-term effects on anxiety-like behavior, depression-like behavior, memory damage, dysregulation of HPA axis function and central CRFR1 receptor expression in adulthood.(2) Long-term regular treadmill exercise had anxiolytic effect, antidepressant effect; it could improve memory and HPA axis functions compared with chronic administration of CP-154,526.(3) Long-term regular treadmill exercise and chronic administration of CP-154,526 lead to adaptive alterations in central CRFR1 receptor expression.(4) Adolescent traumatic stress induced anxiety-like behavior and memory damage, higher expression of miR-34c and CRFR1 mRNA, and lower protein CRFR1 expression in adolescent hypothalamus. While adolescent traumatic stress induced prolonged negative behaviors and increased CRFR1 expression in adult hypothalamus.(5) Chronic administration of CP-154,526 led to higher expression of miR-34c in both adolescent and adult hypothalamus.4. Conclusion(1) Early adolescent severe traumatic stress induced lasting effects on negative emotions, spatial memory, functional state of the HPA axis and central CRFR1 expression during the adult period.(2) Abnormal central CRFR1 expression in brain and increased expression of miR-34c in hypothalamus play important roles in susceptibility to PTSD when individual experience traumatic stress during early adolescence.(3) Long-term regular treadmill exercise and antagonist CP-154,526 can help to effectively alleviate early adolescent stress-induced behavior and spatial memory abnormalities and CRFR1 expression, whereas antagonist CP-154,526 performed less effectively than treadmill exercise in this research.(4) The result that CRFR1 antagonist induced increased expression of miR-34c both in adolescent and adult hypothalamus indicated that the miR-34c and CRFRl may be the target points of CRFRl antagonist.