The Role Of Medial Prefrontal Klotho In The Development Of Stress-induced Depression-like Behavior In Rats And Gender Differences

Depression is a devastating mental illness that causes a high personal and social burden of economic and disease.The pathogenesis of depression involves social,psychological and biological factors.At present,there is no clear pathological theory that can independently clarify the pathogenesis of depression.Currently,the pathogenesis of depression mainly involves genetic factors,biochemical factors,neuroinflammation and neuroplasticity.It has been proved that neuroplasticity disorder is one of the important causes of depression.Therefore,this experiment studied dendritic spines medial prefrontal cortex(mPFC)during the development of depression-like behaviors.Sex differences plays an important role in the depression,clinical studies have shown that depression is twice more prevalent in women than in men,but the mechanistic underpinnings of sex differences in depression is poorly understood.The current treatment of depression does not take into account sex differences,so the clinical efficacy of treatments for depression is not very ideal.Therefore,it is particularly important to study the sex differences in depression.Chronic unpredictable mild stress(CUMS),an established animal model of depression,induces depression-like behavior in animals,which can mimics depression symptoms in human.The mPFC,which is the processing and integration center of cognitive and emotion-related information,is sensitive to stress and is one of the major brain regions closely associated with depression.A large number of literature have confirmed that the occurrence of depression is related to the dysfunction of the mPFC,so the change of mPFC structure and function may be one of the important causes of depression.Klotho,an anti-aging protein,is widely expressed in the rat brain,has the obvious neuroprotective function,and could enhance neuroplasticity.Previous studies show that Klotho is associated with a variety of neurological disorders.These studies suggest that Klotho plays an important role in stress response and sex differences in depression.The main aim of this thesis was to investigate the role of Klotho in the rat mPFC in CUMS-induced depression-like behaviors and sex difference.First experiment was to determine whether reduction of endogenous Klotho in the mPFC of the male and female rats induce depression-like behaviors.Our results showed that expression of adeno-associated virus pHBAAV(adeno-associated virus)-U6-Klotho-shRNA-CMVeGFP in the bilateral mPFC caused a decrease in the levels of Klotho protein compared with the mPFC expressing pHBAAV-U6-scrambled shRNA-CMV-eGFP.Behavioral tests including sucrose preference,open field,elevated plus maze,tail suspension and novelty-suppressed feeding were conducted to evaluate depression-like behaviors.The results showed that decreased levels of mPFC Klotho did not induce depression-like behaviors in rats of both sexes.The second experiment was used to determine susceptibility of male and female rats in which endogenous Klotho protein levels were decreased.CUMS was then applied to rats until the animals showed depression-like behaviors.Immuno-fluorescence staining was used to determine the accuracy of the injection of AAV and the efficiency of Klotho protein knockdown by expression sh-Klo.Dendritic spines in the apical dendrites of pyramidal neurons in the PL region of the mPFC was labeled by a gene gun,CUMS-mediated alterations of synaptic proteins in the mPFC was analyzed by Western blot.The results showed:1.Expression of sh-Klo significantly reduced the level of Klotho protein in the rat mPFC.Immunofluorescence staining and Western blot results showed that the AAV injection site was accurate,and expression of sh-Klo successfully decreased the Klotho protein level in the mPFC,respectively.2.Decreased levels of the Klotho protein in the rat mPFC,did not directly induce depression-like behavior in animals.The male rats showed more active behaviors,while the female rats did not show a significant difference in depression-like behaviors compared with the control group anlimals expressing control sh-Scr.3.Reduction of the levels of Klotho protein in the mPFC resulted in a different susceptibility to CUMS between male and female animals.Decreased expression of klotho protein in the mPFC increased the sensitivity of female mice to stress,but did not affect the sensitivity of male to stress.4.Reduction the Klotho protein level of the mPFC had a different effect on dendrite spine density of mPFC pyramidal neurons in male and female animals.CUMS caused a significant decrease in spine density in the mPFC of male expressing sh-Scr and sh-Klo.After CUMS,the density of dendritic spines of female rats injected with sh-Klo decreased significantly,and the dendritic spines of female rats injected with sh-Scr did not change significantly.5.Reduction the Klotho levels in the rat mPFC had a different effect on the levels of synaptic protein in male and female animals.CUMS caused a significant decrease in the levels of Kal7 and PSD95 in the mPFC of rats which received injection of sh-Scr or sh-Klo.The protein levels of Kal7 and SYN in mPFC expressing sh-Klo were significantly decreased in female rats after CUMS comparing with female rats expressing control sh-Scr.Conclusion:The results of this study proved that reduction of the level of Klotho protein in the mPFC did not directly induce depression-like behavior in rats of both sexes,but caused a significant increase the sensitivity of female rats to stress and alterations of spine density and the levels of synaptic proteins.These results suggest that Klotho plays an important role in sex differences in depression,and these results may enhance our understanding of the mechanism underlying the sex differences in depression and provide a theoretical basis for development of new approaches for treating depression.