| It is well-known that androgens have important role on brain structure and function,but the specific mechanisms is unclear. Aging related to reduction of androgen levels isassociated with cognitive impairment and is a risk factor for Alzheimer’s disease.Epidemiological studies have found that low levels of testosterone in the elderly populationhad worse cognitive function, and they were easier to developed Alzheimer’s disease.Animal experimental evidences suggest that androgen has been profoundly involved in theregulation of the brain structure and function, especially the emotional, cognitive, learningand memory, reproductive function etc. In the brain, the pathway of androgens’ role havetwo ways, one direct through its receptor, another indirect transformed into estrogen byaromatase (aromatase, AROM). The latter is often referred to as the local estrogen(estradiol,E2). According to aromatase hypothesis, the indirect way mainly determines the sexdifferences in brain development. In the direct way, androgen combine with correspondingreceptors namely androgen receptors(AR) and regulate the transcription of target genes. Inthe indirect way, local E2derived from androgen via AROM perfors chronic classicalgenomic regulationwhich is mediated by its nuclear receptor (estrogen receptor alpha andbeta, ERα and ERβ), or through the rapid nongenomic regulation which is mediated byG-protein coupled estrogen receptor(GPER) via intracytoplasmic second messenger system.Classic steroids receptor including AR, ERα and ERβ, belong to the members of the nuclearreceptor superfamily, is located in the nucleus. However, recent studies found that classicnuclear receptors in some cases can also be located in the cytoplasm or cell membrane andmediated rapid nongenomic regulation through the second messenger systems. So no matterandrogens or estrogens, the specific mechanism is very complicated. Steroids regulatetarget gene transcription by corresponding nuclear receptors, which is the main mediationway. In the process of regulation, steroids combine with nucler receptors and recruit coactivators or corepressors, then capture many other elements (e.g. transcriptionalregulation factors, RNA polymerase) for transcription.At present, a great number of coactivators have been discovered, which function indifferent tissues, organs or cells. The coactivators which have been studied most and deeplyare p160family. This family includes three member with molecular weight are all160kDa,i.e. steroid receptor coactivator-1(SRC-1), SRC-2and SRC-3. SRC-1mostly locates inbrain, whereas SRC-2and SRC-3mostly locate in peripheral tissue. SRC-1was firstdiscovered and most widely distributed in brain among the family. SRC-1knockout resultedin motor learning disorder and developmental retard of cerebellar Purkinje cells. Theinjection of SRC-1antisense oligonucleotides into specific brain region leaded to thechange of reproductive function. However, the function of SRC-1in brain is unclear ingeneral up to date. Previous study showed high protein expression of SRC-1inhippocampal neurons whereas little or no expression in glial cells. Our previous studyshowed obvious gender differences about the expression of SRC-1in brain, which washigher in male major brain regions than in female. Moreover, we found its expressiondecreased in some regions related to learning and memory and motor with aging, such ashippocampus and substantia nigra. Hippocampus is the closest to learning and memory,emotion and recognition and its synaptic plasticity is the base of learning and memory. It’sproposed that SRC-1may participate in the modulation of hippocampal synaptic plasticityby sex hormones. Afterwards we found transient change in hippocampal SRC-1expressionafter ovariectomy whereas steady loss after orchiectomy, which suggested SRC-1wasmostly mediated the modulation of hippocampal structure and function by orchic androgenbut not ovarian estrogen. And whether SRC-1mediates androgen effect on hippocampalstructure and function are not reported in any literatures.In the present study, we used immunohistochemistry, Western blot, transmissionelectron microscope, Morris water maze, RNA interference and co-IP for the examination.First, the change in the expression of SRC-1in every brain region was detected followingorchiectomy or aromatase inhibitor administration, which provided information for thefunction of androgen or local estrogen mediated by SRC-1. Then orchiectomy and/or LETadministration were used to observe the correlation between hippocampal SRC-1expression and synaptic plasticity. Afterwards, RNAi method was used to repress the protein expression of hippocampal SRC-1, and to observe the change in hippocampalsynaptic plasticity and learning and memeory. At last, mice hippocampal neuronal cell linewas used to validate the inhibitory effect of SRC-1on synaptic proteins and investigate thepossible mechanism.Main results:1. SRC-1immunoreactivity was observed in cerebrum, diencephalon, brain stem andcerebellum of adult male mice, which was mostly located in nucleus. However, itsignificantly decreased in piriform cortex, olfactory tubercle, hippocampus, hypothalamus,dorsolateral nucleus, ventral lateral nucleus and substantia nigra one month afterorchiectomy. And its immunoreactivity significantly decreased in hippocampus, medialseptal nucleus, dorsal medial nucleus, ventromedial nucleus, arcuate nucleus, superiorcolliculus, the periaqueductal gray and nucleus parolivaris superioris one month after LETadministration.2. The expression of hippocampal SRC-1and synaptic plasticity related proteins (e.g.GluR1, PSD-95, Spinophilin) in ORX group and LET group were significantly lower thanthat in control group. And the changing pattern of these proteins was highly correlative. Thedecrease in SRC-1and synaptic proteins expression was more significant in LET groupcompared with ORX group. The result of TEM showed ORX and LET can both regulateCA1synaptic desity and morphology in mice hippocampus, whereas the effect of LET wasstronger than ORX. In the test of MWM, LET significantly increased the latency of miceand lowered the retention period in target quadrant, whereas ORX didn’t markedly changethe latency and retention period in target quadrant.3. The differences of synaptic protein expression, synaptic density and space memoryimpairment between ORX plus LET treatment and LET treatment were not significant.4. Stereotaxic injection of lentivirus carrying SRC-1shRNA significantly repressed theexpression of hippocampal SRC-1, GluR1, PSD-95, Spinophilin as well as ERα, ERβ andAR. Hippocampal CA1spine synapse density and the thickness of postsynaptic densitywere also significantly decreased. Moreover, mice space memory declined dramatically inMWM test.5. Mice hippocampal neuronal cell line, mHippoE-14, expressed a variety of neuronalmarkers synaptic proteins, nuclear receptors and SRC-1. GluR1, PSD-95, Spinophilin and pCREB levels were all downregulated after SRC-1shRNA interfering in vitro. The resultfrom co-IP showed SRC-1can directly combine with pCREB and the binding capacitybetween the two significantly decreased after inhibition of SRC-1expression.Main conclusions:1. SRC-1can mediate the effect of orchic androgen and local estrogen on brainstructure and function because ORX and LET all significantly reduce SRC-1expression inmany brain regions. In some brain regions (such as the basal forebrain, hippocampus,hypothalamus, midbrain) related to learning and memory, motor, neuroendocrine function,reproduction and neural information integration, ORX and LET all significantly decreaseSRC-1level, which indicates that SRC-1mediated the direct or indirect effect of androgenon these nuclei.2. ORX or LET has a role in the regulation of SRC-1expression and synaptic plasticity,and the effect of LET is stronger. The influence of local estrogen on SRC-1expression andlearning and memory may be more significant than circulating androgen due to obviousspace memory impairment in MWM test only after LET administration.3. ORX plus LET treatment can’t induce further reduction in synaptic plasticity. Itmight be because LET treatment reduces local estrogen or SRC-1level to a threshold, thenORX plus LET treatment can’t further reduce synaptic proteins expression and synapticdensity and aggravate space memory ability. And this also suggest important role of localestrogen in the modulation of hippocampal SRC-1expression, synaptic plasticity as well aslearning and memory.4. Synaptic plasticity as well as learning and memory reduced following the inhibitionof hippocampal SRC-1expression by the injection of lentivirus carrying SRC-1shRNA invivo. The study in vitro further demonstrates the inhibition of SRC-1can result in thereduction in the expression of synaptic proteins and pCREB. The result of co-IP showedthat SRC-1and pCREB directly reacted with each other and the reaction can be regulatedby the expression level of SRC-1. And these suggest SRC-1can recruit pCREB andmodulate the transcription of synaptic proteins gene, then influence learning and memory.The present study used a series of experiments to demonstrate that SRC-1can mediatethe effect of androgen on hippocampal synaptic plasticity as well as learning and memoryand to investigate possible mechanism. Meanwhile, local estrogen in hippocampus may have a more important role in modulating hippocampal synaptic plasticity as well aslearning and memory than androgen. This study provide new experimental evidences forfurther investigation of the expression in the brain and function of SRC-1. Furthermore, thisstudy may suggest a new target for the development of the drug preventing and curing thediseases about learning and memory as well as recognition. Because androgen levelreduction with aging is related to cognition disorder and is a risk factor of Alzheimer’sdisease (AD), hormone replacement treatment has been used in clinical experiments. Ourstudy will provide new hope for the strategic development of prevetion and treatment ofneurodegenerative diseases. |
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