| BackgroundPrevious studies reported that neurodegeneration was presented in themice with heterozygous Grb10Interacting GYF Protein2(GIGYF2) geneknockout. This phenomenon indicated that GIGYF2plays an important rolein the central nervous system. And the level of GIGYF2expression couldalter insulin-like growth factor1receptor (IGF1R) trafficking and activateIGF1stimulated signaling. Recently, accumulating evidence hasdemonstrated a role of IGF1R signaling pathway in the neuropathogenicprocess of cognitive impairment. Studies have shown that Growth factorreceptor-bound protein10(Grb10) functioned as a suppressor of IGF1-IRpathway, which may represented a critical mechanism for regulatingcognitive impairment in diabetes. Recently, the identification of GIGYF2as a gene linked to neurodegeneration was of interest as a consequence ofemerging evidence for its role in human familial Parkinson’s disease. Thus,GIGYF2is emerging as a molecule that plays an important role in neuronaldegradation in the CNS. However, little is known about the expression and function of endogenous GIGYF2in the mice with diabetes-associatedcognitive impairment.ObjectivesThe objective of this study was to detect the level of GIGYF2inhippocampus of diabetic mice and research the effect ofhippocampus-specific downregulation of GIGYF2on neurobehavioral andpathological changes, as so to determine the function of GIGYF2indiabetic encephalopathy and the possible underlying mechanisms.MethodsMice were treated with a single intraperitoneal injection ofstreptozotocin (STZ)(180mg/kg), which dissolved in0.1M sodiumcitrate-hydrochloric acid buffer solution (pH4.5), to induce diabetes. Threedays later, the mice with fasting blood-glucose of above16.7mmol/L wereconsidered to be diabetes. Those mice in the experiment group weredivided into three groups: diabetes mellitus group (DM), sham-knockeddown (DM+0) group and the test (DM+shRNA), and mice in the controlgroup were divided into two groups: control group (Con) and Con+shRNAgroup (n=12per group). To study the function of GIGYF2in diabetes, weapplied lentiviral vector-delivered short hairpin RNA to the hippocampus invivo using stereotactic technology to down-regulating GIGYF2expression.Ten weeks later, we used real time PCR and Western Blot to detect theexpression of GIGYF2, Grb10and IGF1R. Meanwhile, we evaluated the cognition function of mice (examined by Morris water maze test) andobserved the changes of pathological and ultrastructure in hippocampus ineach group.Results1. In this study, we built an effective animal model of diabetesmellitus. The glucose level of mice, who were treated with intraperitonealinjection of STZ, was increased significantly than the normal control group(P <0.05), while the weight of them was significantly decreased (P <0.05).2. Effect of stereotaxic surgery on the result of morris water maze wasnegligible. High level of blood glucose for ten weeks, the mean escapelatency of DM and DM+0groups was apparently increased, the time intarget quadrant and platform crossing were declined and all had asignificant difference compared to control group (p<0.01). But the meanescape latency of DM+shRNA group was not increased and showed noobvious difference compared to control group (p=0.376), but significantlower than that of DM and DM+0groups (p<0.05).3. We found that DM and DM+0group, but not DM+shRNA group,had a significant increase in the level of GIGYF2expression compared tocon group (p<0.01). These results demonstrated that GIGYF2expressionwas specifically knocked down at both mRNA and protein levels bysite-application of special GIGYF2-shRNA. In hippocampus of diabetic mice, the level of Grb10expression was significant increased (p<0.05), butthe level of IGF1R expression was significant decreased (p<0.01), and theexpression of Grb10and IGF1R in DM+shRNA group was no obviouschange compared with DM group (respectively p=0.172, p=0.651), buthigher than that of two non-diabetic groups (p<0.05). Nevertheless, thelevel of IGF1R phosphorylation was significantly increased in DM+shRNAgroup as compared with DM and DM+0groups (p<0.05).4. High level of blood glucose for ten weeks could lead to a series ofpathophysiological changes in hippocampus of diabetic mice. HE stainingresults show that the disorder arrangement in the pyramidal cell layer ofhippocampus, an increase in partly neuronal apoptosis, a large number ofglial hyperplasia. The results of electron microscopy indicated that thenumber of spine synapses in the CA1region of hippocampus of DM groupwas significant less than that of control group. Down-regulation ofGIGYF2expression in hippocampus of diabetic mice, the cell morphologyand ultrastructural pathology of hippocampal tissue was not significantlyaltered, which was similar to normal mice hippocampal structure.ConclusionsIn this study, we established an effective mice model of diabetesmellitus through intraperitoneal injection of STZ. The high level of bloodglucose for a long time in mice could induce GIGYF2over-expression in hippocampus tissue of diabetic mice and cause diabetic mice cognitivedysfunction. Lentiviral vector delivered shRNA targeting GIGYF2to thehippocampus in vivo using stereotactic technology could efficientlyknockdown GIGYF2expression. Down-regulation of GIGYF2expressionin diabetic hippocampus had an improvement in cognitive dysfunction aswell as the amelioration of ultrastructural pathology and abnormalneurobehavioral changes, and had no obvious effect on Grb10over-expression and IGF1R low-expression, but the level ofphosphorylated IGF1R was comparable to normal level, which can beactivated IGF1R-Grb10signaling pathways. Those findings suggest thatendogenous over-expression GIGYF2interacted with N-terminus ofGrb10，possibly has a suppressive role in the regulation of cognitivedisorder in diabetes via modulating IGF1R signaling pathway. The effectsof low-GIGYF2expression in diabetes indicate that it is promising todown-regulate the level of GIGYF2expression to treat diabetes-associatedcognitive impairment caused by abnormal IGF1R signaling pathway. |
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