Grb10-mediated Negative Regulation Of IGF1-activated Signaling Pathway Results In Cognitive Disorder In Diabetic Rats

Background:Growth factor receptor-bound protein10(Grb10)is a Src homology2(SH2) domain-containing protein and one of the binding partners for severaltrans-membrane tyrosine-kinase receptors, including the insulin-like growthfactor receptor(IGF1-R) and the insulin receptor(IR). In brain, one of themain distribution areas of Grb10is hippocampus, which is involved incognitive progress. In recent years, diabetic encephalopathy has beendefined as a third type of diabetes and IGF1-IR pathway has been shown tobe critical for neuropathogenic process of cognitive disorder in diabetes.However, the role of endogenous Grb10in regulating IGF1-IR pathway andneurobehavioral changes is inexplicit.Objectives:The objective of this study was to observe the effect of downregulationof Grb10on neurobehavioral changes and and pathological changes inhippocampus, as so to determine the in vivo function of endogenous Grb10in diabetes encephalopathy and the underlying mechanisms. Methods:Diabetes was induced through a single i. p. injection (70mg/kg bodyweight) of Streptozotocin in0.1M sodium citrate-hydrochloric acid buffersolution (pH4.5). as soon as the rats with capillary blood fastingblood-glucose of over18mmol/L were considered to be diabetes mellitusgroup, they were sham-knocked down (DM+0and con+0) and non-operated(DM and con)as comparisonforthetest (DM+shRNAand con+shRNA)groups (n=10per group). Using short hairpin RNA (shRNA) carried bylentiviral vector and stereotaxic surgery, we demonstrated an effective wayto down regulate the target gene in hippocampus steadily in vivo. After3months, the cognition ability (examined by Morris water maze test) and theexpression of Grb10(evaluated by qPCR and Western Blot) were matchedwith the Ultrastructure changes and pathological alteration.Results:1. Effect of Grb10knocked down on diabetic encephalopathy-relatedbehavioral pathologies and protein expressionLevels of Grb10mRNA and protein were examined by using qPCR andWestern Blotting, respectively, to evaluate the down-regulated effectivenessof local application of Grb10-shRNA in vivo. In the presence ofGrb10-shRNA, significant lower Grb10mRNA level and protein levelswere confirmed (P＜0.05or0.01), while surgery alone had no effectson the expression of Grb10. 2. Effect of Grb10-shRNA on the Morris Water Maze testThe Stereotaxic surgery had no significant effect on the Morris watermaze (P＞0.1). No significant difference in the mean EL among the groupswas observed when the diabetes groups were just induced (P=0.13). Thedecline in DM+shRNA group and control groups occurred on three monthsafter the i.p. injection of STZ, confirmed the successful establishment ofdiabetic encephalopathy. However, no obvious difference was everobserved between the control and the con+shRNA groups, thus ruling outthe possibility that the decline was due to surgery procedures.Regarding probe trials, a significant difference was found in timespent in the target quadrant between the groups (P＜0.05or0.01). Timespent was decreased in DM group and the DM+0groups, but with nosignificant differences between them. Meanwhile, there were no differencesbetween the DM+shRNA group and the control groups.3. The immunohistochemical and ultrastructure in hippocampusThe continuous exposure to hyperglycemia resulted in the relativepathological changes such as a decrease in neuron numbers and an increasein neuron apoptosis in the hippocampal examined. Quantitatively andmorphologically, we showed that neurons in hippocampal areas of Grb10knocked-down group remained regular. However, there was significantdifference on direct comparisons revealed, which was correlated with theGrb10expression level. The morphological ordinary of the tissue was not altered in response toGrb10knocked-down. As expected, results from electron microscopeindicated that the number of spine synapses of DM+shRNA group wasobviously more than that of DM group, since DM rats revealed a significantloss in the number of spine synapses in the CA1region.Conclusions:Using the methods of this study, we found an effective model ofdiabetic encephalopathy. The modulation of Grb10protein level in brain ledto a prominent remission of cognitive disorder, including improvement inboth ultrastructural pathology and abnormal neurobehavioral changes. Ourfindings indicate that endogenous overexpression of Grb10functions as asuppressor of IGF1-IR pathway, which may represent an importantmechanism that regulates cognitive disorder in diabetes. Moreover, wedemonstrate that lack of IGF-1support caused by overexpress of Grb10indiabetes induces pathological changes from ultrastructural to animalbehavioral characteristics. Together with previous observations that IGF1-IRand downstream regulation of Grb10regulator are related to the ADpathogenesis, the neuroprotective effects from low-Grb10expression indiabetes may represent a new and crucial therapeutic target for the treatmentof IGF1-responsive cognitive disorder.