The Potential Role Of GRK5 Deficiency In The Pathogenesis Of Early Alzheimer's Disease

Part 1 Worsened Alzheimer-like pathology in female GRK5 deficient micebackground: Recent studies suggested that G protein-coupled receptor kinase 5 (GRK5) deficiency plays a significant role in early Alzheimer's disease (AD) pathogenesis, and that GRK5 knockout/deficiency (GRK5KO) mouse displays an early Alzheimer-like cognitive deficit associated with increased hippocampal axonal defects and synaptic degenerative changes. Gender is known to play a role in AD, with female showing more extensive pathologic changes in brain as compared to male. Although GRK5 deficiency is linked to AD, it is unknown whether the pathologic changes solely driven by the GRK5 deficiency are gender-differential. Objective(s): The objective of this study is to determine whether the Alzheimer-like pathologic changes solely driven by the GRK5 deficiency are gender-differential and the potential molecular mechanism. Methods: Campbell-Switzer silver staining and immunofluorescent (IF) staining methods were used to observe the pathology in the hippocampus. Western blotting was used to determine the hippocampal levels of synaptic proteins. The extent of the pathologic changes in aged GRK5KO mice was compared between genders. Results: It was found that the female GRK5KO mice had a 2.5-fold increase in hippocampal swollen axonal clusters compared to male GRK5KO mice. Moreover, hippocampal levels of several synaptic proteins, including synaptophysin, were significantly lower in the female than the male. In addition, although increased Luteinizing hormone (LH) activity is believed to play a significant role in the gender phenomenon in AD, the results in our experiment showed that desensitization of LH receptor was not affected by the GRK5 deficiency. Therefore, the worsened pathologic changes in the female mice cannot be attributed to an impaired LH receptor desensitization. Conclusions: Taken together, this study demonstrates a synergistic interaction between GRK5 deficiency and gender in promoting early AD-like pathologic changes in the female GRK5KO mice, although the underlying molecular mechanisms remain to be elucidated.Part 2 GRK5 deficiency exaggerates inflammatory changes in Tg2576 miceBackground: Deficiency of membrane G-protein coupled receptor (GPCR) kinase-5 (GRK5) has been recently linked to early AD pathogenesis, and has been suggested to contribute to augmented microglial activation in vitro by sensitizing relevant GPCRs. Although the GRK5 deficiency indeed caused moderate increase in axonal defects and synaptic degenerative changes and a mild increase ofβ-amyloid (Aβ) level, no fibrillar Aβdeposits or any significant signs of microglial activation were observed in the GRK5 knockout/deficiency (GRK5KO) mice. One possible reason for the absence of microgliosis in these animals we proposed previously is that deficiency of GRK5 in vivo may be largely compensated by other GRK members; another reason might be due to lack of inflammatory initiators, such as fibrillar Aβ, because that GRK only acts when GPCR is activated. Objective(s): The objective of this study was to determine whether the microgliosis is exaggerated in TgAPPsw (Tg2576) mice deficient in GRK5, in which fibrillarβ-amyloid (Aβ) and an active inflammatory process involving activated GPCR signaling are present, and to investigate the potential mechanism why inflammation is exaggerated. Methods: The heterozygotes of GRK5KO mice with a C57/BL6 background were bred with Tg2576 mice also with a C57/BL6 background to produce wild type (WT), GRK5KO, amyloid precursor protein (APP) and the double mice for this study. Both quantitative and qualitative immunofluorescent (IF) staining methods were used to evaluate the microgliosis and astrogliosis in these animals. Qualitative IF staining and Western blotting were used to evaluate the changes of Aβin these animals. Results: Inactivation of one copy of the GRK5 gene in the TgAPPsw mice resulted in approximately doubled extent of microgliosis and Aβplaque deposits (including Aβburden and number), along with significantly exaggerated astrogliosis, in both hippocampus and cortex of the aged animals. Consistent with previous observations, the activated microglia and astrocytes were located primarily near or surrounding the fibrillar Aβdeposits. However, IF staining revealed no significant increase of the gliosis and fibrillar Aβdeposits in hippocampus or cortex of WT or GRK5KO mice. The determination of Aβby Western Blotting was consistent with the result of IF staining. Spearman correlation analyses showed a highly significantly positive correlation between fibrillar Aβplaque burden/number and microglia/astrocyte number respectively. Conclusions: The results demonstrate that knockdown GRK5 gene by 50% in the Tg2576 mice significantly exaggerates inflammatory reaction and Aβdeposits in the brain of these animals. The reason why GRK5 deficiency exaggerates inflammatory changes in TgAPPsw mice is unlikely that GRK5 deficiency mediate inflammatory regulators but inflammatory initiators (fibrillar Aβ).Part3 GRK5 deficiency acceleratesβ-amyloidogenesis in Tg2576 mice by enhancing amyloid precursor protein processingObjective: The objective of this study was to investigate the molecular mechanisms that the membrane G-protein coupled receptor (GPCRs) kinase-5 (GRK5) deficiency in vivo acceleratesβ-amyloid peptide (Aβ) deposition in TgAPPsw (Tg2576) mice. Methods: The heterozygotes of GRK5/deficient mice (GRK5KO) with a C57/BL6 background were bred with TgAPPsw (Tg2576) mice also with a C57/BL6 background to produce amyloid precursor protein (APP, APPsw+/-/GRK5+/+), and the double (APPsw+/-/GRK5+/-) mice for this study. The soluble APP alpha (sAPPα) and full length APP (flAPP) were observed in 6-month-old female APP and Double mice and the primary culture cortical neuron of 17-day-old APP and Double embryo mice. Results: The results revealed a significant decrease of sAPPαin cortex of the 6-month-old female Double mice as compared to APP mice (p<0.05). But the flAPP levels in the cortex were not significantly different between APP and Double mice. The changes of sAPPαand flAPP in primary culture cortical neuron of 17-day-old APP and Double embryo mice were consistent with the results in 6-month-old APP and Double mice. However, specific antagonist against acetylcholine muscarinic receptor 2 (M2) can remarkably reverse the decrease of sAPPαin primary culture cortical neuron of 17-day-old Double embryo mice. Conclusions: The lack of membrane (functional) GRK5 in vivo results in an impaired M2 receptor desensitization and acceleratesβ-amyloidogenesis in Tg2576 mice by enhancing the APP processing.