前脑特异敲除PDK1抑制阿尔茨海默病小鼠淀粉样斑块形成的机理研究

Aβ accumulation and plaque deposition are believed to play a central role in the pathogenesis of Alzheimer’s disease (AD), characterized by massive neuron loss and the presence of amyloid plaques and neurofibrillary tangles. It has been controversial how PDK1 is involved in the pathogenesis of AD. In our preliminary study, we found that conditional inactivation of PDK1 in forebrain neurons and glia of 5×FAD or 2×AD resulted in no formation of amyloid plaques in the cortex and the hippocampus. This finding has prompted us to propose the following hypothesis: inhibition of the PDK1 signaling may diminish AD pathology and provide beneficial effects on cognitive function. To test it, we generated forebrain mature neuron specific PDK1 cKO mice and crossed them to 5×FAD model to obtain PDK1 cKO/5×FAD animals. We are investigating whether plaque burden is reduced and whether learning and memory is improved by inactivation of PDK1. To study whether PDK1 affects Aβ production via the PDK1-Akt signaling pathway, we generated mature neuron specific Akt cTKO/5×FAD mutant mice. We will examine whether Akt cTKO/5×FAD mutants phenocopy PDK1 cKO/5×FAD mice. Finally, we will take advantage of cell lines expressing human mutant APP and primary neuron cultures from newborn mice of PDK1 cKO/5×FAD and Akt cTKO/5×FAD to investigate molecular mechanisms by which PDK1 regulates APP expression and Aβ production/accumulation. In summary, this study may provide insights on the pathogenesis for AD and help identify novel therapeutic targets for the prevention and treatment of this disorder.

最近研究发现在AD大脑中PDK1的活性增强，但PDK1参与衰老及AD病发的机制尚不清楚。在前期实验中我们发现胚胎期条件敲除AD小鼠前脑中PDK1可以完全预防淀粉样斑块形成而成年阶段条件敲除PDK1也能够显著降低皮层中淀粉样斑块数目。因此我们提出“条件敲除PDK1可以预防和治疗AD”。为了进一步验证该假说，我们将在本项目中完成下述工作。1.研究衰老对成年阶段敲除了PDK1的AD小鼠病理、突触功能和认知能力的影响。2.利用培养的细胞系、原代神经元和N2a细胞研究PDK1调控APP表达和Aβ产生的分子机制。3.研究Akt抑制剂是否对AD病理有改善作用。4.研究衰老对成年前脑条件敲除Akt三个亚型的AD小鼠病理的影响。通过利用分子、生化、电生理和行为学等手段，我们将从分子、细胞和整体水平来研究PDK1/Akt在AD病发过程中的作用。上述研究有助于深入理解AD的致病机理并可能发现治疗AD的新靶点。

尽管目前很多证据表明AD大脑中PDK1活性与对照相比明显增强，但是PDK1参与AD病发的机制还不清楚。本项目旨在研究抑制PDK1功能对AD小鼠模型的病理及认知功能的影响。首先，我们利用CaMKIIα-Cre小鼠介导了PDK1在成年5×FAD小鼠皮层及海马中的条件敲除。我们发现在兴奋性神经元中条件敲除PDK1显著地减少了淀粉样斑块在皮层及海马中的累积并明显地改善了小鼠的空间工作记忆。其次，我们利用Emx1-Cre小鼠介导了PDK1在胚胎期5×FAD小鼠前脑神经元中的条件敲除。我们发现胚胎期开始的PDK1条件敲除极显著地抑制了淀粉样斑块在皮层中的形成。再次，我们发现在5×FAD小鼠皮层中条件敲除PDK1可以显著地减轻大脑中神经炎症反应。另外，我们还发现条件敲除PDK1导致5×FAD小鼠皮层中的APP表达水平显著降低。最后，我们开展的分子生物学分析显示PDK1通过核糖体途径来调节APP的表达。上述结果提示利用遗传学手段使PDK1失活可能是一种潜在的用于预治AD的策略。本项目研究有助于深入理解AD的致病机理并发现AD治疗的新靶点。