The influence of Kallikrein-8 on Alzheimer disease-related neuroplasticity defects and tauopathy

我们先前发现激肽释放酶-8(KLK8)在阿尔茨海默病(AD)的发病机制中具有潜在的关键作用。我们可以在AD患者和转基因(TG)CRND8小鼠受AD影响和几乎不受影响的脑区检测到AD早期阶段过高的KLK8mRNA和蛋白水平。此外，我们可以证明，在中度疾病阶段用抗KLK8抗体短期抑制大脑KLK8(仅为期四周)足以缓解TG小鼠阿尔茨海默病的多种病理特征。独立研究表明，KLK8通过底物周转(处理EPHB2、L1-CAM、NeuRegin-1、FN)、调节突触标记过程或通过与结合伙伴(如PEBP-1)形成复合体来调节结构和电化学神经可塑性。上述所有KLK8互动合作伙伴也都参与了AD。我们自己的实验室工作表明，在TG脑中阻断KLK8可以增加可塑性控制分子的表达，增加树突棘密度和树突复杂性，最终改善TG小鼠的记忆。微管相关蛋白tau参与AD相关的神经退行性变和结构可塑性，也与KLK8信号有关。配体介导的KLK8底物EPHB2的激活抑制了tau的过度磷酸化，而KLK8下游靶标FKBP5则引发tau的寡聚化并加速了神经毒性。我们自己实验室的工作证明，KLK8抑制可以保护EPHB2不被降解，并减少TG小鼠的tau病理。通过目前的建议，我们首先旨在验证KLK8抑制在AD中的治疗潜力。因此，我们将通过与KLK8基因敲除小鼠杂交，然后进行行为表型(空间和识别记忆、激动和焦虑)和淀粉样β病理的量化，从基因上永久性地降低Tg小鼠中KLK8的表达。其次，我们将重点研究KLK8在AD相关神经可塑性障碍中的作用。因此，我们将在体内和体外研究短期抑制KLK8、永久性减少KLK8或诱导KLK8对神经元的增殖、分化和存活、树突的复杂性和棘突密度，以及对KLK8底物的更新、突触标记和复合体形成的影响。第三，我们将研究KLK8在tau代谢中的作用，从而测试KLK8抑制、KLK8还原和KLK8诱导对生理性和病理性tau磷酸化、神经元内tau分布以及tau调节EPHB2依赖和非EPHB2信号通路的体内和体外影响。该项目有可能验证KLK8作为治疗阿尔茨海默病的靶点，并在KLK8、神经可塑性和tau代谢之间的相互作用方面获得新的机制见解。

We previously identified Kallikrein-8 (KLK8) as a potential key player in the pathogenesis of Alzheimer disease (AD). We could detect excessive KLK8 mRNA and protein levels at incipient stages of AD, in both AD-affected and scarcely-affected brain areas of AD patients and transgenic (Tg) CRND8 mice. Moreover, we could show that short-term inhibition of cerebral KLK8 (for only four weeks) by an anti-KLK8 antibody at a moderate disease stage was sufficient to mitigate multiple features of Alzheimer pathology in Tg mice. Independent studies have shown that KLK8 modulates structural and electrochemical neuroplasticity by substrate turnover (processing of EPHB2, L1-CAM, neuregulin-1, fibronectin), modulation of synaptic tagging processes or by complex formation with binding partners such as PEBP-1. All of the aforementioned KLK8 interaction partners are also involved in AD. Work of our own lab has shown that KLK8 blockade in Tg brain increases expression of plasticity-controlling molecules, dendritic spine density and dendritic complexity, ultimately improving memory in Tg mice. The microtubule-associated protein tau, involved both in AD-related neurodegeneration and structural plasticity, is also connected to KLK8 signaling. Ligand-mediated activation of the KLK8 substrate EPHB2 inhibits tau hyper-phosphorylation, whereas the KLK8 down-stream target FKBP5 provokes tau oligomerization and accelerates neurotoxicity. Work in our own lab demonstrated that KLK8 inhibition protected EPHB2 from degradation and reduced tau pathology in Tg mice. With the present proposal we first aim to verify the therapeutic potential of KLK8 inhibition in AD. Therefore, we will genetically and permanently reduce KLK8 expression in Tg mice by crossbreeding with KLK8-knockdown mice, followed by behavioral phenotyping (spatial and recognition memory, agitation and anxiety) and quantification of amyloid beta pathology. Second, we will focus on the role of KLK8 in AD-related neuroplasticity disturbances. Accordingly, we will examine the in vivo and in vitro effects of short-term KLK8 inhibition, permanent KLK8 reduction or induction of KLK8 on neuronal proliferation, differentiation, and survival, dendritic complexity and spine density, as well as on KLK8 substrate turnover, synaptic tagging and complex-formation. Third, we will investigate the function of KLK8 in tau metabolism and therefore test the in vivo and in vitro effects of KLK8 inhibition, KLK8 reduction and KLK8 induction on physiologic and pathologic tau phosphorylation, intraneuronal tau distribution, and on tau-modulating EPHB2-dependent and EPHB2-independent signaling pathways. This project has the potential to verify KLK8 as a therapeutic target against AD and to gain new mechanistic insights in the interplay between KLK8, neuroplasticity and tau metabolism.