泛素连接酶Hrd1通过“p62-LC3-自噬途径”介导tau降解的机制研究

Intraneuronal accumulation of abnormal phosphorylated tau is a molecular pathology in many neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism-linked to chromosome 17 (FTDP-17). However, the underlying mechanism remains unclear. The dysfunction of tau degradation under some conditions at least partially contributes to abnormal tau accumulation. Therefore, targeting tau protein accumulation, such as increasing tau clearance is a potential therapeutic approach for treating in Alzheimer's disease. Human hrd1 (Hrd1) is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytically active RING finger facing the cytosol, mediating protein ubiquitination and degradation. Our previous study showed that Hrd1 functions as an E3 targeting tau or abnormal p-tau for proteasome degradation，while the mechanism except the proteasome contribution in the process of tau degradation was not further explored. Our preliminary study showed that treatment of autophagy inhibitor NH4CL or 3-MA blocked Hrd1 mediated degradation of tau，which indicates that autophagy maybe involved in Hrd1 mediated tau degradation. To test this hypothesis, we investigated 1.The effect of Hrd1 overexpression or Hrd1 knockdown on degradation of tau through the autophagy pathway; 2.The effect of autophagy inhibition on degradation of tau; 3. Whether p62-LC3-autophagy pathway was involved in Hrd1-mediated tau degradation; 4. Hrd1 could directly ubiqutinate tau via K48-linked ubiquitin chains or k63-linked ubiqutin chains. 5. Effects of autophagy inhibition on Hrd1-dependent protection against tau induced toxicity. Thus it would be helpful not only to further elucidate the molecular mechanisms of Hrd1 mediated tau degradation, but also to offer theoretic basis for drug screening and therapy for patients with tauopathies based on Hrd1 target.

神经细胞内tau异常表达或修饰引起的tau聚集是tau蛋白病共有的病理特征，促进tau降解是减少tau聚集的有效途径。我们首次发现，泛素连接酶Hrd1通过泛素-蛋白酶体通路(UPP)促进tau降解；自噬抑制能稳定Hrd介导的tau降解，推测自噬可能是Hrd1介导tau降解的一个新机制。迄今，本研究思路国内外未见报道。为此，我们拟进行以下研究：过表达Hrd1或用siRNA下调内源性Hrd1，观察Hrd1对tau自噬降解和细胞自噬活性的影响；自噬抑制是否影响Hrd1介导的tau降解；探讨Hrd1介导tau降解是否和p62-LC3-自噬途径有关；弄清Hrd1通过自噬途径介导tau自噬时，Hrd1泛素化tau的主要形式是K48还是K63链接的多聚泛素链;观察自噬抑制对Hrd1的细胞保护作用是否有影响。该研究可能为阐明Hrd1介导tau 降解的机制提供新思路，也为tau蛋白病治疗提供新的候选靶点。

1. 证明自噬参与tau蛋白降解。神经来源的N2a细胞转染tau真核表达质粒，磷酸酯酶抑制剂冈田酸诱导tau磷酸化。在自噬活性抑制的细胞中tau与自噬体、溶酶体共定位增加；自噬抑制剂NH4Cl增加tau和磷酸化tau水平，反之，自噬诱导剂雷帕霉素（Rapamycin，Rapa）显著降低tau和磷酸化tau水平，而对内源性tau无明显影响。CHX实验证实激活自噬促进tau和磷酸化tau降解，减轻tau的细胞毒作用。此外，我们发现不同自噬诱导方式对磷酸化tau降解作用不同。两种经典的自噬诱导剂Rapa和EBSS均能改善表达磷酸化tau细胞形态，减少磷酸化tau聚集，抑制细胞凋亡。但Rapa诱导的自噬主要降解高分子量磷酸化tau寡聚体，而EBSS主要促进低分子量的磷酸化tau降解。2. 证明泛素连接酶Hrd1介导tau的自噬降解；tau和Hrd1质粒转染N2a细胞，Hrd1能促进tau和自噬体的共定位；并增加细胞自噬活性。采用多种自噬抑制剂如Baf A1，3-MA和NH4Cl或自噬诱导剂如Rapa，Torin1，CBZ和EBSS，观察Hrd1对tau和磷酸化tau降解的影响。结果发现自噬抑制能部分逆转Hrd1介导的tau 降解；自噬缺陷的Atg5-/-细胞株中也同样观察到这一现象，提示自噬参与Hrd1介导的tau降解过程。用siRNA下调内源性Hrd1后tau水平保持稳定；同样E3活性缺失的Hrd1突变体不能促进tau的自噬降解，提示Hrd1的E3活性是其介导tau自噬降解的先决条件；但有趣的是，自噬抑制对磷酸化tau降解无明显影响，反之，蛋白酶体抑制能增加磷酸化tau水平，提示Hrd1介导磷酸化tau通过蛋白酶体降解。3. 探讨Hrd1介导tau 自噬降解的机制；采用表达不同泛素突变体的质粒转染N2a细胞，co-IP实验证实Hrd1 主要介导tau的K63 位泛素链修饰；Hrd1增加tau和p62的相互作用，而Hrd1 突变体不能增加tau和p62的相互作用，提示Hrd1介导tau的K63位泛素化并促进其与P62结合，进而通过自噬降解。4. 自噬抑制取消Hrd1对表达tau细胞的保护作用：Hrd1能减轻tau的细胞毒作用，改善表表达tau细胞的形态，减少细胞凋亡；而自噬抑制后，Hrd1的细胞保护作用减弱甚至消失，提示Hrd1促进tau自噬降解进而发挥细胞保护作用。