TDP-43 acetylation, phase separation, aggregation, and clearance by antibody-mediated degradation

据报道，TDP-43在几乎每一种年龄依赖性神经退行性变中都有胞浆聚集现象。 阿尔茨海默病患者海马神经元中的疾病，包括40%的额叶颞叶痴呆(FTD) 疾病(AD)患者，90%的ALS患者，以及最近发现的最老的AD样痴呆患者的~100% 在老年人中，2019年发现了一种AD样综合征，并将其命名为边缘主导的年龄相关TDP-43 脑病(晚期)。我们已经证明了TDP-43的相分离和聚集可以驱动 与RNA结合、应激颗粒形成和TDP-43与应激相关的神经元死亡无关 颗粒。我们随后发现TDP-43的乙酰化(它取消了其RNA结合) 驱使其分离成液体球形环状物体。这些核环有液体环壳。 富含TDP-43，液体中心高度富含HSP70家族分子伴侣。使用 已知的脱乙酰酶或蛋白酶体的抑制剂(以模拟已知的年龄依赖性脱乙酰酶下降 和蛋白酶体活性)引起细胞质TDP-43聚集。我们建议确定生物学上的 乙酰化TDP-43的病理作用(S)以及热休克蛋白70分子伴侣活性如何调节核TDP-43 功能及其在细胞质中的聚集。我们将确定其调节和生物学后果 通过确定关键的调节酶(乙酰转移酶和脱乙酰酶)在神经元中乙酰化TDP-43 乙酰化TDP-43和Alter TDP-43‘S在核糖核酸剪接及其亚细胞定位/聚集中的作用。 为了了解HSP70家族分子伴侣如何调节TDP-43的相变，我们将使用Hsc70 (由HSPA8基因和神经元中含量最丰富的HSP70编码)，并确定Hsc70如何相互作用 使用TDP-43。我们还将确定是否增强HSP70的活性(如HSPA8，它是高度 在神经元中表达)改善TDP-43的病理。我们还将开发一种潜在的治疗方法 蛋白酶体快速降解聚集的TDP-43的TDP-43蛋白病 通过与人工进化的纳米体(单链)连接的工程E3泛素连接酶 来自抗体重链的抗体)识别乙酰化或磷酸化的TDP-43。 这些努力的结果将为理解TDP-43生物学的基本方面和 常见痴呆症的病理生物学，并开发一种专门针对TDP的新治疗概念- 43可能使患有TDP-43相关痴呆的老年患者受益的病理学。

Cytoplasmic aggregation of TDP-43 has been reported in almost every age-dependent neurodegenerative disease, including in >40% of frontal temporal dementia (FTD), in the hippocampal neurons of Alzheimer's disease (AD) patients, in >90% of ALS, and in ~100% of a recently recognized AD-like dementia in the oldest of the elderly, an AD-like syndrome identified in 2019 and named Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). We have demonstrated that TDP-43 phase separation and aggregation can drive neuronal death independent of RNA binding, stress granule formation, and TDP-43 association with stress granules. We have subsequently identified that acetylation of TDP-43 (which abolishes its RNA binding) drives its separation into liquid spherical annular bodies. These nuclear annuli have liquid annular shells enriched in TDP-43 and liquid centers highly enriched in HSP70 family molecular chaperones. Use of inhibitors of known deacetylases or the proteasome (to mimic the known age-dependent declines in deacetylase and proteasome activities) provokes cytoplasmic TDP-43 aggregation. We propose to determine the biological and pathological role(s) of acetylated TDP-43 and how HSP70 chaperone activity regulates nuclear TDP-43 function and its aggregation in the cytoplasm. We will determine the regulation and biological consequences of acetylated TDP-43 in neurons by identifying the key regulatory enzymes (acetyltranferases and deacetylases) of acetylated TDP-43 and alter TDP-43’s function in RNA splicing and its subcellular localization/aggregation. To understand how HSP70 family molecular chaperones regulates phase transition of TDP-43, we will use Hsc70 (encoded by the HSPA8 gene and the most abundant HSP70 in neurons) and determine how Hsc70 interacts with TDP-43. We will also determine if enhancing the activity of HSP70 (such as HSPA8, which is highly expressed in neurons) ameliorates TDP-43 pathology. We will also develop a potential therapeutic approach for TDP-43 proteinopathies in which rapid proteasome-mediated degradation of aggregated TDP-43 is achieved through an engineered E3 ubiquitin ligase linked to a synthetically evolved nanobody (a single chain antibody derived from an antibody heavy chain) recognizing either acetylated or phosphorylated TDP-43. Outcomes of these efforts will provide key insights for understanding basic aspects of TDP-43 biology and pathobiology in common dementia, and for developing a new concept of therapy that specifically targets TDP- 43 pathology that could potentially benefit aged patients with TDP-43-related dementia.