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

TDP-43的细胞质聚集已被报道在几乎所有年龄依赖性神经退行性变 疾病，包括>40%的额颞叶痴呆（FTD），阿尔茨海默病的海马神经元 在>90%的ALS患者中，以及在最年长的老年人中最近认识到的AD样痴呆的~100%中， 在老年人中，2019年发现了一种AD样综合征，并命名为边缘优势型AD相关TDP-43 脑病（晚期）。我们已经证明，TDP-43相分离和聚集可以驱动 不依赖于RNA结合、应激颗粒形成和TDP-43与应激相关的神经元死亡 颗粒。我们随后发现TDP-43的乙酰化（消除其RNA结合） 驱动其分离成液体球形环状体。这些核环有液态的环形壳 富含TDP-43和高度富含HSP 70家族分子伴侣的液体中心。使用 已知的脱乙酰酶或蛋白酶体的抑制剂（以模拟已知的脱乙酰酶的年龄依赖性下降 和蛋白酶体活性）引起胞质TDP-43聚集。我们建议确定 乙酰化TDP-43的病理作用以及HSP 70伴侣活性如何调节核TDP-43 功能及其在细胞质中的聚集。我们将确定的监管和生物后果， 通过鉴定关键调节酶（乙酰转移酶和脱乙酰酶）， 乙酰化的TDP-43和改变TDP-43在RNA剪接及其亚细胞定位/聚集中的功能。 为了了解HSP 70家族分子伴侣如何调节TDP-43的相变，我们将使用Hsc 70 （由HSPA 8基因和神经元中最丰富的HSP 70编码）并决定Hsc 70如何相互作用 TDP-43我们还将确定是否增强HSP 70（如HSPA 8，其高度表达）的活性， 在神经元中表达）改善TDP-43病理学。我们还将开发一种潜在的治疗方法， TDP-43蛋白病，其中实现了聚集的TDP-43的快速蛋白酶体介导的降解 通过工程化的E3泛素连接酶连接到合成进化的纳米抗体（单链 衍生自抗体重链的抗体）识别乙酰化或磷酸化TDP-43。 这些努力的结果将为理解TDP-43生物学的基本方面提供关键的见解， 常见痴呆症的病理生物学，并开发一种新的治疗概念，专门针对TDP- 43病理学可能使TDP-43相关痴呆的老年患者受益。

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.