Disease Mechanism and Therapy in TDP-43 Proteinopathies and Dementias

TDP-43的核清除和胞浆聚集几乎在每个年龄相关的人中都有报道 神经退行性疾病，包括作为最近发现的痴呆症的定义特征 老年人，一种类似AD的综合征，名为边缘占优势的年龄相关性TDP-43脑病(晚期)，a 阿尔茨海默病(AD)海马神经元的比例占额叶颞叶痴呆的40% (Ftd)和90%的ALS病例。我们先前的努力(我们现在寻求新的支持)已经 建立了瞬时应力可以诱导细胞质TDP-43的液-液相分离(LLP)进入 液滴随后转变为固态，缓慢耗尽核TDP-43，并在一年内引发细胞死亡 周的时间刻度。我们还确定了部分或完全的蛋白酶体抑制(模仿已建立的 正常衰老期间蛋白小体活性下降)引发TDP-43错误定位/在 细胞质。定量质谱学(用邻近标记和等压标记)已经确定了小分子 热休克蛋白HSPB1是胞质TDP-43相分离及其后续调控因子 聚合。HSPB1分解成TDP-43小滴，抑制TDP-43组装成纤维，并介导 应力诱导的TDP-43液滴的解体。在我们先前和继续工作的基础上，我们现在提议 确定1)蛋白酶体活性的年龄相关性降低如何导致TDP-43功能丧失， 细胞质错误定位、相分离和聚集以及2)蛋白质伴侣HSPB1是如何在 与HSP70结合，影响细胞质TDP-43的相分离，抑制TDP-43组装成纤维， 并介导TDP-43液滴的解体。我们还开始开发一种方法，以生成 一过性抑制RNA结合蛋白在老年成年小鼠脑内的新神经元/替代神经元 使用由A细胞递送的反义寡核苷酸的多嘧啶结合蛋白-1(PTB) 脑脊液(CSF)一次注射。放射状胶质样细胞(可能还有其他表达GFAP的细胞 细胞)在两个月内转化为新的神经元，获得成熟的神经元特征，并在功能上 整合到改变鼠标行为的内源性回路中。在这里，我们将系统地识别 经历身份转换的细胞的功能、定位、细胞来源、时间和分子途径， 空间标记单细胞RNA标记的初步开发和利用 转录学(多重错误-稳健的荧光原位杂交[MerFish])。

Nuclear clearance and cytoplasmic aggregation of TDP-43 have been reported in almost every age-dependent neurodegenerative disease, including as the defining feature of a recently recognized dementia in the oldest of the elderly, an AD-like syndrome named Limbic-predominant Age-related TDP-43 Encephalopathy (LATE), a proportion of the hippocampal neurons in Alzheimer's disease (AD), >40% of frontal temporal dementia (FTD), and >90% of instances of ALS. Our prior efforts (for which we now seek renewed support) have established that transient stress can induce Liquid-Liquid Phase Separation (LLPS) of cytoplasmic TDP-43 into liquid droplets that then transition to a solid state, slowly deplete nuclear TDP-43, and provoke cell death over a timescale of weeks. We also determined that partial or complete proteasome inhibition (to mimic the established decline in proteosome activity during normal aging) provokes TDP-43 mislocalization/accumulation within the cytoplasm. Quantitative mass spectrometry (with proximity-labeling and isobaric-tagging) has identified the small heat shock protein HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and subsequent aggregation. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and mediates disassembly of stress-induced, TDP-43 droplets. Building on our prior and continuing work, we now propose to determine 1) how the age-dependent decrease in proteasome activity drives TDP-43 loss of function, cytoplasmic mislocalization, phase separation, and aggregation and 2) how protein chaperone HSPB1, in conjunction with HSP70, affects cytoplasmic TDP-43 phase separation, inhibits TDP-43 assembly into fibrils, and mediates disassembly of TDP-43 droplets. We have also initiated development of an approach to generate new/replacement neurons in the aged adult mouse brain by transiently suppressing the RNA binding protein Polypyrimidine Tract Binding Protein-1 (PTB) using an antisense oligonucleotide (ASO) delivered by a single injection into cerebral spinal fluid (CSF). Radial glial-like cells (and possibly other GFAP-expressing cells) convert into new neurons over a two month period, acquire mature neuronal character, and functionally integrate into endogenous circuits that modify mouse behavior. Here we will systematically identify the functionality, localization, cell origin, timing, and molecular pathways of cells undergoing identity conversion, with a primary assay the development and utilization of single cell RNA signatures obtained with spatial transcriptomics (Multiplexed Error-Robust Fluorescence In Situ Hybridization [MERFISH]).