Disease Mechanism and Therapy in TDP-43 Proteinopathies and Dementias

TDP-43的核清除和细胞质聚集已被报道在几乎所有年龄依赖性 神经退行性疾病，包括作为最近认识到的老年痴呆症的定义特征， 老年人，一种AD样综合征，称为边缘优势型脑血管病相关TDP-43脑病（LATE）， 阿尔茨海默病（Alzheimer's disease，AD）海马神经元的比例> 40%，额颞叶痴呆（front-temporal dementia，AD）海马神经元的比例>40%， (FTD)，和>90%的ALS实例。我们先前的努力（我们现在寻求新的支持） 建立了瞬时应力可以诱导细胞质TDP-43的液-液相分离（LLPS）， 然后转变为固态的液滴，缓慢耗尽细胞核TDP-43，并在一段时间内引起细胞死亡。 周的时间尺度。我们还确定了部分或完全的蛋白酶体抑制（模拟已建立的 正常衰老过程中蛋白体活性的下降）引起TDP-43在细胞内的错误定位/积累。 细胞质定量质谱法（邻近标记和同量异位素标记）已经确定了小的 热休克蛋白HSPB 1作为胞质TDP-43相分离和随后的相分离的调节剂 聚合来HSPB 1分配到TDP-43液滴中，抑制TDP-43组装成原纤维，并介导 应力诱导的TDP-43液滴的分解。在我们先前和持续开展的工作的基础上，我们现提议 确定1）蛋白酶体活性的年龄依赖性降低如何驱动TDP-43功能丧失， 细胞质错误定位，相分离和聚集，以及2）蛋白伴侣HSPB 1如何在 与HSP 70结合，影响胞质TDP-43相分离，抑制TDP-43组装成原纤维， 并介导TDP-43液滴的分解。我们还开始制定一种方法， 通过瞬时抑制RNA结合蛋白在老年成年小鼠脑中的新/替代神经元 使用由反义寡核苷酸（阿索）递送的聚嘧啶道结合蛋白-1（PTB） 单次注射入脑脊液（CSF）。放射状胶质样细胞（以及可能的其他GFAP表达细胞） 细胞）在两个月的时间内转化为新的神经元，获得成熟的神经元特征，并在功能上 整合到改变小鼠行为的内源性回路中。在这里，我们将系统地识别 功能性、定位、细胞起源、时间和经历身份转换的细胞的分子途径， 一个主要的分析，开发和利用单细胞RNA签名获得的空间 转录组学（多重误差稳健荧光原位杂交[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]).