Mitra Joy, Kodavati Manohar, Dharmalingam Prakash, Guerrero Erika N, Rao K S, Garruto Ralph M, Hegde Muralidhar L
Division of DNA Repair Research, Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX, 77030, USA.
Gorgas Memorial Institute for Health Studies, Avenida Justo Arosemena y Calle 35, Panama City, Republic of Panama.
Acta Neuropathol Commun. 2025 Mar 8;13(1):54. doi: 10.1186/s40478-025-01962-9.
TDP-43 mislocalization and aggregation are key pathological features of amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD). However, existing transgenic hTDP-43 WT or ∆NLS-overexpression animal models primarily focus on late-stage TDP-43 proteinopathy. To complement these models and to study the early-stage motor neuron-specific pathology during pre-symptomatic phases of disease progression, we generated a new endogenous knock-in (KI) mouse model using a combination of CRISPR/Cas9 and FLEX Cre-switch strategy for the conditional expression of a mislocalized Tdp-43∆NLS variant of mouse Tdp-43. This variant is expressed either in the whole body (WB) or specifically in the motor neurons (MNs) in two distinct models. These mice exhibit loss of nuclear Tdp-43, with concomitant cytosolic accumulation and aggregation in targeted cells, leading to increased DNA double-strand breaks (DSBs), signs of inflammation, and associated cellular senescence. Notably, unlike WT Tdp-43, which functionally interacts with Xrcc4 and DNA Ligase 4, the key DSB repair proteins in the non-homologous end-joining (NHEJ) pathway, the Tdp-43∆NLS mutant sequesters them into cytosolic aggregates, exacerbating neuronal damage in mouse brain. The mutant mice also exhibit myogenic degeneration in hindlimb soleus muscles and distinct motor deficits, consistent with the characteristics of motor neuron disease (MND). Our findings reveal progressive degenerative mechanisms in motor neurons expressing endogenous Tdp-43∆NLS mutant, independent of Tdp-43 overexpression or other confounding factors. Thus, this unique Tdp-43 KI mouse model, which displays key molecular and phenotypic features of Tdp-43 proteinopathy, offers a significant opportunity to characterize the early-stage progression of MND further and also opens avenues for developing DNA repair-targeted approaches for treating TDP-43 pathology-linked neurodegenerative diseases.
TDP-43的错误定位和聚集是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的关键病理特征。然而，现有的转基因hTDP-43野生型或∆NLS过表达动物模型主要关注晚期TDP-43蛋白病变。为了补充这些模型并研究疾病进展的症状前期早期运动神经元特异性病理，我们使用CRISPR/Cas9和FLEX Cre开关策略的组合，生成了一种新的内源性敲入（KI）小鼠模型，用于条件性表达小鼠Tdp-43的错误定位Tdp-43∆NLS变体。在两种不同的模型中，该变体在全身（WB）或特异性地在运动神经元（MNs）中表达。这些小鼠表现出核Tdp-43的丧失，同时在靶细胞中出现胞质积累和聚集，导致DNA双链断裂（DSB）增加、炎症迹象和相关的细胞衰老。值得注意的是，与在非同源末端连接（NHEJ）途径中与关键DSB修复蛋白Xrcc4和DNA连接酶4发生功能相互作用的野生型Tdp-43不同，Tdp-43∆NLS突变体将它们隔离到胞质聚集体中，加剧了小鼠脑中的神经元损伤。突变小鼠在后肢比目鱼肌中还表现出肌源性变性和明显的运动缺陷，这与运动神经元疾病（MND）的特征一致。我们的研究结果揭示了表达内源性Tdp-43∆NLS突变体的运动神经元中的进行性退化机制，独立于Tdp-43过表达或其他混杂因素。因此，这种独特的Tdp-43 KI小鼠模型展示了TDP-43蛋白病变的关键分子和表型特征，为进一步表征MND的早期进展提供了重要机会，也为开发针对DNA修复的方法来治疗与TDP-43病理相关的神经退行性疾病开辟了道路。
