Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

肌萎缩侧索硬化症 (ALS) 是运动神经元疾病 (MND) 的一种形式，其特征是脊髓、脑干和运动皮层内的运动神经元逐渐丧失。尽管 ALS 临床上表现为一种异质性疾病，发病率和生存率各不相同，但一个共同特征是存在含有 TDP-43 的泛素化细胞质蛋白包涵体聚集体。然而，目前人们对蛋白质内含物和聚集与神经元损失之间联系的确切机制知之甚少。双分子荧光互补 (BiFC) 利用附着在感兴趣的易于聚集的蛋白质上的荧光团片段（本身不发出荧光）的关联。感兴趣的蛋白质的相互作用使得荧光报告蛋白折叠成其天然状态并发出荧光信号。在这里，我们将 BiFC 的强大功能与斑马鱼系统的优势相结合，在脊椎动物模型中实时验证、优化和可视化 ALS 相关聚集体的形成。我们进一步提供了该技术选择性的体内验证，并证明通过引入荧光团突变减少了体内非荧光片段的自发自组装。此外，我们使用 BiFC 报告了 ALS 相关标志蛋白 Fus 和 TDP-43 在其相应核和细胞质区室中动态聚集的初步发现。总体而言，我们的数据证明了这种 BiFC 方法适用于研究和表征体内 ALS 相关聚集体的形成。 重要的是，相同的原理可以应用于其他神经退行性疾病，因此对于增进我们对异常蛋白质聚集背后的病理学的理解具有重要意义。