Abstract
Given the mounting evidence implicating TDP-43 dysfunction in several neurodegenerative diseases, there is a pressing need to establish accessible tools to sense and quantify TDP-43 loss-of-function (LOF). These tools are crucial for assessing potential disease contributors and exploring therapeutic candidates in TDP-43 proteinopathies. Here, we develop a sensitive and accurate real-time sensor for TDP-43 LOF: the CUTS (CFTR UNC13A TDP-43 Loss-of-Function) system. This system combines previously reported cryptic exons regulated by TDP-43 with a reporter, enabling the tracking of TDP-43 LOF through live microscopy and RNA/protein-based assays. We demonstrate CUTS' effectiveness in detecting LOF caused by TDP-43 mislocalization and RNA binding dysfunction, and pathological aggregation. Our results highlight the sensitivity and accuracy of the CUTS system in detecting and quantifying TDP-43 LOF, opening avenues to explore unknown TDP-43 interactions that regulate its function. In addition, by replacing the fluorescent tag in the CUTS system with the coding sequence for TDP-43, we show significant recovery of its function under TDP-43 LOF conditions, highlighting CUTS' potential for self-regulating gene therapy applications. In summary, CUTS represents a versatile platform for evaluating TDP-43 LOF in real-time and advancing gene-replacement therapies in neurodegenerative diseases associated with TDP-43 dysfunction.