CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function.

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. Highlights: CUTS is a cryptic exon RNA biosensor enabling real-time detection of TDP-43 loss of splicing function.CUTS exhibits a significant linear relationship with TDP-43 loss.CUTS can deliver an autoregulated gene payload in response to TDP-43 loss-of-function.TDP-43 homotypic phase transitions induce loss of splicing function via CUTS detection.

Disrupting pathologic phase transitions in neurodegeneration.

Solid-like protein deposits found in aged and diseased human brains have revealed a relationship between insoluble protein accumulations and the resulting deficits in neurologic function. Clinically diverse neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis, exhibit unique and disease-specific biochemical protein signatures and abnormal protein depositions that often correlate with disease pathogenesis. Recent evidence indicates that many pathologic proteins assemble into liquid-like protein phases through the highly coordinated process of liquid-liquid phase separation. Over the last decade, biomolecular phase transitions have emerged as a fundamental mechanism of cellular organization. Liquid-like condensates organize functionally related biomolecules within the cell, and many neuropathology-associated proteins reside within these dynamic structures. Thus, examining biomolecular phase transitions enhances our understanding of the molecular mechanisms mediating toxicity across diverse neurodegenerative diseases. This Review explores the known mechanisms contributing to aberrant protein phase transitions in neurodegenerative diseases, focusing on tau and TDP-43 proteinopathies and outlining potential therapeutic strategies to regulate these pathologic events.