TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A.

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Transport Selectivity of Nuclear Pores, Phase Separation, and Membraneless Organelles.

Nuclear pore complexes (NPCs) provide a selective passageway for receptor-mediated active transport between nucleus and cytoplasm, while maintaining the distinct molecular compositions of both compartments at large. In this review we discuss how NPCs gain a remarkable sorting selectivity from non-globular FG domains and their phase separation into dense polymer meshworks. The resulting sieve-like FG hydrogels are effective barriers to normal macromolecules but are at the same time highly permeable to shuttling nuclear transport receptors, which bind to FG motifs as well as to their designated cargoes. Phase separation driven by disordered protein domains was recently also recognized as being pivotal to the formation of membraneless organelles, making it an important emerging principle in cell biology.

Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease.

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

Oxaliplatin disrupts nucleolar function through biophysical disintegration.

Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.

Aberrant Phase Separation of FUS Leads to Lysosome Sequestering and Acidification.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.

High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function.

Mutations in RNA-binding proteins (RBPs) such as TDP43 are associated with transcriptome-wide splicing defects and cause severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The impact of RBP mutations on splicing function is routinely studied using PCR-based bulk measurements. However, the qualitative and low-throughput nature of this assay make quantitative and systematic analyses, as well as screening approaches, difficult to implement. To overcome this hurdle, we have developed a quantitative, high-throughput flow cytometry assay to investigate TDP43 splicing function on a single-cell level.