The resultant data from this clinical trial did not support the prestudy hypothesis that chamomile could decrease 5-FU-induced stomatitis.

Nuclear clearance of the DNA/RNA-binding protein TDP-43 is a pathologic hallmark of amyotrophic lateral sclerosis and frontotemporal dementia that remains unexplained. Moreover, our current understanding of TDP-43 nucleocytoplasmic shuttling does not fully explain the predominantly nuclear localization of TDP-43 in healthy cells. Here, we used permeabilized and live-cell models to investigate TDP-43 nuclear export and the role of RNA in TDP-43 localization. We show that TDP-43 nuclear efflux occurs in low-ATP conditions and independent of active mRNA export, consistent with export by passive diffusion through nuclear pore channels. TDP-43 nuclear residence requires binding to GU-rich nuclear intronic pre-mRNAs, based on the induction of TDP-43 nuclear efflux by RNase and GU-rich oligomers and TDP-43 nuclear retention conferred by pre-mRNA splicing inhibitors. Mutation of TDP-43 RNA recognition motifs disrupts TDP-43 nuclear accumulation and abolishes transcriptional blockade-induced TDP-43 nuclear efflux, demonstrating strict dependence of TDP-43 nuclear localization on RNA binding. Thus, the nuclear abundance of GU-rich intronic pre-mRNAs, as dictated by the balance of transcription and pre-mRNA processing, regulates TDP-43 nuclear sequestration and availability for passive nuclear export.

HighlightsMulti-omic analysis of differentiated C9ORF72 iPSC-derived motor neurons Network-based integrative computational analysis Pathogenic versus compensatory pathways elucidated using C9ORF72 Drosophila model Pathways confirmed with alternative differentiation protocol and postmortem data

Nucleocytoplasmic transport, controlled by the nuclear pore complex, has recently emerged as a pathomechanism underlying neurodegenerative diseases including C9orf72 ALS/FTD. However, little is known about the underlying molecular events and the underlying biology in human neurons. Using super resolution structured illumination microscopy of twenty three nucleoporins in nuclei from C9orf72 iPSC derived neurons and postmortem human tissue we identify a unique subset of eight nucleoporins lost from human neuronal nuclei. POM121, an integral transmembrane nucleoporin, appears to coordinate the composition of the nucleoporins within human neuronal nuclei ultimately impacting nucleocytoplasmic transport, and subsequent cellular toxicity in C9orf72 iPSNs. These data suggest that POM121 is a critical nucleoporin in the maintenance of the nuclear localization of specific nucleoporins in human neurons. Moreover, loss of nuclear POM121, as a result of expanded C9orf72 ALS/FTD repeat RNA, initiates a pathological cascade affecting nucleoporin composition within neuronal nuclei, nuclear pore complex function, and overall downstream neuronal survival.

Neurodegenerative diseases present a challenge for systems biology, due to the lack of reliable animal models and the difficulties in obtaining samples from patients at early stages of disease, when interventions might be most effective. Studying induced pluripotent stem cell (iPSC)-derived neurons could overcome these challenges and dramatically accelerate and broaden therapeutic strategies. Here we undertook a network-based multi-omic characterization of iPSC-derived motor neurons from ALS patients carrying genetically dominant hexanucleotide expansions in C9orf72 to gain a deeper understanding of the relationship between DNA, RNA, epigenetics and protein in the same pool of tissue. ALS motor neurons showed the expected C9orf72-related alterations to specific nucleoporins and production of dipeptide repeats. RNA-seq, ATAC-seq and data-independent acquisition mass-spectrometry (DIA-MS) proteomics were then performed on the same motor neuron cultures. Using integrative computational methods that combined all of the omics, we discovered a number of novel dysregulated pathways including biological adhesion and extracellular matrix organization and disruption in other expected pathways such as RNA splicing and nuclear transport. We tested the relevance of these pathways in vivo in a C9orf72 Drosophila model, analyzing the data to determine which pathways were causing disease phenotypes and which were compensatory. We also confirmed that some pathways are altered in late-stage neurodegeneration by analyzing human postmortem C9 cervical spine data. To validate that these key pathways were integral to the C9 signature, we prepared a separate set of C9orf72 and control motor neuron cultures using a different differentiation protocol and applied the same methods. As expected, there were major overall differences between the differentiation protocols, especially at the level of in individual omics data. However, a number of the core dysregulated pathways remained significant using the integrated multiomic analysis. This new method of analyzing patient specific neural cultures allows the generation of disease-related hypotheses with a small number of patient lines which can be tested in larger cohorts of patients.

The clinical presentation of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, varies widely across patients, making it challenging to determine if potential therapeutics slow progression. We sought to determine whether there were common patterns of disease progression that could aid in the design and analysis of clinical trials. We developed an approach based on a mixture of Gaussian processes to identify clusters of patients sharing similar disease progression patterns, modeling their average trajectories and the variability in each cluster. We show that ALS progression is frequently nonlinear, with periods of stable disease preceded or followed by rapid decline. We also show that our approach can be extended to Alzheimer’s and Parkinson’s diseases. Our results advance the characterization of disease progression of ALS and provide a flexible modeling approach that can be applied to other progressive diseases.

15Disruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin b 16 cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a 17 hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. 18Importin b and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-19 containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline 20 arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs 21 interact with importin b, disrupt its cargo loading, and inhibit nuclear import in permeabilized 22 mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs 23 induce widespread protein aggregation in this in vitro system, transport disruption is not due to 24 NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore 25 complex. Our results support a model in which R-DPRs interfere with nuclear transport 26 receptors in the vicinity of the nuclear envelope. 27 101 importin β and compete with the IBB. Synthetic GP10, GA10, and PA10 peptides did not affect 102 6Rango FRET even at high concentrations ( Figure 1D). However, we observed a dose-103 dependent increase in FRET with low-nanomolar PR10 and GR10, indicating these DPRs are 104 capable of binding to importin β and displacing the sensor. To further validate these 105 observations, we used GFP nanobody-coated beads to bind Rango and probe for co-106 immunoprecipitation of importin β in the presence of increasing concentrations of GR10 and 107 PR10 ( Figure 1E-F). Again, we observed the dose-dependent displacement of importin β from 108 the sensor at low nanomolar concentrations, confirming that Rango release was responsible for 109 the increases in FRET. 110To test the functional consequence of R-DPR-importin β interactions for nuclear import, 111we performed the permeabilized cell assay (Adam et al., 1990), in which the plasma membrane 112 of cultured cells is selectively permeabilized, leaving the nuclear membrane intact as verified by 113 nuclear exclusion of 70 kD dextran ( Figure 1G). Fluorescent transport cargo is then added, with 114 energy regeneration mix and cell lysate to provide a source of importins and Ran for nuclear 115 import, which is measured by increasing nuclear fluorescence. Traditionally, this method uses 116 digitonin for permeabilization; however, when attempted with primary mouse cortical neurons, 117 we repeatedly found that even minimal concentrations of digitonin opened both the plasma and 118 nuclear membranes. Since the nuclear envelope is devoid of the digitonin target cholesterol 119 (Colbeau et al., 1971;Adam et al., 1990), we reasoned that its rupture in permeabilized 120 neuronal cells was caused by mechanical perturbation upon removal of cytoplasmic proteins. 121Therefore, we developed a new protocol involving hypotonic cell opening in the presence of a 122 high concentration of BSA as a cushion, which faci...

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