TDP-43 represses cryptic exon inclusion in FTD/ALS gene<i>UNC13A</i>

Xr, Ma; Prudencio, Mercedes; Koike, Yasuhiro; Sc, Vatsavayai; G, Kim; Harbinski, Fred; Cm, Rodriguez; Hb, Schmidt; Bb, Cummings; Dw, Wyatt; Kurylo, Katherine; Miller, G.; Mekhoubad, Shila; Sallee, Nathan A; Jansen-West, Karen; Cn, Cook; Pickles, Sarah; Oskarsson, Björn; Graff-Radford, NR; Boeve, BF; Ds, Knopman; Rc, Petersen; Dw, Dickson; Em, Green; Ww, Seeley; Petrucelli, Leonard; Ad, Gitler

doi:10.1101/2021.04.02.438213

Cited by 22 publications

(34 citation statements)

References 45 publications

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“…Since we used an antibody to exon 2 epitopes near the N-terminal in both the neuropathological and biochemical studies, it seems reasonable to conclude that the shortening is after exon 2. One possibility is inclusion of a cryptic exon from reduction of TDP-43 such as has been identified in STMN2 48 and UNC13A 58,59 . Indeed, a cryptic exon due to TDP-43 downregulation has been identified at intron 3 of ELAVL3 48 .…”

Section: Discussionmentioning

confidence: 99%

Nuclear depletion of RNA binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

Diaz-Garcia

Vázquez-Sánchez

et al. 2021

Preprint

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Amyotrophic lateral sclerosis is a progressive fatal neurodegenerative disease caused by loss of motor neurons and characterized neuropathologically in almost all cases by nuclear depletion and cytoplasmic aggregation of TDP-43, a nuclear RNA binding protein (RBP). We identified ELAVL3 as one of the most downregulated genes in our transcriptome profiles of laser captured microdissection of motor neurons from sporadic ALS nervous systems and the top dysregulated RBPs. Neuropathological characterizations showed ELAVL3 nuclear depletion in a great percentage of remnant motor neurons, sometimes accompanied by cytoplasmic accumulations. These abnormalities were common in sporadic cases with and without intermediate expansions in ATXN2 and familial cases carrying mutations in C9orf72 and SOD1. Depletion of ELAVL3 occurred at both the RNA and protein levels and a short protein isoform was identified but it is not related to a TDP-43-dependent cryptic exon in intron 3. Strikingly, ELAVL3 abnormalities were more frequent than TDP-43 abnormalities and occurred in motor neurons still with normal nuclear TDP-43 present, but all neurons with abnormal TDP-43 also had abnormal ELAVL3. In a neuron-like cell culture model using SH-SY5Y cells, ELAVL3 mislocalization occurred weeks before TDP-43 abnormalities were seen. We interrogated genetic databases but did not identify association of ELAVL3 genetic structure associated with ALS. Taken together, these findings suggest that ELAVL3 is an important RBP in ALS pathogenesis acquired early and the neuropathological data suggest it is involved by loss of function rather than cytoplasmic toxicity.

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Section: Discussionmentioning

confidence: 99%

Nuclear depletion of RNA binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

Diaz-Garcia

Vázquez-Sánchez

et al. 2021

Preprint

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“…ALS is characterized by widespread occurrence of cytoplasmic aggregates of TDP-43, an RNA binding protein preventing aberrant inclusion of cryptic exons 11 . Interestingly, decreased TDP-43 function potentiates the inclusion of a cryptic exon in the UNC13A risk allele identified in GWAS, leading to decreased levels of the synaptic vesicle protein UNC13A, selectively in carriers of this allele 12,13 . Whether other variants associated to ALS are also related to splicing events remains to be determined.…”

Section: Introductionmentioning

confidence: 99%

Loss of nucleoporin Nup50 is a risk factor for amyotrophic lateral sclerosis

Megat

Mora

Sanogo

et al. 2021

Preprint

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The genetic basis of amyotrophic lateral sclerosis (ALS) is still incompletely understood. Using two independent genetic strategies, we show here that a large part of ALS heritability lies in genes expressed in inhibitory and excitatory neurons, especially at splicing sites regulated by a defined set of RNA binding proteins including TDP-43 and FUS. We conducted a transcriptome wide association study (TWAS) and identified 59 loci associated with ALS, including 14 previously identified genes, some of them not previously reaching significance in genome wide association studies. Among the 45 novel genes, several genes are involved in pathways known to be affected in ALS such as mitochondrial metabolism (including ATP5H, ATP5D, BCS1L), proteostasis (including COPS7A, G2E3, TMEM175, USP35) or gene expression and RNA metabolism (including ARID1B, ATXN3, PTBP2, TAF10). Interestingly, decreased expression of NUP50, a constrained gene encoding a nuclear pore basket protein, was associated with ALS in TWAS (Zscore = -4, FDR = 0.034). 11 potentially pathogenic variants (CADD score > 20) in 23 patients were identified in the NUP50 gene, most of them in the region of the protein mediating interaction with Importin alpha, and including 2 frameshift mutations. In cells from two patients carrying NUP50 variants, we showed decreased levels of NUP50 protein. Importantly, knocking down Nup50 led to increased neuronal death associated with p62 and nucleoporin inclusions in cultured neurons, and motor defects in Drosophila and zebrafish models. In all, our study identifies alterations in splicing in neurons as a critical pathogenic process in ALS, uncovers several new loci potentially contributing to ALS missing heritability, and provides genetic evidence linking nuclear pore defects to ALS.

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“…Furthermore, a small number of amino acid differences within highly conserved mouse and human protein orthologues can significantly affect properties such as propensity to aggregate, for example (Crown et al, 2020; DuVal et al, 2019; Nagano et al, 2015; Perri et al, 2020). Including non-coding gene regions is also important since several known mutations and risk variants exist in such regions (Brown et al, 2021; Dini Modigliani et al, 2014; Ma et al, 2021; Sabatelli et al, 2013). Our genomically humanised strains are ‘knock-in’ animals and therefore express proteins from the endogenous locus at physiological levels.…”

Section: Discussionmentioning

confidence: 99%

“…Looking to the future, single humanised alleles may not be sufficient to study disease mechanisms involving binding partners of humanised proteins, or to assess human specific splicing events (Brown et al, 2021; Ma et al, 2021), and new bespoke humanised alleles may need to be engineered and cross-bred for these purposes. In turn these may aid in more faithfully recapitulating later stage pathology and phenotypes, for further mechanistic insight in vivo and for improved read outs for testing therapeutic strategies.…”

Section: Discussionmentioning

confidence: 99%

“…The RNA binding proteins TDP-43 and FUS are dosage-sensitive and phenotypes arise from even mild overexpression of the wildtype human gene (Ling et al, 2019; Mitchell et al, 2013; Wils et al, 2010; Xu et al, 2010) and even low copy transgenics have to be bred to mice with the endogenous gene knocked out, in order to keep dosage as normal as possible (Lopez-Erauskin et al, 2018). While cross-breeding to endogenous gene Knock Out (KO) mice is an added complication for transgenic models, it is necessary in order to model loss of function, which has recently gained traction as being important for pathomechanism in forms of ALS (Briese et al, 2020; Brown et al, 2021; Humphrey et al, 2020; Klim et al, 2019; Ma et al, 2021; Melamed et al, 2019; Saccon et al, 2013). Finally, transgenic models that ectopically express a wildtype human ALS gene are used as controls for transgenics that express the mutant protein; however, since transgenics integrate at random in different copy numbers, comparisons between control and mutant transgenics are confounded.…”

Section: Introductionmentioning

confidence: 99%

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Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

Devoy

Price

Giorgio

et al. 2021

Preprint

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Amyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS. We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan. These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

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TDP-43 represses cryptic exon inclusion in FTD/ALS geneUNC13A

Cited by 22 publications

References 45 publications

Nuclear depletion of RNA binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

Nuclear depletion of RNA binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

Loss of nucleoporin Nup50 is a risk factor for amyotrophic lateral sclerosis

Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

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