Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43

Abstract

Cross-linking and immunoprecipitation coupled with high-throughput sequencing was used to identify binding sites within 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein which when mutated causes Amyotrophic Lateral Sclerosis (ALS). Use of massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs are changed (including Fus/Tls, progranulin, and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events are detected (including in sortilin, the receptor for progranulin), following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels are most depleted by reduction in TDP-43 are derived from genes with very long introns and which encode proteins involved in synaptic activity. Lastly, TDP-43 was found to auto-regulate its synthesis, in part by directly binding and enhancing splicing of an intron within the 3′ untranslated region of its own transcript, thereby triggering nonsense mediated RNA degradation. (147 words)

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

“…Exon 18 inclusion was increased in RRM2mut , as previously described in TDP‐43 knockdown (Polymenidou et al , 2011), but had an opposite change in LCDmut , further supporting the splicing GOF (Fig 1C).…”

“… In RNA‐seq data from LCDmut , genes were ranked for gene expression based on signed z‐score ( x ‐axis), and the mean intron length for groups of 200 genes was plotted ( y ‐axis), showing no enrichment of long intron genes in downregulated transcripts (A), unlike in RRM2mut where data show an enrichment of long intron genes in downregulated transcripts from RRM2mut (blue) and previously published data from adult striatum TDP‐43 knockdown (grey; Polymenidou et al , 2011) (B). Ribbon plots show mean ± standard error of the mean (shaded regions).…”

“…TDP‐43 is extremely dosage sensitive and is tightly autoregulated by a mechanism that involves TDP‐43 protein binding to its own 3′UTR; these properties have proven challenging for studying the effects of TARDBP mutations in vivo (Ayala et al , 2011; Polymenidou et al , 2011; Avendaño‐Vázquez et al , 2012). Thus, the main approach to studying LOF, by using null mice, has been hampered because TDP‐43 knockout (KO) causes very early embryonic lethality, and heterozygous mice have normal levels of TDP‐43 due to autoregulation (Kraemer et al , 2010; Sephton et al , 2010; Wu et al , 2010; Ricketts et al , 2014).…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…TDP43 binds to and regulates thousands of RNA targets [13,14,[36][37][38], and is thus in a unique position to dramatically affect gene expression on a global scale. The RNA-binding domains of TDP43 and FUS are essential for toxicity in ALS model systems, testifying to the importance of RNA dysregulation in ALS pathogenesis [34,[39][40][41].…”

“…Mounting genetic evidence suggests that motor neuron degeneration in ALS represents a final common pathway initiated by ≥1 conserved upstream mechanisms, including protein misfolding/aggregation [5][6][7][8][9][10][11][12], RNA misprocessing [10,[13][14][15][16][17][18][19], and disruptions in axonal transport [12,[20][21][22][23][24]. These mechanisms are not mutually exclusive, and instead may enhance one another, thereby accentuating neurodegeneration in ALS.…”