Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones.

Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor neurone-like cell model; and (2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurones obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. We found altered expression of spliceosome components in motor neurones and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43-depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, which were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurones, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism.

Highley JR ，Kirby J ，Jansweijer JA ，Webb PS ，Hewamadduma CA ，Heath PR ，Higginbottom A ，Raman R ，Ferraiuolo L ，Cooper-Knock J ，McDermott CJ ，Wharton SB ，Shaw PJ ，Ince PG ... -  《-》

Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder. In motor neurons of ALS, TAR DNA binding protein-43 (TDP-43), a nuclear protein encoded by TARDBP, is absent from the nucleus and forms cytoplasmic inclusions. TDP-43 auto-regulates the amount by regulating the TARDBP mRNA, which has three polyadenylation signals (PASs) and three additional alternative introns within the last exon. However, it is still unclear how the autoregulatory mechanism works and how the status of autoregulation in ALS motor neurons without nuclear TDP-43 is. Here we show that TDP-43 inhibits the selection of the most proximal PAS and induces splicing of multiple alternative introns in TARDBP mRNA to decrease the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. When TDP-43 is depleted, the TARDBP mRNA uses the most proximal PAS and is increased in the cytoplasm. Finally, we have demonstrated that in ALS motor neurons-especially neurons with mislocalized TDP-43-the amount of TARDBP mRNA is increased in the cytoplasm. Our observations indicate that nuclear TDP-43 contributes to the autoregulation and suggests that the absence of nuclear TDP-43 induces an abnormal autoregulation and increases the amount of TARDBP mRNA. The vicious cycle might accelerate the disease progression of ALS.

Koyama A ，Sugai A ，Kato T ，Ishihara T ，Shiga A ，Toyoshima Y ，Koyama M ，Konno T ，Hirokawa S ，Yokoseki A ，Nishizawa M ，Kakita A ，Takahashi H ，Onodera O ... -  《-》

Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis.

Ishihara T ，Ariizumi Y ，Shiga A ，Kato T ，Tan CF ，Sato T ，Miki Y ，Yokoo M ，Fujino T ，Koyama A ，Yokoseki A ，Nishizawa M ，Kakita A ，Takahashi H ，Onodera O ... -  《-》

Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS).

Volkening K ，Leystra-Lantz C ，Yang W ，Jaffee H ，Strong MJ ... -  《-》

Downstream Effects of Mutations in SOD1 and TARDBP Converge on Gene Expression Impairment in Patient-Derived Motor Neurons.

Dash BP ，Freischmidt A ，Weishaupt JH ，Hermann A ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》