Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish.

Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.

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.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Novel genotype-phenotype and MRI correlations in a large cohort of patients with SPG7 mutations.

OBJECTIVE: To clinically, genetically, and radiologically characterize a large cohort of SPG7 patients. METHODS: We used data from next-generation sequencing panels for ataxias and hereditary spastic paraplegia to identify a characteristic phenotype that helped direct genetic testing for variations in SPG7. We analyzed MRI. We reviewed all published SPG7 mutations for correlations. RESULTS: We identified 42 cases with biallelic SPG7 mutations, including 7 novel mutations, including a large multi-exon deletion, representing one of the largest cohorts so far described. We identified a characteristic phenotype comprising cerebellar ataxia with prominent cerebellar dysarthria, mild lower limb spasticity, and a waddling gait, predominantly from a cohort of idiopathic ataxia. We report a rare brain MRI finding of dentate nucleus hyperintensity on T2 sequences with SPG7 mutations. We confirm that the c.1529C>T allele is frequently present in patients with long-standing British ancestry. Based on the findings of the present study and existing literature, we confirm that patients with homozygous mutations involving the M41 peptidase domain of SPG7 have a younger age at onset compared to individuals with mutations elsewhere in the gene (14 years difference, p < 0.034), whereas c.1529C>T compound heterozygous mutations are associated with a younger age at onset compared to homozygous cases (5.4 years difference, p < 0.022). CONCLUSIONS: Mutant SPG7 is common in sporadic ataxia. In patients with British ancestry, c.1529C>T allele represents the most frequent mutation. SPG7 mutations can be clinically predicted by the characteristic hybrid spastic-ataxic phenotype described above, along with T2 hyperintensity of the dentate nucleus on MRI.