Impairment of the tRNA splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of Pontocerebellar Hypoplasia

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterised by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesised that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the morpholino with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown, however an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, ENU mutant zebrafish homozygous for a tsen54 premature stop codon mutation die within 9 days post-fertilisation. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of rars2 (PCH1, PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54 and RARS2-related PCH, which may involve a tRNA processing related mechanism.