rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA

Abstract

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.

Fig. 1.

AO127 mutant animals are deficient in rnaset2. (A) AO127 mutants displayed a higher uptake of AO compared with WT embryos, quantifiable by flow cytometry. (Note the intense fluorescence common to both animals is a result of AO within the lumen of the pharynx and intestine, autofluorescence of residual yolk, and ready uptake of AO by olfactory neurons and hair cells within neuromasts.) (Scale bars: 200 μm.) (B) Sequencing of the gene zgc:113369, coding for the zebrafish orthologue of human RNASET2, identified a T-to-A transversion in the mutant sample. (C) RT-PCR indicates a dramatic down-regulation of rnaset2 mRNA in AO127 mutants compared with WT siblings. β-Actin serves as a control for RNA integrity and equal input cDNA. (D) Suppression of excess AO staining in AO127 mutants by injection of WT rnaset2 mRNA but not mutant mRNA.

Fig. 2.

Subcellular localization of RNASET2. HEK293 cells were transfected with myc-epitope tagged WT RNASET2 (A) and the mutated version, RNASET2 C184R (B). Immunofluorescence for endoplasmic reticulum (PDI), Golgi, and late endosome (LAMP-1) markers show partial colocalization with WT RNASET2, suggesting that RNASET2 enters the secretory pathway. The mutated version colocalized completely with the PDI, suggesting ER retention. (Scale bar: 10 μm.)

Fig. 3.

Brain neurons of AO127 mutants contain lysosomes engorged with rRNA. Brain transverse sections from embryos 5 d after fertilization. EM revealed abundant electron-dense vesicles in the neurons of AO127 mutants (some of these are indicated by red arrowheads; Inset, electron-dense material is bounded by a limiting membrane). (Scale bars: 5 μm). In situ hybridization revealed accumulation of rRNA aggregates (white arrows) in the cytoplasm of AO127 mutant neurons. (Scale bars: 10 μm.)

Fig. 4.

White matter anomalies in AO127 mutants. T₂-weighted MR images of the brain of WT and mutant AO127 adult zebrafish. Sagittal images indicate the presence of a white matter anomaly (white arrow) in telencephalon of mutant, which was not seen in WT. Coronal images show the presence of lesions adjacent to the ventricles (white arrow). IHC was carried out on the same samples used for MRI. Axonal disruptions around ventricles were seen using APP as a marker, as well as a higher number of astrocytes using GFAP as a marker, suggesting the presence of damaged neurons and inflammation around the lesions. (Scale bars: 1 mm for MR sagittal images, 500 μm for coronal images, 100 μm for immunohistochemistry.)