A Pilot Study Assessing the Effect of Betaine and Diosmin on Disease Biomarkers in a Mouse Model for Human Zellweger Spectrum Disorder

Catherine Argyriou1, Erminia DiPietro1, Hyba Bessaklia2, Steven Steinberg3, Pierre Lachapelle2, Nancy Braverman1

1. Dept. of Human Genetics, McGill University, Montreal, QC, Canada; 2. Dept. of Ophthalmology, McGill University, Montreal, QC, Canada; 3. Dept. of Neurology, John’s Hopkins University, Baltimore, MD, USA

Introduction. Zellweger Spectrum Disorder (ZSD) is a heterogeneous autosomal recessive disease caused by mutations in any one of 14 PEX genes whose protein products are required for peroxisome assembly1. A common mutation encodes PEX1-p.Gly843Asp (G843D), a misfolded protein that has residual activity. Patients with this allele exhibit a progressive disease course that could be ameliorated by early therapy. Currently, management for ZSD is supportive only. To test if PEX1-G843D activity could be recovered by drug therapy, a high-throughput cell-based drug screen was developed that measures downstream recovery of peroxisome functions2. The results of this screen and subsequent structure-activity experiments indicated that betaine, a chemical chaperone, and disometin, a pharmacological chaperone, effectively recovered peroxisomal import in PEX1-G843D patient cell lines, and that combination therapy was synergistic. To facilitate testing candidate therapies in vivo, a PEX1-G843D equivalent knock-in mouse model was created (Pex1-G844D). Pex1-G844D homozygous mice exhibit many of the typical ZSD manifestations, including growth retardation, fatty livers with cholestasis, retinopathy, and elevated very long chain fatty acids (VLCFAs) and decreased plasmalogens (PLs)3.

Methods. We performed a pilot study testing the effect of our candidate drugs in Pex1-G844D homozygous mice. Starting at 5 weeks of age, the mice received either no treatment, 800mg/kg/day betaine, 100mg/kg/day diosmin, or a combination of the two, administered in the drinking water for 7-8 weeks.

Results. (1) Physiological analysis of the rod and cone visual systems by elecroretinography showed that drug therapy dramatically preserved the rod-mediated visual pathway, and that the combination treatment produced the best effect. In contrast, the cone-mediated visual pathway remained severely impaired in both the treated and untreated mice, suggesting that significant degeneration may have already occurred before the onset of therapy. (2) Peroxisome metabolic functions were measured at baseline, 1 month, and 2 month intervals from dried blood spots. Total PL levels as well as the C26:0 and C26/C22 ratio of VLCFAs did not improve in the animals that received betaine or diosmin alone. Although combination therapy produced the desired, albeit mild, trend of increased PLs and reduced C26/C22 ratio over time, there was no obvious effect of treatment on PLs and C26:0 VLCFAs in the terminal bleed. We noted however, that in the red blood cells of untreated mice, the biochemical phenotype in the mutants worsens with time. We do not have red blood cells from treated animals at different ages to observe whether treatment might affect this progression. (3) We are now determining drug concentrations, VLCFA and PL levels in liver, brain and other clinically relevant tissues. Histology will also be performed on fixed mouse livers to assess fat deposition and cholestasis.

Conclusion. We will optimize these methods and perform the next experiment with an expanded cohort. We will also engineer an immortalized Pex1-G844D homozygous hepatocyte cell line. Since hepatocytes are enriched in peroxisomes this will provide a useful tool for identifying additional candidate drug therapies.


1. reviewed in Braverman et al.(2012) Dev Disabil Res Rev; 17(3):187-96; 2. Zhang et al. (2010) ProcNatlAcadSci USA; 107(12):5569-74 ;3. Hiebler et al. (2014) Mol Genet Metab; 111(4):522-32.