Rett Syndrome is a severe X-linked disorder with a prevalence of approximately one per 10,000 live female births. It presents with developmental regression, including loss of speech, motor impairments and autistic features. Loss-of-function mutations in methyl CpG binding protein 2 (MECP2) cause more than ninety-five percent of cases. Mecp2-null mouse models recapitulate many aspects of the human disease. Symptoms can be reversed by restoration of Mecp2 function in symptomatic mice, and partially rescued with other factors. This provides substantial evidence that therapeutic intervention in Rett Syndrome is possible. Unfortunately, as a widespread epigenetic factor, MECP2 levels are extremely dosage sensitive, making direct manipulation a poor treatment option.
MECP2 has multiple binding partners and its mutation impacts many biological pathways in Rett Syndrome, but it is unclear which are relevant to symptom progression. Our use of forward genetics allowed us to dispense with a priori beliefs about MECP2 function. In this way, we are able to identify novel binding partners and downstream pathways that, when altered, effect the amelioration of symptoms. Any such pathway could contain potential targets for the development of new pharmacological treatments for Rett Syndrome.
Studies were carried out in the Mecp2tm1.1Bird deletion mouse model. We employed a dominant ENU mutagenesis screen to identify biological pathways important for symptom suppression. We capitalized on genetic variation between the C57BL/6J and 129S6/SvEv mouse strains to locate five suppressing mutations through a combination of SNP linkage mapping and whole exome sequencing strategies. One is a loss-of-function mutation in squalene epoxidase (Sqle), a rate-limiting enzyme in committed cholesterol biosynthesis. Cholesterol and lipid concentrations were assessed by gas-liquid chromatography, synthesis was assessed from saponified tissue after tritium incorporation, and sterol intermediates were measured by tandem mass spectrometry.
The loss-of-function Sqle mutation increased longevity and improved motor functioning, activity levels and overall health in Mecp2-null mice. Based on the biochemical role of SQLE, we examined cholesterol and lipid metabolism in the Mecp2-null male mice and found perturbations in both the brain and liver. Similar, but delayed perturbations were found in Mecp2 heterozygous females. Accordingly, we treated Mecp2 mutant mice with cholesterol-lowering statin drugs and found that they also alleviate motor symptoms and confer increased longevity in both males and females.
The discovery of a Rett Syndrome suppressing mutation in the cholesterol biosynthesis pathway was unexpected and unlikely to have been found using the reverse genetics approach that is more common in mouse research. Cholesterol metabolism represents a potential pathway for new therapeutic targets to treat the syndrome. Our data add to a growing body of evidence that cholesterol plays an important role in many neurological diseases. More broadly, the results of this study suggest researchers working on autism-related disorders would benefit from the use of a systems biology approach to identify targetable downstream pathways involved in their pathogenesis.
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