Rett Syndrome (RTT), a devastating neurological disorder, strikes 1 in 10,000 young children, almost all of them girls. In fact, RTT is the leading genetic cause of severe impairment in girls. Symptoms include neuromuscular problems, autonomic dysregulation, seizures and seizure-like episodes, stereotypical hand movements and the inability to speak. Many children are wheelchair-bound, scoliosis is common, and though the majority live to adulthood, they require total care for every aspect of life.
RTT, which is an autism-spectrum disorder, is caused by mutations in a gene called MECP2. Previous research has shown that the protein made by MECP2 is a master controller of other genes, turning them on and off, and scientists have been searching for these genes. Several have been identified, the best known being brain-derived neurotrophic factor, or BDNF, which normally promotes neuronal growth. To find other genes that MECP2 controls, Bird, Kriaucionis, and other colleagues turned to male mice in which MECP2 has been "knocked out", meaning they lack the protein completely. These animals are born healthy but begin to walk and breathe abnormally around 6 weeks of age, and start dying off at about 10 weeks (the average lifespan of a mouse is 2 years). The team compared the mutant mice to normal mice in search of genes that were off when they should be on, or vice-versa.
First, Kriaucionis purified the messenger RNA made by about 10,000 different genes from the brains of mutant mice that were almost 10 weeks old. Compared to normal animals of the same age, the team found more RNA produced by seven genes and less RNA made by three. Then Kriaucionis examined the messenger RNA produced by those 10 genes in the brains of mice that had only just started coming down with symptoms, at about 7 to 8 weeks of age. Three of the genes made too much of their protein product in the mutant brains as compared to normal brains.
The team reasoned that these three genes played greater roles in the disorder because they went awry earliest. They decided to focus on Uqcrc1 because much was already known about it, including its role in the mitochondria generating energy.
Mitochondria make energy in four steps, and researchers can test to determine which step malfunctions. The team purified mitochondria from the brains of mice lacking Mecp2 and normal mice. They fed energy precursors to the mitochondria and measured how much energy the factories put out.
Uqcrc1 protein works at the third step in the chain, called complex III. If the researchers supplied precursors that are used by mitochondria before the third step, the mitochondria from the mutant animals made significantly more energy than the normal mitochondria. If they supplied precursors that are used after complex III, the mutant mitochondria made the same amount of energy as normal organelles.
This suggested that overabundance of Uqcrc1 in complex III resulted in mitochondria cranking out more energy than the mutant animals needed. "More sounds better but it isn't necessarily that way," says Bird. "Mitochondria are exquisitely regulated machines, so any deviation from normality is likely to be bad."
To find out if Uqcrc1 was to blame for the mitochondrial defects, the team overproduced the protein in cultured neuronal cells. They isolated mitochondria and repeated the energy production tests. The mitochondria from the cultured cells behaved like the mutant brain mitochondria. "What we wanted to know is if the overexpression of this gene was solely responsible for the overactive mitochondria. And the data said it's likely to be," says Bird. He adds that additional experiments are needed to link the mitochondrial abnormalities to the symptoms found in the mutant mice. The researchers also caution that similar defects need to be looked for in humans.
"Our findings provide a mechanism for how a mutation in MECP2 could result in abnormal mitochondrial function," says Kriaucionis.
"I'm very excited about this paper," says Bruce Cohen, a pediatric neurologist who specializes in mitochondrial diseases at the Cleveland Clinic in Cleveland, Ohio. Dr. Cohen, who did not take part in the work, says it validates the suspicions of a few researchers who have argued that malfunctioning mitochondria play a role in the disorder.
"For over 15 years there have been clinical reports of mitochondrial abnormalities in Rett Syndrome patients. Dr. Bird's research provides the first genetic link between MECP2 and mitochondria," states Monica Coenraads, Director of Research for RSRF.
Founded in late 1999, RSRF is the world's largest private source of funds for biomedical research for RTT. For more information on RTT or the Foundation please visit our website at www.rsrf.org.
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