Ameliorating Rett syndrome

In 1999, two researchers from the Baylor College of Medicine in the US, Huga Zoghbi and Ruthie Amir, discovered that mutations in the MECP2 gene, found on the X chromosome, caused Rett syndrome, the second most common form of severe intellectual disability in girls.

MECP2 codes for the MeCP2 protein, which binds to methylated CpG dinucleotides and is essential for normal neuronal function. The gene was first identified by Scottish researcher Adrian Bird, and it is thought to be the cause of Rett syndrome in between 85 and 90 per cent of cases. A mutation in another gene, CDKL5, which is thought to have a role in the phosphorylation of MECP2, has been linked to the remaining cases.

That discovery was made by a group of Australian researchers, some of whom have now participated in a study of whether environmental enrichment helps ameliorate some of the symptoms of Rett syndrome, in particular a motor co-ordination deficit commonly seen in Rett girls.

The study, published in the European Journal of Neuroscience, found that environmental enrichment improves motor co-ordination in a mouse model of Rett, and also boosted levels of BDNF, the brain-derived neurotrophic factor that supports the survival of neurons and encourages new growth and differentiation, and which is thought to have a major role in neuronal development.

MeCP2 is primarily expressed in mature neurons and has a role in regulating the activity of specific targets, including BDNF. Last year, two studies looked at whether re-expression of MeCP2 in mice could reverse the symptoms of Rett. One, published in Science by Adrian Bird and Jacky Guy of the University of Edinburgh, found that restoration of a silenced gene saw a dramatic reversal in symptoms over a month.

The other, by Rudolph Jaenisch and Emanuala Giacometti from the Whitehead Institute at MIT, found a partial rescue of MeCP2 defiency by post-natal activation. Jaenisch’s team had earlier found that hyper-expression of BDNF dramatically reduced Rett symptoms in knockout mice.

According to the authors of the new Australian study, this earlier work provides compelling evidence suggesting that neurons are not irrevocably damaged by the absence of MeCP2 during development, and, furthermore, that amelioration of symptoms may be achieved by restoring normal neuronal functionality post-natally.

So the Australian team – involving PhD student Mari Kondo, Dr Laura Gray and Associate Professor Anthony Hannan from the Howard Florey Institute; Dr Gregory Pelka and Professor Patrick Tam from the Children’s Medical Research Institute (CMRI) in Sydney; and Professor John Christodoulou from the Children’s Hospital at Westmead – decided to test out whether environmental enrichment (EE), which has been shown beneficial effects on behaviourial phenotype as well as BDNF expression, may influence the disease outcome in a Rett mouse model.

They found that enrichment significantly improves motor co-ordination in female mutant mice– although not male mice, which usually die very young, as with boys with the mutation – and boosts levels of BDNF.

“Enrichment has already been shown to be very effective in other mouse models and other neurological diseases such as Huntington’s, Alzheimer’s and fragile X mental retardation,” Mari Kondo says. “So this is another one to add to the list of diseases where enrichment has a positive effect.”

---PB--- Life in the playhouse

The mouse model used in the study was created by Greg Pelka, who was part of the team that discovered the role of CDKL5, during his PhD. The MECP2-mutant female mice are heterozygous, having one functional copy and one defective gene, while the males are hemizygous, with no MeCP2 protein at all.

In environmental enrichment, the mice live in a much larger cage, more of a standard size for rats, and are provided with running wheels, paper tunnels, wooden and plastic toys of lots of different colours and ropes hanging from the roof, Kondo says.

“Basically, it’s just something that gets them interested and makes them move more, so they get sensory, motor and cognitive stimulation,” she says. “The way they use their whiskers to feel – they have different textures in there. And they get that on top of the social interaction with their cage mates.

“The standard-housed heterozygous females had 75 per cent of the level of BDNF of standard-housed wild-type controls, and the enriched had 85 per cent, so it’s not a massive increase, but the key point is that the enriched heterozygous didn’t differ statistically from the wild-type. That was quite interesting considering this was done at 30 weeks of age, which is quite advanced.

“The most interesting result was the co-ordination rescue – the behavioural rescue was quite impressive and I’ve been able to replicate that since in another cohort.”

Patrick Tam says he was surprised by the rise in BDNF levels only in one respect. “No, because we know that BDNF is a target gene for this particular protein, and it has been demonstrated both in our analysis and others, that with this particular growth factor, the level of expression is dependent on the activity of the Mecp2 gene, so the association is not that surprising,” he says.

“What is a surprise is where we compare the two types of mutant animal. Enrichement only works in the female mutant mice with a normal copy of the gene. That provides the strongest evidence that this activity is mediated through the gene onto the BDNF. That is clinically significant because the patients are always girls and they are heterozygous.”

One of the unusual behaviours of Rett girls is their stereotypical hand-wringing. The mutant mice also show a strange behaviour – clasping of the hind limbs. “The hind-leg clasping behaviour is very atypical of mice,” Tam says.

“When you gently hold the mice up by the tail, usually the mice will splay their legs to try to balance themselves, anticipating they’ll fall down, whereas these animals without the gene don’t seem to realise that they are being held up and they try to hold on tight into a little ball. If you watch the animals in the cage, very often they’ll stand on their hind legs and put their forelegs together and they are twisting around all the time.

“It’s a very good tell-tale sign that the animal is already progressing in the disease status.”

---PB--- Cognitive function

The rapid progression of the disease – girls appear normal and pass the milestones of early infancy, including walking and talking before they begin to regress, losing functions they had earlier learned – is one of the most distressing aspects of Rett syndrome.

Not only do motor co-ordination problems appear, but cognitive function is also lost. The next step for this project is to see whether environmental enrichment assists in improving cognitive function and the ability to learn and memorise.

As part of this project, Kondo will bring her mouse playhouse up to the CMRI, which has the instruments required to test for cognitive function. Tam says this may prove the most exciting part, because if enrichment works for cognitive function, it could have a major effect on the quality of life of Rett girls and their families.

The success of enrichment in mice obviously can’t be claimed for girls unless clinical trials are carried out, and part of that problem is the rapid progression of the disease, Tam says. Most families dealing with a child affected by the condition provide intensive parental care, but the quality and quantity of the enrichment is the key.

“An example is that some girls affected with this problem love to watch DVDs but they always love to watch one in particular and nothing else. Or they enjoy listening to a particular piece of music. Enrichment is very person-specific so simply giving a lot of toys to play doesn’t mean they will feel enriched, because they may not like these toys.

“Together with Professor John Christodoulou, we have been considering the feasibility of running a trial. An important pre-requisite is that we will have design a proper regimen where you can tell the difference between a group which is not enriched versus the one that is.

“The enrichment has to be matching in quality and quantity so that we can learn some lesson from it. In practice, a behavioural clinical trial is quite a complex exercise.”