1- Genes involved in a new ataxia syndrome: ataxia + ocular apraxia (AOA).

Ocular motor apraxia is a difficulty or inability to make lateral eye movement on command. When requested, the patients turn their head to look sideways (head thrust). Exaggerated eye blinking is often associated.

When associated with ataxia, ocular motor apraxia is better described as slow eye movement (slow saccade or hypometric saccade). It is also described as "viscosity of eyeballs as if they were floating in oil".

A previously known ataxic syndrome with ocular motor apraxia is ataxia telangiectasia (AT). However this condition is dominated by chronic infections, immune deficiency and susceptibility to leukaemia.

We have identified the gene of one form of AOA (AOA1) and we have localised on chromosome the map position of a second form (AOA2). These genetic studies have revealed that not all patients have ocular motor apraxia (which is sometimes difficult to recognise at late stages of the disease). Therefore, the AOA1 and AOA2 forms may account for a substantial proportion of all non-Friedreich recessive ataxias.

The disease seems particularly frequent in Japan and Portugal. We also identified affected individuals in France, Italy, Tunisia and India.

The gene defines a new protein that we and the Japanese group agreed to name aprataxin. The function of aprataxin is not known but it appears to be a mosaic of pieces sharing similarity with known proteins. These hints led us to speculate that aprataxin is involved in DNA single strand break repair. If correct, there may be a link with ataxia-telangiectasia which is due to a defect in DNA double strand break repair.

The identification in 1996 of the gene defective in Friedreich ataxia has prompted numerous work aimed at understanding the mechanism of the disease, construct mouse models and design tentative therapies. Friedreich ataxia is caused by a GAA trinucleotide repeat expansion located outside from the coding region of the gene. For this reason, the encoded protein, which we named frataxin is of normal size but is produced in very reduced amounts due to the expansion mutation. We have shown in a mouse model that having no frataxin at all is lethal very early in embryonic development. The disease in patients is therefore the consequence of having very small amounts of the normal frataxin protein.

What do we know about frataxin and the consequences of its almost complete absence? Frataxin is a protein of the mitochondria and some mitochondrial proteins are secondarily defective in affected tissues of Friedreich ataxia patients. These proteins contain iron in so-called iron-sulfur centers and many of them are part of the mitochondrial electron transport chain, which is involved in the major function of mitochondria, i.e. produce energy (ATP for a cell). Iron centers and Coenzyme Q10 are the direct transporters of electron in this chain). If only one electron leaves the chain too early and encounters oxygen, it will result in the formation of O2^.-, called superoxide ion or anion, which is an oxidant molecule toxic mostly for neuronal cells. Free iron is also known to be able to produce superoxide ions by transfer of one electron to oxygen. It seems today that the iron-sulfur proteins are defective in Friedreich ataxia because of a primary production of oxidant molecules (also called free radicals, such as the superoxide ion) (Chantrel-Groussard et al. Hum. Mol. Genet. 2001; 10:2061-2067) rather than because of iron accumulation, as previously suggested.

One way to protect cells from oxidant molecules is to use antioxidant molecules. Coenzyme Q10, one of the component of the electron transport chain can also act as a membrane antioxidant. Idebenone is an analogue of Coenzyme Q and can act both as a soluble and a membrane antioxidant, i.e. it is believed to be able to take the free electron from a soluble free radical and bring it back to the electron transport chain (at the mitochondrial membranes). For this and other reasons (Rustin et al. 1999; Lancet 354:477-479), idebenone was chosen for a one year therapeutic trial in two major hospitals in Paris (hopital Necker, Pr Arnold Munnich and hopital de la Salpetriere, Pr Alexis Brice. So far, the clearest beneficial effect was seen on the cardiomyopathy, with a progressive reduction of hypertrophy (reduction of the heart walls thickness), seen in most but not all patients. Some subjective improvements were also noted on fatigue, dysarthria and fine hand movements (writing). No improvement was noted on gait ataxia and peripheral neuropathy. From the preliminary results, it is not possible to prove or exclude that idebenone is blocking or slowing down the progression of these last two symptoms. An independent group has shown that idebenone reduces oxidative stress in Friedreich ataxia patients (Schulz et al., Neurology 2000; 55:1719-1721).

In order to gain insights into the mechanisms of the disease and to try to get independent confirmation of the human drug trials (an important issue for drug approval and commercial distribution), we have developed mouse models that somehow mimic the human disease. In order to avoid the embryonic lethality due to early complete absence of frataxin, we thought to induce the mutation in our mice models after embryonic development and to restrict the occurrence of the mutation in some tissues, namely neurones and muscle (including heart), with the strategy called conditional knock-out (Puccio et al. 2001 Nat. genet. 27,181-186). The "muscle" mice initially develop normally, then develop cardiomyopathy and die at about 10 weeks of age. The "neuronal"mice has an induced mutation in many non-neuronal tissues, including heart, liver and thymus, they start to loose weight from birth onward and die at about 3 weeks of age. In all cases we found (in collaboration with the laboratory of Pierre Rustin) mitochondrial iron-sulfur protein deficiency in parallel to the occurrence of the disease in the affected tissues, as in the human disease. However, mitochondrial iron accumulation and deposits were not seen in the "neuronal"mice (presumably because they died too early) and were seen in the heart of the "muscle"mice only after the onset of the cardiomyopathy and after the occurrence of iron-sulfur protein deficiencies. This represent to us another argument suggesting that Friedreich ataxia is initially caused by free radical production, which would in turn cause iron-sulfur protein deficiencies and then intra-mitochondrial iron accumulation (released from the defective iron-sulfur centers). If correct, this knowledge reinforces the rationale of using antioxidants as a treatment directed to one of the early steps of Friedreich ataxia pathology. We have started to test idebenone on our "cardiac" mutant mice at three different doses. We indeed found a statistically significant 10% survival increase for the highest dose (90 mg/kg. These results indicate that idebenone is indeed protective in frataxin deficiency and presumably acts at an early step of the pathogenesis. We now plan to test idebenone on a new "neuronal-only", slowly progressive, model that we recently developed in the laboratory and plan to test additional antioxidant compounds on the "cardiac" model. These results should help accelerating the delivery of a market authorisation for the use of idebenone in Friedreich ataxia therapy, since idebenone recently obtained the orphan drug status at the European level.