Restless legs syndrome is a common neurological disorder characterized by an urge to move the legs associated with uncomfortable sensations that occur or worsen with rest, are relieved by movements, and are worse in the evening [1
]. Motor restlessness is evident when awake and periodic limb movements frequently occur during sleep, resulting in sleep fragmentation and non-restorative sleep. When the syndrome is mild it may be a mere nuisance, but when severe it can be life-altering, resulting in profound sleep disruption, impaired daytime functioning, a global decrease in quality of life and possibly increased cardiovascular disease risk [2
]. The diagnosis is made by clinical symptoms and there is no definitive confirmatory testing available. Although clinical research has demonstrated that restless legs syndrome is associated with iron deficiency [3
] and can be treated pharmacologically with dopamine agonists [4
], the underlying mechanisms are largely unknown and even localization of the primary pathology within the nervous system is uncertain. Population and family studies indicate that restless legs syndrome has a strong genetic component, so several groups have carried out genome-wide association studies to identify genes that confer susceptibility to restless legs syndrome [5
]. These studies have successfully identified a number of genetic variants suspected to be involved in restless legs syndrome, including one named BTBD9
]. While additional loci continue to be identified [8
], it has been difficult to demonstrate that these susceptibility genes are causative for restless legs syndrome. In this issue of Current Biology
, Freeman et al.
] report how they used the molecular genetic tools available in the model genetic organism Drosophila melanogaster
to functionally characterize the fly homologue of BTBD9
) as a causal factor in restless legs syndrome.
is a poorly characterized gene that accounts for approximately 50% of the population attributable risk for restless legs syndrome and is therefore an ideal candidate for functional analysis using Drosophila
genetics. To begin, Freeman et al.
] identified the fly homologue of BTBD9
and created two null mutations in the gene. When they examined sleep in these mutants, they found that both mutant alleles caused severely fragmented nighttime sleep and an increase in the amount of waking after sleep onset, as is seen in human patients with restless legs syndrome. Because sleep fragmentation is only one component of restless legs syndrome, the authors needed to evaluate aspects of movement that have face-validity with symptoms seen in human patients with restless legs syndrome. In humans, the diagnosis of restless legs syndrome is facilitated by the ‘suggested immobilization test’, in which periodic limb movements and leg discomfort are worsened when subjects are asked to sit in bed with their legs outstretched for an hour [10
]. Intriguingly, when mutants in dBTBD9
were confined to a small space, to simulate the suggested immobilization test, they showed significantly more locomoter behavior than controls. Moreover, when locomotor behavior was precisely quantified in an independent assay, the mutants were found to spend more time in longer uninterrupted bouts of walking than controls. Together, these data indicate that dBTB9
mutant flies recapitulate key behavioral aspects of restless legs syndrome, including sleep fragmentation and motor restlessness.
To determine whether dBTB9
would also show physiological changes found in restless legs syndrome patients, Freeman et al.
] examined dopamine signaling and iron homeostasis in the mutant flies. Interestingly, they found that flies mutant for dBTBD9
show dramatically reduced brain dopamine levels and that the abnormal sleep phenotype was completely rescued by administering mutant flies the dopamine D2 receptor agonist Pramipexole, which is used clinically in humans to treat restless legs syndrome. Importantly, Freeman et al.
] used RNA interference (RNAi) to show that they could recapitulate restless legs syndrome symptoms by knocking-down dBTBD9
expression in a subset of dopaminergic neurons. Finally, the authors over-expressed BTBD9
in HEK cells to confirm that the gene does indeed play a role in iron homeostasis, acting via regulation of iron regulatory protein-2 (IRP2). It is worth noting that a homologue of another restless legs syndrome associated variant, MEIS1
, has also been shown to affect iron homeostasis in Caenorhabditis elegans
How does BTBD9
regulate dopamine signalling, iron homeostasis and sleep? Although much more work needs to be done, Freeman et al.
] propose that the BTBD9 protein belongs to a family of BTB-domain containing substrate adaptors for the Cullin-3 (Cul3) class of E3 ubiquitin ligases. Interestingly, Cul3
has recently been shown to regulate sleep in flies [12
], further supporting the idea that Cul3 has a role in sleep regulation. In the case of iron homeostasis, BTBD9 may act together with Cullin-3 to regulate levels of iron regulatory protein-2 (IRP2), which controls ferritin expression and thus iron metabolism. BTBD9 also seems to play a role in dopamine biosynthesis by unknown mechanisms. Several questions remain but they are now experimentally tractable. Are iron regulation and dopamine regulation mechanistically related, or are they independent processes? Is the specificity of dBTBD9
within dopaminergic neurons due to restricted expression of dBTBD9
in a defined set of dopaminergic neurons? Or, is the specificity of dBTBD9
due to differences in the presence of Cul3 substrates in particular neurons? Is the ability of dBTBD9
to influence sleep regulation confined to dopaminergic neurons or does dBTBD9 play a more fundamental role in regulating sleep and waking throughout the nervous system? Finally, which are the substrates for BTBD9 and Cul3, and how might they influence restless legs syndrome?
Genome-wide association studies have succeeded in identifying a large number of disease susceptibility genes. Nonetheless, it is frequently difficult to collect additional data that can provide causative or functional insight into the underlying molecular mechanisms and this is only complicated further when evaluating complex traits that are differentially influenced by environmental conditions. With this in mind, one of the most compelling aspects of the approach of Freeman et al.
] is the successful use of a model system to functionally characterize a susceptibility gene that was originally identified in human genome-wide association studies. A similar strategy has recently been used to functionally examine genes identified in a genome-wide association study for loci influencing the burden of Alzheimer’s disease [13
] and to extend the results from a small genetic study on short sleeping humans [14
]. In this regard, it is important to note that tools have been developed so that orthologous genes can be quickly identified in Drosophila
and other model organisms [15
]. These tools will certainly expedite the ability of labs to identify and then test candidate genes from genome-wide association studies to provide critical insight into mechanism and causation of human disease states. Thus, the use of model systems to functionally characterize genes identified with genome-wide association studies is a move in the right direction.