Dr Ko-Fan Chen and Prof Charalambos P. Kyriacou
Dystonia is the third most common neurological movement disorder, and is characterised by involuntary twisting and repetitive movements or abnormal postures. Sleep disruption has been reported in dystonia despite the easing of motor symptoms during sleep1, suggesting that the involuntary regulation of arousal and motor control is also perturbed in dystonia. Interestingly, dystonia and sleep disturbances are associated with alterations in the common neuronal pathway of synaptic plasticity2,3. Yet it remains unclear whether sleep abnormalities and motor phenotypes in dystonia stem from overlapping synaptic mechanisms. Moreover, it is unclear whether reduced sleep quality influences the severity of dystonic movements. The induction of synaptic plasticity results in well-characterized changes in neuronal transcription4. In contrast, the transcriptional signatures of dystonia pathogenesis remain elusive, despite the recent identification of several dystonia-associated genes (DAGs).
One of these DAGs encodes the neuronal calcium sensor, Hippocalcin. Loss-of-function mutations in Hippocalcin are linked to autosomal recessive torsion dystonia type 2 (DYT2) and increased excitability in cultured neurons5. However, it is unclear whether reduced Hippocalcin function perturbs neuronal transcription, and if so, how such alterations impact sleep and motor phenotypes in dystonia. Recently we generated knock-out (NcaKO) and neuronal knock-down (NcaKD) fly lines for the Drosophila Hippocalcin homologue, Neurocalcin (Nca). These flies show sleep loss and reduced locomotor activity. Also, the sleep loss is associated with enhanced synaptic neurotransmitter release in the AMMC, a subset of the Drosophila sensorimotor system6. These flies therefore successfully model the motor/sleep defects and the synaptic abnormality in dystonia.
This PhD project will use these novel fly models of DYT2 dystonia, combined with the versatile Drosophila genetic toolkit, to investigate the potential role of sleep in dystonia pathogenesis, answering following key research questions:
• What is the molecular pathway underlying altered synaptic activity and sleep in Drosophila DYT2 dystonia models?
• Do the identified mechanism also impact motor control?
• Does sleep modulate motor defects in DYT2 and other dystonia models?
1) Identifying pathogenic molecular pathways in DYT2 flies
A recent study demonstrated that the differentially expressed transcriptome in a Huntington’s disease mutant background contains pathogenic molecular pathways linked to disease phenotypes7. By applying the same strategy, we used RNA-Seq to define the differentially expressed transcriptome in NcaKO fly heads. Focusing on the role of these transcriptional targets in sleep control, we have performed an RNAi screen to knock down the top 50 differentially expressed genes in the nervous system of NcaKD flies. Among these genes, we have identified five candidate genes as sleep modifiers in the following cellular pathways: piRNA synthesis, telomere maintenance, and GPCR signalling. These data suggest that Nca functions via these pathways to regulate sleep. The PhD student will further verify these genetic interactions using hypomorphic and hypermorphic somatic CRISPR alleles of candidate genes. They will also investigate whether these candidate genes regulate Nca-mediated synaptic release in AMMC neurons using a fluorescent reporter of synaptic neurotransmitter exocytosis (spH)6.
2) Identifying shared pathways underlying motor and sleep phenotype
Using a video tracking tool, we found that NcaKO flies show reduced locomotion upon stimuli. Although this is an indicator of motor defects, the abnormal movement akin to dystonia has not been explored further in the fly models. Drosophila requires fine control over sensorimotor circuitry to perform the male’s courtship song, which represents an ideal readout of precise motor control. The student will thus test for alterations in the courtship song of DYT2 models and whether candidate genes identified (Objective.1) also modulate song and locomotion motor phenotypes in DYT2 flies
3) Testing whether sleep can ameliorate motor phenotypes
To test if increases in sleep improve motor phenotypes in dystonia models, the student will optogenetically activate known sleep-promoting circuits in DYT2 and four other previously established dystonia fly models (collaboration with Dr James Jepson, UCL). They will then monitor subsequent changes in motor phenotypes. Conversely, constant vibration throughout the night via the DART system6 will be applied to test whether sleep deprivation exacerbates motor phenotypes.
1. Sleep Med. Rev. 26, 95-107 (2016).
2. Neurobiol. Dis. 42, 162-170 (2011).
3. Science 355.6324: 507-510. (2017).
4. Neuron 66, 337-351 (2010).
5. Am. J. Hum. Genet. 96, 657-665 (2015).
6. Elife 8, 38114 (2019).
7. Cell Syst. 7(1), 28-40 (2018).
8. Mol. Cell 44, 572-584, (2011)