Rhythmic data are ubiquitous in the life sciences. Biologists need reliable statistical tests to identify whether a particular experimental treatment has caused a significant change in a rhythmic signal. When these signals display nonstationary behaviour, as is common in many biological systems, the established methodologies may be misleading. Therefore, there is a real need for new methodology that enables the formal comparison of nonstationary processes. As circadian behaviour is best understood in the spectral domain, here we develop novel hypothesis testing procedures in the (wavelet) spectral domain, embedding replicate information when available. The data are modelled as realisations of locally stationary wavelet processes, allowing us to define and rigorously estimate their evolutionary wavelet spectra. Motivated by three complementary applications in circadian biology, our new methodology allows the identification of three specific types of spectral difference. We demonstrate the advantages of our methodology over alternative approaches, by means of a comprehensive simulation study and real data applications, using both published and newly generated circadian datasets. In contrast to the current standard methodologies, our method successfully identifies differences within the motivating circadian datasets, and facilitates wider ranging analyses of rhythmic biological data in general.MSC 2010 subject classifications: Primary 62M10, 60G18; secondary 60-08.

Background: Inherited mutations in the LRRK2 protein are the common causes of Parkinson’s disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. Objective: To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. Methods: LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern (‘sleep’), and courtship memory determined in aged flies. Results: Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. Conclusion: We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson’s disease.

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Acknowledgements. We are grateful for the gifts of flies from Kristin Scott,

BackgroundInherited mutations in the LRRK2 protein are the most common known cause of Parkinson’s, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. Which Rabs interact with LRRK2 in dopaminergic neurons to facilitate normal and pathological physiological responses remains to be determined. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. We now ask if Rab10 is required for LRRK2-induced physiological responses in DA neurons.MethodsLRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, vision, circadian activity pattern and courtship memory determined in aged flies.ResultsRab10 loss-of-function rescued bradykinesia of the Proboscis Extension Response (PER) and visual defects of aged flies expressing LRRK2-G2019S in DA neurons. Rab10 knock-down however, did not rescue the marked sleep phenotype which results from dopaminergic expression of LRRK2-G2019S. Courtship memory is not affected by LRRK2 expression, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons.ConclusionsWe conclude that, in Drosophila dopaminergic neurons, Rab10 is involved differentially in LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of dopaminergic neurons to neurodegeneration seen in people with Parkinson’s.Graphical AbstractRab10 depletion ameliorates the proboscis extension bradykinesia and loss of synaptic signalling in the retina induced by LRRK2-G2019S expression (magenta arrows/orange crosses). Rab10 manipulation does not affect the ‘sleep’ phenotype from LRRK2-G2019S (magenta arrow). Reduction of Rab10 facilitates conditioned courtship memory, but LRRK2 has no effect (yellow arrow). All manipulations of Rab10 and G2019S in dopaminergic neurons, shown in the outline of the brain (filled cells have high levels of Rab10). We conclude that Rab10 and LRRK2 interact in some, but not all dopaminergic neurons. This may underlie differences in the susceptibility of different human striatal dopaminergic cells to Parkinson’s.

Circadian behavioural deficits, such as increased daytime naps and reduced night-time sleep, are common in Alzheimer’s disease and other tauopathies. But it has remained unclear whether these circadian abnormalities arise from tau pathology in either the master pacemaker or downstream neurons. Here we study this question by selectively expressing different human tau proteins in specific Drosophila brain circuits and monitoring locomotor activity under light-dark (LD) and in “free-running” dark-dark (DD) conditions. We show that expressing human tau proteins in the fly brain recapitulates faithfully several behavioural changes found in tauopathies. We identify discrete neuronal subpopulations within the clock network as the primary target of distinct circadian behavioural disturbances in different environmental conditions. Specifically, we show that the PDF-positive pacemaker neurons are the main site for night-activity gain and -sleep loss, whereas the non-PDF clock-neurons are the main site of reduced intrinsic behavioural rhythmicity. Bioluminescence measurements revealed that the molecular clock is intact despite the behavioural arrhythmia. Our results establish that dysfunction in both the central clock- and afferent clock-neurons jointly contribute to the circadian locomotor activity rhythm disruption in Drosophila expressing human tau.Significance StatementThis study directly links in vivo human tau protein expression in region-specific Drosophila clock-neurons with the resulting sleep and circadian rhythm deficits to extract new knowledge of how Alzheimer’s disease and other tauopathies perturb the balance of activity and sleep. We anticipate that this novel approach will provide a useful general template for other studies of neurodegeneration in model organisms, seeking to dissect the impact of neurodegenerative disease on circadian behaviour, and further deepening our understanding of how the clock-neuron network works.

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