Alterations in sleep-activity cycles and clock gene expression across the synucleinopathy spectrum

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Grunstein, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4717144/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Neurodegenerative synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are strongly associated with sleep disturbances. Furthermore, isolated Rapid Eye Movement Sleep Behaviour Disorder (iRBD) is now established as the strongest risk factor for developing PD or DLB, often preceding the clinical diagnosis by several years. Objectives We aimed to investigate sleep/wake cycles and circadian rhythms in patients with early PD and DLB, along with ‘at risk’ prodromal subjects diagnosed with iRBD. Methods Fifteen healthy controls, 20 iRBD, 16 PD and 17 DLB patients within 5 years of diagnosis, underwent assessment. Sleep/wake cycles were evaluated using questionnaires and actigraphy. Salivary and oral mucosa samples were collected every 3 hours to measure melatonin levels and Bmal1 clock gene expression over 24-hours. Results Both subjective and objective measures of sleep/wake cycles demonstrated that the DLB group exhibited the most significant sleep/wake cycle disruption. In the DLB group, no fundamental sine wave could be fitted to the level of melatonin secretion, indicating a severe disruption in the daily rhythm of this hormone. There was a statistically significant pattern of decreasing median Bmal1 amplitude from HC, to iRBD, to PD and then to DLB ( p = 0.037). Conclusions This work highlights a differential gradient of objective disruption in the daily circadian rhythms from iRBD to established PD and DLB and is the first to directly demonstrate disruption of clock gene expression and melatonin in DLB. The findings support sleep/wake disruption as a marker of neuropathological severity and potentially a novel therapeutic target across the synucleinopathy spectrum. Dementia with Lewy bodies Parkinson’s disease rapid eye movement sleep behaviour disorder circadian melatonin Bmal1 actigraphy sleep Figures Figure 1 Figure 2 BACKGROUND Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative disorders defined at post-mortem by the presence of abnormal intraneuronal aggregates containing α-synuclein, known as Lewy bodies and Lewy neurites, alongside regional cellular loss [ 1 , 2 ]. Whilst similar, the pathology of DLB is typically more extensive than that seen even in the advanced stages of PD, when dementia often becomes evident (Parkinson’s disease dementia). The clinical features of PD and DLB are highly convergent and include motor parkinsonism, cognitive dysfunction, neuropsychiatric (e.g., hallucinations) and autonomic symptoms [ 2 , 3 ]. Interestingly, patients with PD and DLB also experience a wide range of sleep disturbances including longer sleep onset latency, increased night-time sleep fragmentation, early morning awakenings and daytime sleepiness [ 4 , 5 ]. This suggests that sleep disturbances may be central to the pathophysiology and progression of Lewy body disorders. Furthermore, around 90% of DLB and 50% of PD patients will suffer from Rapid Eye Movement (REM) sleep behaviour disorder (RBD), which manifests as dream enactment accompanying the loss of the normal paralysis (atonia) of REM sleep [ 6 , 7 ]. This parasomnia frequently precedes the clinical diagnosis of PD and DLB by many years where it is referred to as ‘isolated’ RBD (iRBD), before overt cognitive or motor symptoms occur [ 6 , 7 ]. Isolated RBD is the strongest clinical predictor for developing a synucleinopathy with 75% of patients converting to either PD or DLB (and a small proportion to multiple system atrophy, another synucleinopathy) over a 12-year period [ 8 ]. Thus, iRBD is now widely considered a ‘prodromal’ synucleinopathy, which can be placed early on the disease spectrum of both PD and DLB. Given the implications for prognosis and recruitment for disease modifying trials, there is a need for clinical and biological markers that highlight the risk of phenoconversion in patients with iRBD, and their trajectory towards either DLB or PD [ 9 ]. It is well recognised that the biological clock regulates the circadian oscillations of processes such as sleep, alertness, immune and metabolic functions, as well as the secretion of critical regulating hormones, such as melatonin [ 10 , 11 ]. At the molecular level, the mammalian circadian clock consists of a set of clock genes (e.g., Bmal1, Clock, Per1, Per2,Cry1, Cry2 ), which operate via oscillating levels in mRNA and protein abundance across most mammalian cells and tissues over the daily cycle [ 12 , 13 ]. The levels of melatonin increase during the evening and this hormone plays a key role in sleep regulation and the synchronisation of the circadian clock [ 14 , 15 ]. Melatonin levels [ 16 – 19 ] and peripheral blood clock gene expression [ 16 , 19 – 21 ] have previously been reported for iRBD and PD patients and most studies show dysregulation of these rhythms. To our knowledge, there are no publications showing clock gene expression or melatonin patterns in DLB patients. There is a bidirectional regulation between the circadian and the dopamine systems in terms of their gene expression [ 22 – 26 ], as well as the synthesis and release of dopamine and melatonin [ 27 , 28 ]. This suggests that the loss of dopamine cells in PD or the dopamine medication they take may have a deregulatory effect on the circadian clock. The aim of the current study was to investigate the function of the circadian clock in patients with early but established PD and DLB, along with an ‘at risk’ prodromal iRBD group and an age matched healthy control (HC) group. Participants underwent standardised clinical assessments and an analysis of their daily profiles of activity/rest recorded by actigraphy, as well as having salivary and oral mucosa samples collected around the clock to measure melatonin secretion and Bmal1 expression, respectively. It was hypothesized that disruption in daily rhythms would occur in a graded manner across the continuum, with least disruption in healthy controls, followed by further disruption in patients with iRBD, then PD and the greatest disruption in DLB, reflecting the perceived severity of pathology. Methods Participants Sixty-eight participants with a diagnosis of PD (N = 16), DLB (N = 17), iRBD (N = 20) or age matched healthy controls (HC) (N = 15) were recruited from the Parkinson’s Disease Research Clinic at the Brain and Mind Centre, University of Sydney and via community advertisements. All participants underwent a detailed neurological assessment by S.J.G.L. and E.M. at the Brain and Mind Centre, in Sydney (Australia). Video polysomnography (PSG) of all participants was performed at the Woolcock Institute of Medical Research. The presence of RBD was confirmed by clinical diagnosis using video polysomnography and met the diagnostic criteria according to the International Classification of Sleep Disorders-3 [ 29 ]. All PD patients had been diagnosed within 5 years of the study according to the Movement Disorders Society (MDS) PD diagnostic criteria [ 30 ]. The DLB patients satisfied the consensus criteria of the Fourth report of the DLB Consortium and were all within 3 years of diagnosis at the time of the study [ 31 ]. Motor function was assessed using the motor section of the MDS Unified Parkinson’s Disease Rating Scale (MDS-UPDRS-III) [ 32 ]. Healthy controls underwent the same procedures as patients, including neurological assessment. The study was approved by Royal Prince Alfred Hospital Ethics Review Committee, Sydney, Australia Protocol No X15-0207 & HREC/15/RPAH/272. All participants provided written informed consent prior to commencement of the study. Procedures Participants completed general health and lifestyle questionnaires, as well as neurological and sleep questionnaires detailed in Table 1 . Sleep-wake activity was objectively assessed via actigraphy and all participants stayed in the sleep laboratory at the Woolcock Institute of Medical Research for 24 hours, where they underwent overnight PSG following the SINBAR protocol [ 33 ]. During this time, salivary and oral mucosa samples were collected every three hours (over the 24-hour period) to measure melatonin and Bmal1 clock gene expression. Illuminance in the room during daytime hours (~ 7am-10pm) was ~ 100 lux and at lights-out < 1 lux. Table 1 Average and standard deviation of demographic, clinical and sleep measures. Key: Data presented as mean (standard deviation). F, female. MoCA, Montreal Cognitive Assessment. MMSE, Mini-Mental State Exam. HADS-A, Hospital Anxiety and Depression Scale-Anxiety. HADS-D, Hospital Anxiety and Depression Scale-Depression. MEQ, Morningness-Eveningness Questionnaire; ESS, Epworth Sleepiness Scale. SCOPA-Sleep (NS), Scales for Outcomes in Parkinson’s Disease-Sleep (Nocturnal Sleep). SCOPA-Sleep (DS), Scales for Outcomes in Parkinson’s Disease (Sleep-Daytime Sleep). Bolded values denote significant difference in ANOVA (* p < 0.05; ** p < 0.01, *** p < 0.001; a represents HC, b iRBD, c PD and d DLB in the Tukey’s posthoc analysis). Variable HC n = 15 (6F) iRBD n = 20 (4F) PD n = 16 (6F) DLB n = 17 (2F) ANOVA F p value Age 67.3 (9.5) 66.6 (7.3) bd* 63.7 (10.5) cd** 74.3 (6.7) 4.631 0.0054 Disease duration (years) - 7.9 (7.2) bc**, bd*** 2.7 (1.8) 1.3 (1.1) 10.45 0.0002 Years of education 13.1 (2.5) 14.6 (3.1) bd* 14.7 (3.4) cd* 11.5 (3.4) 4.034 0.0109 MoCA 27.3 (2.35) ad*** 27.9 (1.6) bd*** 28.8 (1.3) cd*** 16.9 (6.6) 37.91 < 0.0001 MMSE 29.2 (0.9) ad*** 29.05 (1.02) bd*** 29.3 (1.3) cd*** 20.6 (6.4) 27.12 < 0.0001 HADS-A 2.9 (2.2) ad* 3.6 (2.6) bd* 3.9 (3.2) 6.5 (4.2) 3.899 0.0129 HADS-D 1.8 (2) ad*** 2.5 (3.7) bd*** 3.4 (3) cd*** 8.7 (5.1) 11.32 < 0.0001 HADS-Total 4.7 (3.9) ad*** 6.1 (5.7) bd*** 7.3 (5.9) cd** 15.2 (8.1) 9.206 < 0.0001 MEQ 60.9 (6.3) 58.1 (7.7) 59.5 (9.1) 62.8 (9.2) 1.089 0.3604 ESS 6.7 (3.1) ad** 5.6 (4.5) cd* 7.25 (3.7) bd*** 11.8 (5.2) 7.046 0.0004 SCOPA-Sleep NS 4.3 (2.3) 2.9 (2.5) 3.6 (3.1) 4.5 (4.9) 0.6656 0.5767 SCOPA-Sleep DS 3.3 (2.6) ad** 2.4 (2.1) bd*** 3.6 (3.1) cd*** 8.9 (4.9) 12.25 < 0.0001 UPDRS-III 3.1 (4) ac***, ad*** 8.8 (8.5) bc***, bd*** 24.2 (10.2) ac*** bc***, cd** 36.7 (15.3) ad*** bd***, cd** 32.816 < 0.001 DDE (mg) 0 (0) ac** 0 (0) ac** 359.4 (262.2) ac** bc** 238.2 (509.1) 6.479 < 0.001 Cholinergic (mg) 0 (0) 0 (0) 0 (0) 6.0 (4.3) 32.6 < 0.001 Questionnaires A series of questionnaires evaluating mood and sleep were completed as shown in Table 1 . Cognitive performance was evaluated with the Montreal Cognitive Assessment (MoCA) [ 34 ] and the Mini-Mental State Examination (MMSE) [ 35 ]. The Hospital Anxiety and Depression Scale (HADS) was used to assess anxiety (HADS-A) and depressive symptoms (HADS-D) [ 36 ]. The Morningness-Eveningness Questionnaire (MEQ) was used to assess chronotype [ 37 ]. The Epworth Sleepiness Scale (ESS) was used to derive an estimate of daytime somnolence [ 38 ]. The Scales for Outcomes in PD-Sleep Scale (SCOPA-S) was used to estimate both night-time sleep (NS) and day-time sleep (DS) quality [ 39 ]. Actigraphy Participants wore a Philips Respironics Actiwatch 2 (Koninklijke Philips N.V., Amsterdam, Netherlands) on the non-dominant wrist for at least one week. Manual scoring of actigraphy and analysis of sleep parameters was performed using Philips Respironics Actiware-5 software (Koninklijke Philips N.V.). Salivary melatonin Melatonin was sampled every three hours for a period of 24 hours. Sampling started at 16:00 and continued every 3 hours until 13:00 on the next day. During night-time hours (10 pm-7 am) saliva sampling occurred in dim light conditions (< 10 lux). Samples were stored immediately in a -80 0 C freezer until analysis. Salivary melatonin concentrations were determined by radioimmunoassay (Buhlmann Laboratories; Allschwil, Switzerland). These assays have a limit of detection of 1 pg/mL with an inter-assay coefficient of variations of 7.4% at 4.41 pg/mL and 10.7% at 48.14 pg/mL. Bmal1 gene expression Oral mucosa samples were collected at the same times as saliva samples. They were obtained by ‘scratching’ the inner cheek for one minute with a cytological brush. The samples were immediately placed into RNAlater reagent (Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia) and stored at -20 0 C until analysis. The mRNA was isolated using the Isolate II RNA Micro kit (Bioline, London, UK). SuperScript VILO cDNA Synthesis kit (Thermofisher) was used to reverse-transcribe the mRNA samples. Quantitative PCR was performed using the ABI Step one Plus detection system and data were analyzed with StepOne software (Applied Biosystems, Melbourne, VIC, Australia). Assays were carried out using Taqman Universal MMIX II with UNG and TaqMan Gene Expression assays for Bmal1 (Hs00154147_m1) and Gapdh (Hs99999905_m1) according to protocol of manufacturer (Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia). Data analysis The averaged activity levels measured by actigraphy were plotted for each patient group in 30 seconds-bins of the 24-hour interval averaged over 7 days of recording. Significance of the rhythmicity of activity levels, melatonin and Bmal1 expression was determined with the CircWave v 1.4 software developed by R.A. Hut, University of Groningen, NL [ 40 – 42 ], which uses a linear harmonic regression fit with an assumed period of 24 hours. Cosinor analysis was performed and cosine curve parameters (amplitude, acrophase and mesor) obtained using the toolbox developed by [ 43 ] for MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Inc., Natick, Massachusetts, United States. GraphPad Prism 9.0 was used for statistical analysis. Demographic, questionnaire, and actigraphy data were compared by ANOVA. Post hoc analyses were performed using Tukey's honest significant difference post hoc test. Rhythmicity differences between groups (activity-rest rhythms, melatonin and Bmal1 expression) were assessed using the Jonckheere-Terpstra test for ordered alternatives, in line with the a priori hypothesis that circadian abnormalities would differ in accordance with expected degree of neuropathological disease - with controls being least affected, followed by iRBD, PD and then DLB (most affected). Post-hoc partial correlations were performed using SPSS (version 26.0.0, IBM) with controlling variables as specified. Results are shown as mean ± standard deviation or median (interquartile range) as specified. Values of p < 0.05 were considered statistically significant. RESULTS Demographics and clinical variables Table 1 summarizes the demographic, clinical, subjective sleep and circadian measures of the participants. In keeping with the natural history of synucleinopathies there was a predominance of male subjects in all groups: PD (37.5% females), DLB (11.7% females), iRBD (20% females) and HC (40% females). Age did differ significantly between patient groups, which is expected given the established later onset of disease in DLB compared to PD. The post-hoc testing showed the DLB group to be older (74.3 ± 6.7y) than iRBD (66.6 ± 7.3y); and PD (63.7 ± 10.5) who did not differ significantly from each other or the control group. To assist matching, HC were intentionally recruited across a wider age spectrum, as such age did not differ between HC and any of the disease groups on post-hoc testing. In keeping with the goal of recruiting of early disease, disease duration was 2.7 ± 1.8 years in the PD group, and 1.3 ± 1.1 years in the DLB group. Subjective Sleep and Circadian Measures There was no difference in morningness-eveningness between groups, being all moderate morning chronotypes according to the MEQ (score 59–69) except iRBD (58.1 ± 7.7), which was the intermediate chronotype (score 42–58). Excessive daytime sleepiness assessed by the ESS demonstrated lower normal daytime sleepiness (0–5 score) in the iRBD group (5.6 ± 4.5), higher normal daytime sleepiness (scores 6–10) for HC (6.7 ± 3.1) and PD (7.25 ± 3.7) and mild excessive daytime sleepiness (scores 11–12) for the DLB group (11.8 ± 5.2), which was significantly higher than all of the other groups. Subjective night-time sleep disturbance measured by SCOPA-S (cut off for poor sleep quality > 6) showed values below cut off in all groups and no significant difference. Subjective day-time sleep disturbance measured by SCOPA-S (cut off for poor sleep quality > 4) showed values below the cut off for all groups except the DLB group (8.9 ± 4.9), which reported significantly worse daytime somnolence compared to all other groups. We found a statistically significant pattern of increasing median day-time sleep disturbance measured by SCOPA-S from controls, to iRBD, PD and DLB (two sided, T JT = 923.00, z = 4.114, p < 0.001). A similar pattern of increasing daytime sleepiness measured by the ESS was seen across groups (two sided, T JT = 1122.00, z = 3.207, p = 0.001). Actigraphy derived activity and sleep parameters Activity patterns analysed by the Actiwatch software are summarised in Table 2 . There was no significant difference among the groups in actigraphic bed and rise times, average time spent in bed, total sleep time (TST) and sleep time in 24 hours. Wake time after sleep onset (WASO) was significantly shorter in the iRBD (44.7 ± 23.9) and PD (24.5 ± 8.7) groups compared to DLB (73.4 ± 40.4). Percentage of sleep fragmentation was lower in the HC (27.9 ± 6.8), iRBD (30.9 ± 10.1) and PD (19.7 ± 6.6) groups compared to DLB (44.7 ± 16.8). Activity counts over 24 hours were significantly higher in the HC (268,616 ± 49,590) group compared to DLB (146,191 ± 66,488). Sleep efficiency (%) was higher in the PD group (90 ± 5) compared to DLB (81 ± 10). Activity counts during the night were lower in the iRBD (9,066 ± 4,385) and PD (5,276 ± 1,475) compared to DLB (1,5915 ± 9,385). Percentage of time awake during the night was lower in the PD (6.3 ± 2.5) compared to DLB (14.2 ± 7.9) and percentage of wake during the day was higher in the HC (79.3 ± 7.8) compared to DLB (63.5 ± 11.5). Table 2 Average and standard deviation of actigraphy variables. Bolded values denote significant difference in the Tukey Post hoc analyses (* p < 0.05; ** p < 0.01; a represents HC, b iRBD, c PD and d DLB). Sleep variable HC iRBD PD DLB ANOVA F p value Bedtime 22:54 (1:17) 23:01 (0:41) 22:33 (0:56) 22:18 (0:32) 1.697 0.1818 Risetime 7:33 (1:04) 7:27 (01:12) 6:50 (0:43) 7:45 (0:42) 1.886 0.1462 Average Time in bed (min) 519.4 (77.2) 505.5 (75.8) 497.1 (53.3) 567.1 (47.5) 2.772 0.0529 Total Sleep Time (min) 447.1 (80.5) 440.7 (64.2) 447.9 (45.9) 459.3 (68.5) 0.1753 0.9126 Wake after Sleep Onset (min) 45.8 (16.6) 44.7 (23.9) bd* 24.5 (8.7) cd*** 73.4 (40.4) 7.000 0.0006 Fragmentation % 27.9 (6.8) ad** 30.9 (10.1) bd* 19.7 (6.6) cd*** 44.7 (16.8) 10.17 < 0.0001 Sleep time 24 hours (min) 654.2 (117.6) 701.3 (101.5) 754.8 (140.1) 781.6 (119.6) 2.487 0.0732 Activity counts 24 hours 268616 (49589.8) ad** 219963.9 (87545.4) 184697.9 (93230.9) 146191.4 (66487.7) 4.985 0.0047 Sleep efficiency % 85.8 (5.5) 85.4 (9.9) 90.3 (4.8) cd* 80.9 (10.2) 2.474 0.0743 Activity counts during night 10080.4 (2907.9) 9065.5 (4385) bd* 5275.8 (1474.8) cd*** 15915 (9385.2) 7.3666 0.0004 %Wake during night 10.3 (3.7) 9.5 (4.8) 6.3 (2.5) cd** 14.2 (7.9) 4.46 0.0082 %Wake during day active 79.3 (7.8) ad* 71.3 (14.1) 67.4 (14.1) 63.5 (11.5) 3.194 0.0328 Rest-activity rhythms between the groups The averaged rest-activity profiles measured by actigraphy across diagnoses are plotted in Fig. 1 . All four groups had significant activity rhythm profiles. A cosinor analysis was performed to derive the activity mesor, amplitude, and acrophase (Table 3 , Fig. 1 ). Mesor (Midline Estimating Statistic of a Rhythm) is the average level around which the oscillation occurs. We estimated amplitude as the distance between the mesor and the peak of the oscillations. Acrophase is the time (clock time in our case) at which the maximal value of the oscillation occurs (Suppl Fig. 1). The test for ordered alternatives showed that there was a statistically significant pattern of decreasing median mesor of the rest-activity rhythm from controls, to iRBD, to PD and then to DLB (two sided, T JT = 225.00, z = -3.52, p < 0.001). Similarly, a statistically significant trend was found for the amplitude of the rest-activity rhythm, being significantly higher in the HC (87.4 ± 13) group and lowest for the DLB (47.2 ± 27.3) group (two sided, T JT = 205.00, z = -3.90, p < 0.001). No statistically significant trend was seen for the acrophase of the rest-activity rhythm across groups. In view of this result, a post-hoc partial correlation was performed assessing the relationship between rest-activity mesor and motor parkinsonism quantified by UPDRS-III score across iRBD, PD and DLB groups controlling for diagnosis and disease duration. As expected, there was a moderate negative correlation between UPDRS-III score (21.3 ± 14.8) and activity mesor (70.1 ± 30.8) and activity amplitude (58.1 ± 28.4), which were statistically significant ( r (33)=-0.38, p = 0.024; and r (33)=-0.46, p = 0.006, respectively). Table 3 Cosinor analysis of actigraphy, melatonin and Bmal1 expression. Average for all variables with standard deviation in between brackets is given for all variables. Median and interquartile range in brackets is given in square brackets. Rhythmicity differences between groups were assessed using the Jonckheere-Terpstra test for ordered alternatives. The level of statistical significance was set at p < 0.05. Cosinor analysis HC iRBD PD DLB p values Activity Mesor 98.9 (18.7) [98.5 (15.4)] 82.1 (27.5) [78.7(28.5)] 67.8 (34.4) [52.6(52.0)] 57.8 (26.4) [52.2(36.4)] p < 0.001 Activity Amplitude 87.4 (13) [86.8 (6.1)] 69.2 (27.1) [64.4(23.2)] 56.3 (27.3) [47.6(35.8)] 47.2 (27.3) [40.9(23.8)] p < 0.001 Activity Acrophase 14.1 (1.9) [13.5(2.4)] 14.5 (1.3) [14.7(1.4)] 13.8 (0.9) [13.9(1.2)] 13.5 (0.7) [13.4(0.5)] NS Melatonin Mesor 3.1 (1.8) [2.4(2.6)] 3.01 (2.1) [2.7(3.1)] 3.33 (2.4) [2.8(2.6)] 3.01 (1.9) [2.7(2.6)] NS Melatonin Amplitude 3.2 (2.4) [2.4(2.2)] 2.7 (2.4) [2.6(3.2)] 2.5(2.3) [1.2(3.3)] 3.0 (3.0) [1.8(2.5)] NS Melatonin Acrophase 2.2 (2.1) [1.8(2.9)] 3.4 (4.1) [2.9(3.8)] 2.5 (2.06) [2.7(2.3] 7.4 (5.1) [5.9(9.7)] NS Bmal1 Mesor 38.4 (7.8) [39.2(11.4)] 43.1 (15.5) [38.2(24.9)] 43.8 (15.5) [41.9 (19.4)] 42.7(13.9) [44.6(22.4)] NS Bmal1 Amplitude 28.1 (6.2) [27.9(7.2)] 23.1 (11.4) [22.9(14.8)] 23.1 (9.4) [21.1 (11.1)] 20.4 (4.5) [19.9(5.1)] p < 0.05 Bmal1 Acrophase 1.9 (1.8) [2.4(2.7)] 1.2(3.6) [1.5(3.09)] 1.3 (4.5) [0.8(4.9)] 24.1 (4.7) [23.88(3.2)] NS Daily melatonin secretion Across each group, except DLB, one fundamental sine wave contributed significantly to the explained variance (Fig. 2 ). No harmonics could be fitted for the melatonin data obtained from the DLB group, meaning that there was no circadian variation of melatonin in these patients. Analysis of the cosinor analysis outputs (mesor, amplitude and acrophase) of the melatonin daily profiles showed no statistically significant pattern of change trend across groups. There was a numerical, non-significant increase of the acrophase of melatonin secretion within the iRBD group (3.4 ± 4.1) compared to the HC (2.2 ± 2.1) (Table 3 ). Daily Bmal1 expression profiles A fundamental sine wave was significantly fitted to the Bmal1 gene expression profile across all groups (Fig. 2 ). There was a statistically significant pattern of decreasing median Bmal1 amplitude from controls, to iRBD, to PD and then to DLB (two sided, T JT = 393.00, z =-2.09, p = 0.037). No statistically significant trend was seen for the Bmal1 mesor or acrophase across groups. DISCUSSION This study aimed to investigate the circadian system and sleep in patients with synucleinopathy diagnosed with iRBD, PD and DLB, compared to age-matched healthy controls. In addition to subjective findings showing a predilection towards morning chronotypes, and reaffirming excessive daytime sleep, we found a significant trend of worsening objective sleep/wake cycle disruption, along the synucleinopathy spectrum with respect to actigraphy measures of rest-activity profiles and the expression of Bmal1 . Through use of a unique and inclusive approach, regarding all groups along this synucleinopathy spectrum, our work supports the hypothesis of sleep/wake disruption being a marker of neuropathological severity across the synucleinopathy spectrum and is the first to demonstrate disruption of clock gene expression in DLB. Disruption of sleep-wake cycles has been reported previously in iRBD, PD and DLB [ 44 – 46 ]. The high prevalence of clinical sleep disturbances have been hypothesised to reflect the specific pattern of neurodegeneration seen in these disorders, and thus may have diagnostic utility [ 46 ]. Of these, excessive daytime somnolence has been consistently reported in DLB at a greater severity than that seen in Alzheimer’s disease [ 47 – 49 ]. Such disturbance has been found to relate to other core symptoms including cognitive fluctuations [ 50 , 51 ]. This finding was recapitulated in the present study, which showed greatest disturbances in patients with DLB, but with increasing daytime symptoms across the groups. Interestingly, daytime sleepiness in DLB has been associated with cholinergic neuronal loss within the nucleus basalis of Meynert [ 52 ], which is implicated in a number of cognitive functions including memory, perhaps suggesting that this symptom may be closely related to pathological progression towards dementia specifically. Our actigraphy results also demonstrated evidence of sleep-wake daily rhythm disruption in the DLB group across various measures (Tables 2 and 3 ). Rest-activity mesor and amplitude was found to decrease across the groups, which we hypothesized to be related to worsening motor symptoms. This was confirmed in our post-hoc analyses, which demonstrated a significant moderate strength inverse correlation between these measures and motor parkinsonism measured by the MDS UPDRS-III. Even iRBD patients have been shown to demonstrate mild signs of motor parkinsonism, which predicts a higher risk of phenoconversion [ 53 ]. Therefore, our study suggests that actigraphy may be an objective surrogate of worsening motor symptoms and presumed dopaminergic depletion across synucleinopathies, and as such may reflect a marker of phenoconversion in iRBD, which has been suggested elsewhere [ 44 ]. Previous findings from circadian melatonin secretion studies in synucleinopathies have been limited and inconsistent. Two studies on newly diagnosed PD patients have shown opposite results in terms of finding melatonin rhythm alteration [ 16 , 54 ]. In one study evaluating PD patients with a disease duration of less than 2 years, the authors found a reduced amplitude of melatonin secretion [ 16 ]. A second study looking at a similar patient group (1.5 ± 1.2 years since diagnosis), showed no difference in melatonin secretion levels compared to healthy controls [ 54 ]. In our dataset, the duration of disease after diagnosis was relatively short for the PD (2.7 ± 1.8y) group and we could not show any significant difference in melatonin rhythms across healthy control, iRBD and PD groups. There are several reasons for this discrepancy. One may be that our study is underpowered or that there is intrinsic intra-subject variability within this measure. Alternatively, PD itself is a heterogenous disorder with different patients manifesting different disease trajectories and clinical (especially non-motor) manifestations. With greater disease duration and severity, it is generally accepted that circadian variation of melatonin secretion (amplitude) seems to be more affected [ 17 ]. There have been very few studies in iRBD with one suggesting a delay in melatonin secretion of 2 hours in their sample [ 19 ]. This would be in keeping with the numerical but non-significant trend observed in our study. Interestingly, there is little data on melatonin secretion in DLB. Our study found that the melatonin secretion in our DLB group did not show a significant oscillation, suggesting a significant disruption of the circadian clock, at least for this hormonal output (Fig. 2 ). We found a pattern of progressively reduced amplitude of daily Bmal1 expression profiles across the disease groups (Fig. 2 and Table 3 ). Bmal1 is a main driver of the circadian clock in mammals and acts as a component of the positive limb of the transcriptional-translational feedback loop, which underlies the 24 hour autonomous circadian rhythms in nearly all cells of the body ([ 55 ]. Altered Bmal1 rhythmicity (rather than amplitude) has been shown in peripheral blood mononuclear cells (PBMCs) in patients with iRBD compared to healthy controls [ 19 ]. Bmal1 has also been shown to be decreased in PBMCs taken at a single timepoint [ 56 ] and during the dark span over specific timepoints [ 21 ] in PD. Interestingly, the presence of probable RBD in PD patients was associated with reduced Bmal1 expression [ 56 ]. To our knowledge, no previous study has shown Bmal1 expression profiles in patients with DLB. However, a study assessing methylation status on circadian gene promoters including Bmal1 in patients with dementia found DLB patients had the highest frequency of circadian gene CpG island methylation compared to other dementias and age- and gender-matched controls [ 57 ]. Indeed, a variety of Bmal1 related alterations including aberrant cycles of methylation of Bmal1 and phase alterations have been shown in patients with AD [ 58 – 60 ]. However, there are few studies showing direct reductions in the amplitude of Bmal1 rhythmicity in AD, as seen here in DLB. Indeed, one recent study could not find any significant differences in oral mucosa Bmal1 or Per1 expression profiles in AD compared to controls, despite finding disruption in their activity rest cycles [ 61 ]. Based on the findings above, it is possible that such alterations may be greater in patients with DLB. Whether the changes in Bmal1 expression seen in synucleinopathies, and especially in DLB, is a result of neuropathology or directly interacts with the pathological process is unknown. Regardless of the mechanism, alteration of BMAL1 may be inextricably tied to the pathology of α-synucleinopathies, and perhaps DLB especially. Therefore, these changes may represent a biomarker of disease progression or even phenoconversion, specifically to DLB in prodromal patients with iRBD. There are a number of strengths and limitations in the present study. Overall, this is the first study to examine a holistic set of objective and subjective variables of sleep and circadian rhythms across the spectrum of patients with synucleinopathies from prodromal stages through to established PD and DLB. Patients were all comprehensively phenotyped, and the diagnosis of iRBD was confirmed with video polysomnography. For all participants, saliva and oral mucosa samples were collected under the same controlled conditions. The minimally invasive nature and ease of this technique increases the practical utility of this method in clinical and research contexts. Finally, our findings conform to an a -priori statistical model with biological validity. Limitations of this work include the cross-sectional nature of the observations, which cannot be used to derive causality. Actigraphy was only assessed for 7 days due to the limitation of available Actiwatches. Furthermore, although our study samples are comparable or greater than those that have been previously published, it is possible that the study was underpowered for some of the measurements. CONCLUSIONS This study has demonstrated altered sleep and daily rhythms across the synucleinopathy spectrum, in an order reflecting pathological severity with DLB being most affected, and iRBD being least affected. Significant trends were seen especially in changes in rest-activity rhythms reflecting likely motor parkinsonism, as well as in reduced Bmal1 expression. This latter result offers specific insights into the biology and pathobiology of sleep-wake disturbances in synucleinopathies and supports the role of circadian disruption as a potential biomarker of disease progression and phenoconversion. With a growing recognition of the bidirectional influence of sleep in neurodegeneration [ 62 , 63 ], such insights may also present opportunities for future symptomatic and disease modifying treatments. Abbreviations AD Alzheimer’s disease DLB Dementia with Lewy Bodies ESS Epworth Sleepiness Scale HC Healthy Control HADS Hospital Anxiety and Depression Scale HADS A–Hospital Anxiety and Depression Scale–Anxiety HADS D–Hospital Anxiety and Depression Scale–Depression ipRGCs intrinsically photosensitive retinal ganglion cells iRBD Isolated Rapid Eye Movement Sleep Behaviour Disorder MEQ Morning evening questionnaire MDS Movement Disorder Society MDS UPDRS–III–Movement Disorder Society Unified Parkinson’s Disease Rating Scale motor part 3 MMSE Mini–Mental State Examination MoCA Montreal Cognitive Assessment PBMCs peripheral blood mononuclear cells PD Parkinson's Disease PSG Polysomnography REM Rapid Eye Movement SCOPA S Scales for Outcomes in PD–Sleep Scale in night–time sleep (NS) and day–time sleep (DS) SD standard deviation TST total sleep time WASO wake after sleep onset Declarations Ethics approval and consent to participate The study was approved by Royal Prince Alfred Hospital Ethics Review Committee, Sydney, Australia Protocol No X15-0207 & HREC/15/RPAH/272. All participants provided written informed consent prior to commencement of the study. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding MC, XV, OR and RG declare absence of financial support. Authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. EM is supported by a National Health and Medical Research Council Emerging Leadership Fellowship (2008565) and the US department of Defense Congressionally Directed Medical Research Program Early Investigator Grant (PD220061). SL is supported by a National Health and Medical Research Council Leadership Fellowship (1195830) and has received research funding from the Michael J. Fox Foundation and the Australian Research Council, as well as consulting for Pharmaxis Ltd. Authors' contributions MC, EM data collection, MC, XV, EM, OR data analysis, MC, EM, RG, SL designed the research project; MC, OR, RG, SL and EM, contributed to writing the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. References McCann H, Stevens CH, Cartwright H, Halliday GM. α-Synucleinopathy phenotypes. Parkinsonism & Related Disorders. 2014;20:S62-S7. doi: https://doi.org/10.1016/S1353-8020(13)70017-8. Gomperts SN. Lewy Body Dementias: Dementia With Lewy Bodies and Parkinson Disease Dementia. Continuum (Minneap Minn). 2016;22(2 Dementia):435-63. doi: 10.1212/CON.0000000000000309. Jellinger KA. Dementia with Lewy bodies and Parkinson’s disease-dementia: current concepts and controversies. Journal of Neural Transmission. 2018;125(4):615-50. doi: 10.1007/s00702-017-1821-9. Malhotra RK. Neurodegenerative Disorders and Sleep. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4717144","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326853014,"identity":"22058de3-d58b-4c81-a7a5-695891825618","order_by":0,"name":"Maria Comas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBCDBAhVAcTMzA1Ea2FsYDgD0sJIihbGNgaIVnyAv7078eOPPzZ5/NKHnz/8Oa82mr8dqOVHxTacWiTOnN0szduWVizZl2bYzLvteO6Mw0Dbes7cxqnFQCJ3gzRjw+HEDWcYDJsZtx3LbQBqYWZsw6tl888ffw4n7j/D/rHx55xjufOJ0LJNgocNaAsPj2EDb0NN7gZCWoB+2WYN8ovEGZ7C2TzHDuRuBGo5iM8v/O29m2+CQ6yHfcPHHzV1ufPOHz744EcFbi3o4DCYPEC0eiCoI0XxKBgFo2AUjBAAAJHLX5IzsKoyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1368-0274","institution":"Hospital Universitario Araba la Sede Santiago: Hospital Universitario Araba","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Comas","suffix":""},{"id":326853015,"identity":"9feb082b-da98-4256-8822-e7dbee179498","order_by":1,"name":"Xavier Vidal","email":"","orcid":"","institution":"Tecnalia Research \u0026 Innovation Foundation - Campus Derio: Fundacion Tecnalia Research \u0026 Innovation - Campus Derio","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Vidal","suffix":""},{"id":326853016,"identity":"9a1357ba-c500-4408-a78e-93ae479d3961","order_by":2,"name":"Oliver Rawashdeh","email":"","orcid":"","institution":"UQ SBMS: The University of Queensland School of Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Oliver","middleName":"","lastName":"Rawashdeh","suffix":""},{"id":326853017,"identity":"b853be96-0130-44e3-b898-4fb1c4be0829","order_by":3,"name":"Ronald R. 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Solid black lines indicate harmonic regressions (fundamental wave and first harmonic). X axis represents clock time (hours).\u003c/p\u003e","description":"","filename":"Figure1Comasetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4717144/v1/c34e27c0d0e285637a057b06.jpg"},{"id":62219996,"identity":"88edab6c-d42d-47f7-b442-760238d60ba9","added_by":"auto","created_at":"2024-08-11 12:18:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":357757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaily profiles of melatonin and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBmal1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression levels for HC, iRBD, PD and DLB patients. \u003c/strong\u003eSamples were collected every three hours during the 24 hours cycle. Salivary melatonin levels are shown in pg/ml and \u003cem\u003eBmal1\u003c/em\u003e as relative abundance (%). Values are expressed as means (black circles) ± S.E.M. Solid black lines indicate harmonic regressions. X axis represents clock time (hours).\u003c/p\u003e","description":"","filename":"Figure2Comasetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4717144/v1/99fe586d215653678d60fc2d.jpg"},{"id":63348134,"identity":"098b862a-493c-4c89-8ee1-237b87264489","added_by":"auto","created_at":"2024-08-27 07:52:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1746782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4717144/v1/fd2f629f-ac6a-4a4a-beb4-42994da44fe3.pdf"},{"id":62219994,"identity":"23dcfb60-6664-4eba-bf79-036bf4bef1c1","added_by":"auto","created_at":"2024-08-11 12:18:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31991,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4717144/v1/14a74cda037ddb50cbaf87ee.docx"}],"financialInterests":"","formattedTitle":"Alterations in sleep-activity cycles and clock gene expression across the synucleinopathy spectrum","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eParkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative disorders defined at post-mortem by the presence of abnormal intraneuronal aggregates containing α-synuclein, known as Lewy bodies and Lewy neurites, alongside regional cellular loss [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Whilst similar, the pathology of DLB is typically more extensive than that seen even in the advanced stages of PD, when dementia often becomes evident (Parkinson\u0026rsquo;s disease dementia). The clinical features of PD and DLB are highly convergent and include motor parkinsonism, cognitive dysfunction, neuropsychiatric (e.g., hallucinations) and autonomic symptoms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Interestingly, patients with PD and DLB also experience a wide range of sleep disturbances including longer sleep onset latency, increased night-time sleep fragmentation, early morning awakenings and daytime sleepiness [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This suggests that sleep disturbances may be central to the pathophysiology and progression of Lewy body disorders. Furthermore, around 90% of DLB and 50% of PD patients will suffer from Rapid Eye Movement (REM) sleep behaviour disorder (RBD), which manifests as dream enactment accompanying the loss of the normal paralysis (atonia) of REM sleep [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This parasomnia frequently precedes the clinical diagnosis of PD and DLB by many years where it is referred to as \u0026lsquo;isolated\u0026rsquo; RBD (iRBD), before overt cognitive or motor symptoms occur [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Isolated RBD is the strongest clinical predictor for developing a synucleinopathy with 75% of patients converting to either PD or DLB (and a small proportion to multiple system atrophy, another synucleinopathy) over a 12-year period [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, iRBD is now widely considered a \u0026lsquo;prodromal\u0026rsquo; synucleinopathy, which can be placed early on the disease spectrum of both PD and DLB. Given the implications for prognosis and recruitment for disease modifying trials, there is a need for clinical and biological markers that highlight the risk of phenoconversion in patients with iRBD, and their trajectory towards either DLB or PD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is well recognised that the biological clock regulates the circadian oscillations of processes such as sleep, alertness, immune and metabolic functions, as well as the secretion of critical regulating hormones, such as melatonin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. At the molecular level, the mammalian circadian clock consists of a set of clock genes (e.g., \u003cem\u003eBmal1, Clock, Per1, Per2,Cry1, Cry2\u003c/em\u003e), which operate via oscillating levels in mRNA and protein abundance across most mammalian cells and tissues over the daily cycle [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The levels of melatonin increase during the evening and this hormone plays a key role in sleep regulation and the synchronisation of the circadian clock [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Melatonin levels [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and peripheral blood clock gene expression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] have previously been reported for iRBD and PD patients and most studies show dysregulation of these rhythms. To our knowledge, there are no publications showing clock gene expression or melatonin patterns in DLB patients. There is a bidirectional regulation between the circadian and the dopamine systems in terms of their gene expression [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], as well as the synthesis and release of dopamine and melatonin [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This suggests that the loss of dopamine cells in PD or the dopamine medication they take may have a deregulatory effect on the circadian clock.\u003c/p\u003e \u003cp\u003eThe aim of the current study was to investigate the function of the circadian clock in patients with early but established PD and DLB, along with an \u0026lsquo;at risk\u0026rsquo; prodromal iRBD group and an age matched healthy control (HC) group. Participants underwent standardised clinical assessments and an analysis of their daily profiles of activity/rest recorded by actigraphy, as well as having salivary and oral mucosa samples collected around the clock to measure melatonin secretion and \u003cem\u003eBmal1\u003c/em\u003e expression, respectively. It was hypothesized that disruption in daily rhythms would occur in a graded manner across the continuum, with least disruption in healthy controls, followed by further disruption in patients with iRBD, then PD and the greatest disruption in DLB, reflecting the perceived severity of pathology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003e Sixty-eight participants with a diagnosis of PD (N\u0026thinsp;=\u0026thinsp;16), DLB (N\u0026thinsp;=\u0026thinsp;17), iRBD (N\u0026thinsp;=\u0026thinsp;20) or age matched healthy controls (HC) (N\u0026thinsp;=\u0026thinsp;15) were recruited from the Parkinson\u0026rsquo;s Disease Research Clinic at the Brain and Mind Centre, University of Sydney and via community advertisements. All participants underwent a detailed neurological assessment by S.J.G.L. and E.M. at the Brain and Mind Centre, in Sydney (Australia). Video polysomnography (PSG) of all participants was performed at the Woolcock Institute of Medical Research. The presence of RBD was confirmed by clinical diagnosis using video polysomnography and met the diagnostic criteria according to the International Classification of Sleep Disorders-3 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. All PD patients had been diagnosed within 5 years of the study according to the Movement Disorders Society (MDS) PD diagnostic criteria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The DLB patients satisfied the consensus criteria of the Fourth report of the DLB Consortium and were all within 3 years of diagnosis at the time of the study [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Motor function was assessed using the motor section of the MDS Unified Parkinson\u0026rsquo;s Disease Rating Scale (MDS-UPDRS-III) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Healthy controls underwent the same procedures as patients, including neurological assessment. The study was approved by Royal Prince Alfred Hospital Ethics Review Committee, Sydney, Australia Protocol No X15-0207 \u0026amp; HREC/15/RPAH/272. All participants provided written informed consent prior to commencement of the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003eParticipants completed general health and lifestyle questionnaires, as well as neurological and sleep questionnaires detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Sleep-wake activity was objectively assessed via actigraphy and all participants stayed in the sleep laboratory at the Woolcock Institute of Medical Research for 24 hours, where they underwent overnight PSG following the SINBAR protocol [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. During this time, salivary and oral mucosa samples were collected every three hours (over the 24-hour period) to measure melatonin and \u003cem\u003eBmal1\u003c/em\u003e clock gene expression. Illuminance in the room during daytime hours (~\u0026thinsp;7am-10pm) was ~\u0026thinsp;100 lux and at lights-out \u0026lt;\u0026thinsp;1 lux.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAverage and standard deviation of demographic, clinical and sleep measures.\u003c/b\u003e Key: Data presented as mean (standard deviation). F, female. MoCA, Montreal Cognitive Assessment. MMSE, Mini-Mental State Exam. HADS-A, Hospital Anxiety and Depression Scale-Anxiety. HADS-D, Hospital Anxiety and Depression Scale-Depression. MEQ, Morningness-Eveningness Questionnaire; ESS, Epworth Sleepiness Scale. SCOPA-Sleep (NS), Scales for Outcomes in Parkinson\u0026rsquo;s Disease-Sleep (Nocturnal Sleep). SCOPA-Sleep (DS), Scales for Outcomes in Parkinson\u0026rsquo;s Disease (Sleep-Daytime Sleep). Bolded values denote significant difference in ANOVA (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cb\u003ea\u003c/b\u003e represents HC, \u003cb\u003eb\u003c/b\u003e iRBD, \u003cb\u003ec\u003c/b\u003e PD and \u003cb\u003ed\u003c/b\u003e DLB in the Tukey\u0026rsquo;s posthoc analysis).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC n\u0026thinsp;=\u0026thinsp;15\u003c/p\u003e \u003cp\u003e(6F)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eiRBD n\u0026thinsp;=\u0026thinsp;20\u003c/p\u003e \u003cp\u003e(4F)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePD n\u0026thinsp;=\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(6F)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDLB n\u0026thinsp;=\u0026thinsp;17\u003c/p\u003e \u003cp\u003e(2F)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eANOVA F\u003c/p\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.3 (9.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.6 (7.3) \u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.7 (10.5)\u003csup\u003ecd**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.3 (6.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.631\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0054\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease duration (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9 (7.2) \u003csup\u003ebc**, bd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.7 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.3 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10.45\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYears of education\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.1 (2.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6 (3.1)\u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.7 (3.4)\u003csup\u003ecd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.5 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.034\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0109\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.3 (2.35) \u003csup\u003ead***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.9 (1.6)\u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.8 (1.3)\u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.9 (6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e37.91\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMSE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.2 (0.9) \u003csup\u003ead***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.05 (1.02) \u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.3 (1.3) \u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.6 (6.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e27.12\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHADS-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.9 (2.2) \u003csup\u003ead*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6 (2.6) \u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9 (3.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.5 (4.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.899\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0129\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHADS-D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8 (2) \u003csup\u003ead***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5 (3.7) \u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4 (3) \u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.7 (5.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e11.32\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHADS-Total\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.7 (3.9) \u003csup\u003ead***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.1 (5.7) \u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.3 (5.9) \u003csup\u003ecd**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.2 (8.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e9.206\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMEQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.9 (6.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.1 (7.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.5 (9.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.8 (9.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.089\u003c/p\u003e \u003cp\u003e0.3604\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.7 (3.1) \u003csup\u003ead**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6 (4.5) \u003csup\u003ecd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.25 (3.7) \u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.8 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.046\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSCOPA-Sleep NS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.3 (2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9 (2.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.6 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5 (4.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6656\u003c/p\u003e \u003cp\u003e0.5767\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSCOPA-Sleep DS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.3 (2.6) \u003csup\u003ead**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4 (2.1) \u003csup\u003ebd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.6 (3.1) \u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.9 (4.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e12.25\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUPDRS-III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.1 (4) \u003csup\u003eac***, ad***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.8 (8.5) \u003csup\u003ebc***, bd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.2 (10.2) \u003csup\u003eac*** bc***, cd**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.7 (15.3) \u003csup\u003ead*** bd***, cd**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e32.816\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDDE (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0) \u003csup\u003eac**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0) \u003csup\u003eac**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e359.4 (262.2) \u003csup\u003eac** bc**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e238.2 (509.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e6.479\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholinergic (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.0 (4.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e32.6\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eQuestionnaires\u003c/h2\u003e \u003cp\u003eA series of questionnaires evaluating mood and sleep were completed as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Cognitive performance was evaluated with the Montreal Cognitive Assessment (MoCA) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and the Mini-Mental State Examination (MMSE) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The Hospital Anxiety and Depression Scale (HADS) was used to assess anxiety (HADS-A) and depressive symptoms (HADS-D) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The Morningness-Eveningness Questionnaire (MEQ) was used to assess chronotype [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The Epworth Sleepiness Scale (ESS) was used to derive an estimate of daytime somnolence [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The Scales for Outcomes in PD-Sleep Scale (SCOPA-S) was used to estimate both night-time sleep (NS) and day-time sleep (DS) quality [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eActigraphy\u003c/h2\u003e \u003cp\u003eParticipants wore a Philips Respironics Actiwatch 2 (Koninklijke Philips N.V., Amsterdam, Netherlands) on the non-dominant wrist for at least one week. Manual scoring of actigraphy and analysis of sleep parameters was performed using Philips Respironics Actiware-5 software (Koninklijke Philips N.V.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eSalivary melatonin\u003c/h2\u003e \u003cp\u003eMelatonin was sampled every three hours for a period of 24 hours. Sampling started at 16:00 and continued every 3 hours until 13:00 on the next day. During night-time hours (10 pm-7 am) saliva sampling occurred in dim light conditions (\u0026lt;\u0026thinsp;10 lux). Samples were stored immediately in a -80 \u003csup\u003e0\u003c/sup\u003eC freezer until analysis. Salivary melatonin concentrations were determined by radioimmunoassay (Buhlmann Laboratories; Allschwil, Switzerland). These assays have a limit of detection of 1 pg/mL with an inter-assay coefficient of variations of 7.4% at 4.41 pg/mL and 10.7% at 48.14 pg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eBmal1 gene expression\u003c/h2\u003e \u003cp\u003e Oral mucosa samples were collected at the same times as saliva samples. They were obtained by \u0026lsquo;scratching\u0026rsquo; the inner cheek for one minute with a cytological brush. The samples were immediately placed into RNAlater reagent (Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia) and stored at -20 \u003csup\u003e0\u003c/sup\u003eC until analysis. The mRNA was isolated using the Isolate II RNA Micro kit (Bioline, London, UK). SuperScript VILO cDNA Synthesis kit (Thermofisher) was used to reverse-transcribe the mRNA samples. Quantitative PCR was performed using the ABI Step one Plus detection system and data were analyzed with StepOne software (Applied Biosystems, Melbourne, VIC, Australia). Assays were carried out using Taqman Universal MMIX II with UNG and TaqMan Gene Expression assays for \u003cem\u003eBmal1\u003c/em\u003e (Hs00154147_m1) and \u003cem\u003eGapdh\u003c/em\u003e (Hs99999905_m1) according to protocol of manufacturer (Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe averaged activity levels measured by actigraphy were plotted for each patient group in 30 seconds-bins of the 24-hour interval averaged over 7 days of recording. Significance of the rhythmicity of activity levels, melatonin and \u003cem\u003eBmal1\u003c/em\u003e expression was determined with the CircWave v 1.4 software developed by R.A. Hut, University of Groningen, NL [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], which uses a linear harmonic regression fit with an assumed period of 24 hours. Cosinor analysis was performed and cosine curve parameters (amplitude, acrophase and mesor) obtained using the toolbox developed by [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] for MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Inc., Natick, Massachusetts, United States. GraphPad Prism 9.0 was used for statistical analysis. Demographic, questionnaire, and actigraphy data were compared by ANOVA. Post hoc analyses were performed using Tukey's honest significant difference post hoc test. Rhythmicity differences between groups (activity-rest rhythms, melatonin and \u003cem\u003eBmal1\u003c/em\u003e expression) were assessed using the Jonckheere-Terpstra test for ordered alternatives, in line with the \u003cem\u003ea priori\u003c/em\u003e hypothesis that circadian abnormalities would differ in accordance with expected degree of neuropathological disease - with controls being least affected, followed by iRBD, PD and then DLB (most affected). Post-hoc partial correlations were performed using SPSS (version 26.0.0, IBM) with controlling variables as specified. Results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation or median (interquartile range) as specified. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDemographics and clinical variables\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the demographic, clinical, subjective sleep and circadian measures of the participants. In keeping with the natural history of synucleinopathies there was a predominance of male subjects in all groups: PD (37.5% females), DLB (11.7% females), iRBD (20% females) and HC (40% females). Age did differ significantly between patient groups, which is expected given the established later onset of disease in DLB compared to PD. The post-hoc testing showed the DLB group to be older (74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7y) than iRBD (66.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3y); and PD (63.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5) who did not differ significantly from each other or the control group. To assist matching, HC were intentionally recruited across a wider age spectrum, as such age did not differ between HC and any of the disease groups on post-hoc testing. In keeping with the goal of recruiting of early disease, disease duration was 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 years in the PD group, and 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 years in the DLB group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSubjective Sleep and Circadian Measures\u003c/h2\u003e \u003cp\u003eThere was no difference in morningness-eveningness between groups, being all moderate morning chronotypes according to the MEQ (score 59\u0026ndash;69) except iRBD (58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7), which was the intermediate chronotype (score 42\u0026ndash;58). Excessive daytime sleepiness assessed by the ESS demonstrated lower normal daytime sleepiness (0\u0026ndash;5 score) in the iRBD group (5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5), higher normal daytime sleepiness (scores 6\u0026ndash;10) for HC (6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1) and PD (7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7) and mild excessive daytime sleepiness (scores 11\u0026ndash;12) for the DLB group (11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2), which was significantly higher than all of the other groups. Subjective night-time sleep disturbance measured by SCOPA-S (cut off for poor sleep quality\u0026thinsp;\u0026gt;\u0026thinsp;6) showed values below cut off in all groups and no significant difference. Subjective day-time sleep disturbance measured by SCOPA-S (cut off for poor sleep quality\u0026thinsp;\u0026gt;\u0026thinsp;4) showed values below the cut off for all groups except the DLB group (8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9), which reported significantly worse daytime somnolence compared to all other groups. We found a statistically significant pattern of increasing median day-time sleep disturbance measured by SCOPA-S from controls, to iRBD, PD and DLB (two sided, T\u003csub\u003eJT\u003c/sub\u003e = 923.00, \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.114, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A similar pattern of increasing daytime sleepiness measured by the ESS was seen across groups (two sided, T\u003csub\u003eJT\u003c/sub\u003e = 1122.00, \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.207, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eActigraphy derived activity and sleep parameters\u003c/h2\u003e \u003cp\u003eActivity patterns analysed by the Actiwatch software are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There was no significant difference among the groups in actigraphic bed and rise times, average time spent in bed, total sleep time (TST) and sleep time in 24 hours. Wake time after sleep onset (WASO) was significantly shorter in the iRBD (44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;23.9) and PD (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7) groups compared to DLB (73.4\u0026thinsp;\u0026plusmn;\u0026thinsp;40.4). Percentage of sleep fragmentation was lower in the HC (27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8), iRBD (30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1) and PD (19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6) groups compared to DLB (44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8). Activity counts over 24 hours were significantly higher in the HC (268,616\u0026thinsp;\u0026plusmn;\u0026thinsp;49,590) group compared to DLB (146,191\u0026thinsp;\u0026plusmn;\u0026thinsp;66,488). Sleep efficiency (%) was higher in the PD group (90\u0026thinsp;\u0026plusmn;\u0026thinsp;5) compared to DLB (81\u0026thinsp;\u0026plusmn;\u0026thinsp;10). Activity counts during the night were lower in the iRBD (9,066\u0026thinsp;\u0026plusmn;\u0026thinsp;4,385) and PD (5,276\u0026thinsp;\u0026plusmn;\u0026thinsp;1,475) compared to DLB (1,5915\u0026thinsp;\u0026plusmn;\u0026thinsp;9,385). Percentage of time awake during the night was lower in the PD (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5) compared to DLB (14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9) and percentage of wake during the day was higher in the HC (79.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8) compared to DLB (63.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAverage and standard deviation of actigraphy variables.\u003c/b\u003e Bolded values denote significant difference in the Tukey Post hoc analyses (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cb\u003ea\u003c/b\u003e represents HC, \u003cb\u003eb\u003c/b\u003e iRBD, \u003cb\u003ec\u003c/b\u003e PD and \u003cb\u003ed\u003c/b\u003e DLB).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSleep variable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eiRBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDLB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eANOVA F\u003c/p\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBedtime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22:54 (1:17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23:01 (0:41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22:33 (0:56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22:18 (0:32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.697\u003c/p\u003e \u003cp\u003e0.1818\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRisetime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7:33 (1:04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7:27 (01:12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6:50 (0:43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7:45 (0:42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.886\u003c/p\u003e \u003cp\u003e0.1462\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage Time in bed (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e519.4 (77.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e505.5 (75.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e497.1 (53.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e567.1 (47.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.772\u003c/p\u003e \u003cp\u003e0.0529\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Sleep Time (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e447.1 (80.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e440.7 (64.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e447.9 (45.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e459.3 (68.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1753\u003c/p\u003e \u003cp\u003e0.9126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWake after Sleep Onset (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.8 (16.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.7 (23.9)\u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.5 (8.7)\u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.4 (40.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.000\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFragmentation %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.9 (6.8) \u003csup\u003ead**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.9 (10.1) \u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.7 (6.6) \u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.7 (16.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10.17\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSleep time 24 hours (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e654.2 (117.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e701.3 (101.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e754.8 (140.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e781.6 (119.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.487\u003c/p\u003e \u003cp\u003e0.0732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity counts 24 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e268616 (49589.8) \u003csup\u003ead**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e219963.9 (87545.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e184697.9 (93230.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e146191.4 (66487.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.985\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0047\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSleep efficiency %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.8 (5.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.4 (9.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.3 (4.8) \u003csup\u003ecd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.9 (10.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2.474\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0743\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity counts during night\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10080.4 (2907.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9065.5 (4385) \u003csup\u003ebd*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5275.8 (1474.8) \u003csup\u003ecd***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15915 (9385.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7.3666\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%Wake during night\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.3 (3.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.5 (4.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.3 (2.5) \u003csup\u003ecd**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.2 (7.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4.46\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0082\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%Wake during day active\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.3 (7.8) \u003csup\u003ead*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.3 (14.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.4 (14.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.5 (11.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3.194\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0.0328\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRest-activity rhythms between the groups\u003c/h2\u003e \u003cp\u003eThe averaged rest-activity profiles measured by actigraphy across diagnoses are plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All four groups had significant activity rhythm profiles. A cosinor analysis was performed to derive the activity mesor, amplitude, and acrophase (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mesor (Midline Estimating Statistic of a Rhythm) is the average level around which the oscillation occurs. We estimated amplitude as the distance between the mesor and the peak of the oscillations. Acrophase is the time (clock time in our case) at which the maximal value of the oscillation occurs (Suppl Fig.\u0026nbsp;1). The test for ordered alternatives showed that there was a statistically significant pattern of decreasing median mesor of the rest-activity rhythm from controls, to iRBD, to PD and then to DLB (two sided, T\u003csub\u003eJT\u003c/sub\u003e = 225.00, \u003cem\u003ez\u003c/em\u003e= -3.52, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, a statistically significant trend was found for the amplitude of the rest-activity rhythm, being significantly higher in the HC (87.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13) group and lowest for the DLB (47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;27.3) group (two sided, T\u003csub\u003eJT\u003c/sub\u003e = 205.00, \u003cem\u003ez\u003c/em\u003e= -3.90, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No statistically significant trend was seen for the acrophase of the rest-activity rhythm across groups. In view of this result, a post-hoc partial correlation was performed assessing the relationship between rest-activity mesor and motor parkinsonism quantified by UPDRS-III score across iRBD, PD and DLB groups controlling for diagnosis and disease duration. As expected, there was a moderate negative correlation between UPDRS-III score (21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;14.8) and activity mesor (70.1\u0026thinsp;\u0026plusmn;\u0026thinsp;30.8) and activity amplitude (58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;28.4), which were statistically significant (\u003cem\u003er\u003c/em\u003e(33)=-0.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024; and \u003cem\u003er\u003c/em\u003e(33)=-0.46, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.006, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCosinor analysis of actigraphy, melatonin and Bmal1 expression. Average for all variables with standard deviation in between brackets is given for all variables. Median and interquartile range in brackets is given in square brackets. Rhythmicity differences between groups were assessed using the Jonckheere-Terpstra test for ordered alternatives. The level of statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCosinor analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eiRBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDLB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep values\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity Mesor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.9 (18.7)\u003c/p\u003e \u003cp\u003e[98.5 (15.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.1 (27.5)\u003c/p\u003e \u003cp\u003e[78.7(28.5)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.8 (34.4)\u003c/p\u003e \u003cp\u003e[52.6(52.0)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.8 (26.4)\u003c/p\u003e \u003cp\u003e[52.2(36.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity Amplitude\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.4 (13)\u003c/p\u003e \u003cp\u003e[86.8 (6.1)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.2 (27.1)\u003c/p\u003e \u003cp\u003e[64.4(23.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.3 (27.3)\u003c/p\u003e \u003cp\u003e[47.6(35.8)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.2 (27.3)\u003c/p\u003e \u003cp\u003e[40.9(23.8)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity Acrophase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.1 (1.9)\u003c/p\u003e \u003cp\u003e[13.5(2.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.5 (1.3)\u003c/p\u003e \u003cp\u003e[14.7(1.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.8 (0.9)\u003c/p\u003e \u003cp\u003e[13.9(1.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.5 (0.7)\u003c/p\u003e \u003cp\u003e[13.4(0.5)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMelatonin Mesor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.1 (1.8)\u003c/p\u003e \u003cp\u003e[2.4(2.6)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.01 (2.1)\u003c/p\u003e \u003cp\u003e[2.7(3.1)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.33 (2.4)\u003c/p\u003e \u003cp\u003e[2.8(2.6)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.01 (1.9)\u003c/p\u003e \u003cp\u003e[2.7(2.6)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMelatonin Amplitude\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2 (2.4)\u003c/p\u003e \u003cp\u003e[2.4(2.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7 (2.4)\u003c/p\u003e \u003cp\u003e[2.6(3.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5(2.3)\u003c/p\u003e \u003cp\u003e[1.2(3.3)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.0 (3.0)\u003c/p\u003e \u003cp\u003e[1.8(2.5)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMelatonin Acrophase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.2 (2.1)\u003c/p\u003e \u003cp\u003e[1.8(2.9)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4 (4.1)\u003c/p\u003e \u003cp\u003e[2.9(3.8)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5 (2.06) [2.7(2.3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.4 (5.1)\u003c/p\u003e \u003cp\u003e[5.9(9.7)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmal1\u003c/em\u003e Mesor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.4 (7.8)\u003c/p\u003e \u003cp\u003e[39.2(11.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.1 (15.5)\u003c/p\u003e \u003cp\u003e[38.2(24.9)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.8 (15.5)\u003c/p\u003e \u003cp\u003e[41.9 (19.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.7(13.9)\u003c/p\u003e \u003cp\u003e[44.6(22.4)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmal1\u003c/em\u003e Amplitude\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.1 (6.2)\u003c/p\u003e \u003cp\u003e[27.9(7.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.1 (11.4)\u003c/p\u003e \u003cp\u003e[22.9(14.8)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.1 (9.4)\u003c/p\u003e \u003cp\u003e[21.1 (11.1)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.4 (4.5)\u003c/p\u003e \u003cp\u003e[19.9(5.1)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmal1\u003c/em\u003e Acrophase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.9 (1.8)\u003c/p\u003e \u003cp\u003e[2.4(2.7)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2(3.6)\u003c/p\u003e \u003cp\u003e[1.5(3.09)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3 (4.5)\u003c/p\u003e \u003cp\u003e[0.8(4.9)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.1 (4.7)\u003c/p\u003e \u003cp\u003e[23.88(3.2)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDaily melatonin secretion\u003c/h2\u003e \u003cp\u003eAcross each group, except DLB, one fundamental sine wave contributed significantly to the explained variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No harmonics could be fitted for the melatonin data obtained from the DLB group, meaning that there was no circadian variation of melatonin in these patients. Analysis of the cosinor analysis outputs (mesor, amplitude and acrophase) of the melatonin daily profiles showed no statistically significant pattern of change trend across groups. There was a numerical, non-significant increase of the acrophase of melatonin secretion within the iRBD group (3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1) compared to the HC (2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDaily\u003c/b\u003e \u003cb\u003eBmal1\u003c/b\u003e \u003cb\u003eexpression profiles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA fundamental sine wave was significantly fitted to the \u003cem\u003eBmal1\u003c/em\u003e gene expression profile across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There was a statistically significant pattern of decreasing median \u003cem\u003eBmal1\u003c/em\u003e amplitude from controls, to iRBD, to PD and then to DLB (two sided, T\u003csub\u003eJT\u003c/sub\u003e = 393.00, \u003cem\u003ez\u003c/em\u003e=-2.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037). No statistically significant trend was seen for the \u003cem\u003eBmal1\u003c/em\u003e mesor or acrophase across groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to investigate the circadian system and sleep in patients with synucleinopathy diagnosed with iRBD, PD and DLB, compared to age-matched healthy controls. In addition to subjective findings showing a predilection towards morning chronotypes, and reaffirming excessive daytime sleep, we found a significant trend of worsening objective sleep/wake cycle disruption, along the synucleinopathy spectrum with respect to actigraphy measures of rest-activity profiles and the expression of \u003cem\u003eBmal1\u003c/em\u003e. Through use of a unique and inclusive approach, regarding all groups along this synucleinopathy spectrum, our work supports the hypothesis of sleep/wake disruption being a marker of neuropathological severity across the synucleinopathy spectrum and is the first to demonstrate disruption of clock gene expression in DLB.\u003c/p\u003e \u003cp\u003eDisruption of sleep-wake cycles has been reported previously in iRBD, PD and DLB [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The high prevalence of clinical sleep disturbances have been hypothesised to reflect the specific pattern of neurodegeneration seen in these disorders, and thus may have diagnostic utility [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Of these, excessive daytime somnolence has been consistently reported in DLB at a greater severity than that seen in Alzheimer\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Such disturbance has been found to relate to other core symptoms including cognitive fluctuations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This finding was recapitulated in the present study, which showed greatest disturbances in patients with DLB, but with increasing daytime symptoms across the groups. Interestingly, daytime sleepiness in DLB has been associated with cholinergic neuronal loss within the nucleus basalis of Meynert [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], which is implicated in a number of cognitive functions including memory, perhaps suggesting that this symptom may be closely related to pathological progression towards dementia specifically.\u003c/p\u003e \u003cp\u003eOur actigraphy results also demonstrated evidence of sleep-wake daily rhythm disruption in the DLB group across various measures (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Rest-activity mesor and amplitude was found to decrease across the groups, which we hypothesized to be related to worsening motor symptoms. This was confirmed in our post-hoc analyses, which demonstrated a significant moderate strength inverse correlation between these measures and motor parkinsonism measured by the MDS UPDRS-III. Even iRBD patients have been shown to demonstrate mild signs of motor parkinsonism, which predicts a higher risk of phenoconversion [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, our study suggests that actigraphy may be an objective surrogate of worsening motor symptoms and presumed dopaminergic depletion across synucleinopathies, and as such may reflect a marker of phenoconversion in iRBD, which has been suggested elsewhere [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious findings from circadian melatonin secretion studies in synucleinopathies have been limited and inconsistent. Two studies on newly diagnosed PD patients have shown opposite results in terms of finding melatonin rhythm alteration [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In one study evaluating PD patients with a disease duration of less than 2 years, the authors found a reduced amplitude of melatonin secretion [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A second study looking at a similar patient group (1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 years since diagnosis), showed no difference in melatonin secretion levels compared to healthy controls [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In our dataset, the duration of disease after diagnosis was relatively short for the PD (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8y) group and we could not show any significant difference in melatonin rhythms across healthy control, iRBD and PD groups. There are several reasons for this discrepancy. One may be that our study is underpowered or that there is intrinsic intra-subject variability within this measure. Alternatively, PD itself is a heterogenous disorder with different patients manifesting different disease trajectories and clinical (especially non-motor) manifestations. With greater disease duration and severity, it is generally accepted that circadian variation of melatonin secretion (amplitude) seems to be more affected [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There have been very few studies in iRBD with one suggesting a delay in melatonin secretion of 2 hours in their sample [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This would be in keeping with the numerical but non-significant trend observed in our study. Interestingly, there is little data on melatonin secretion in DLB. Our study found that the melatonin secretion in our DLB group did not show a significant oscillation, suggesting a significant disruption of the circadian clock, at least for this hormonal output (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe found a pattern of progressively reduced amplitude of daily \u003cem\u003eBmal1\u003c/em\u003e expression profiles across the disease groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eBmal1\u003c/em\u003e is a main driver of the circadian clock in mammals and acts as a component of the positive limb of the transcriptional-translational feedback loop, which underlies the 24 hour autonomous circadian rhythms in nearly all cells of the body ([\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Altered \u003cem\u003eBmal1\u003c/em\u003e rhythmicity (rather than amplitude) has been shown in peripheral blood mononuclear cells (PBMCs) in patients with iRBD compared to healthy controls [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eBmal1\u003c/em\u003e has also been shown to be decreased in PBMCs taken at a single timepoint [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and during the dark span over specific timepoints [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] in PD. Interestingly, the presence of probable RBD in PD patients was associated with reduced \u003cem\u003eBmal1\u003c/em\u003e expression [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. To our knowledge, no previous study has shown \u003cem\u003eBmal1\u003c/em\u003e expression profiles in patients with DLB. However, a study assessing methylation status on circadian gene promoters including \u003cem\u003eBmal1\u003c/em\u003e in patients with dementia found DLB patients had the highest frequency of circadian gene CpG island methylation compared to other dementias and age- and gender-matched controls [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Indeed, a variety of \u003cem\u003eBmal1\u003c/em\u003e related alterations including aberrant cycles of methylation of \u003cem\u003eBmal1\u003c/em\u003e and phase alterations have been shown in patients with AD [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, there are few studies showing direct reductions in the amplitude of \u003cem\u003eBmal1\u003c/em\u003e rhythmicity in AD, as seen here in DLB. Indeed, one recent study could not find any significant differences in oral mucosa \u003cem\u003eBmal1\u003c/em\u003e or \u003cem\u003ePer1\u003c/em\u003e expression profiles in AD compared to controls, despite finding disruption in their activity rest cycles [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Based on the findings above, it is possible that such alterations may be greater in patients with DLB. Whether the changes in \u003cem\u003eBmal1\u003c/em\u003e expression seen in synucleinopathies, and especially in DLB, is a result of neuropathology or directly interacts with the pathological process is unknown. Regardless of the mechanism, alteration of BMAL1 may be inextricably tied to the pathology of α-synucleinopathies, and perhaps DLB especially. Therefore, these changes may represent a biomarker of disease progression or even phenoconversion, specifically to DLB in prodromal patients with iRBD.\u003c/p\u003e \u003cp\u003eThere are a number of strengths and limitations in the present study. Overall, this is the first study to examine a holistic set of objective and subjective variables of sleep and circadian rhythms across the spectrum of patients with synucleinopathies from prodromal stages through to established PD and DLB. Patients were all comprehensively phenotyped, and the diagnosis of iRBD was confirmed with video polysomnography. For all participants, saliva and oral mucosa samples were collected under the same controlled conditions. The minimally invasive nature and ease of this technique increases the practical utility of this method in clinical and research contexts. Finally, our findings conform to an \u003cem\u003ea\u003c/em\u003e-priori statistical model with biological validity. Limitations of this work include the cross-sectional nature of the observations, which cannot be used to derive causality. Actigraphy was only assessed for 7 days due to the limitation of available Actiwatches. Furthermore, although our study samples are comparable or greater than those that have been previously published, it is possible that the study was underpowered for some of the measurements.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study has demonstrated altered sleep and daily rhythms across the synucleinopathy spectrum, in an order reflecting pathological severity with DLB being most affected, and iRBD being least affected. Significant trends were seen especially in changes in rest-activity rhythms reflecting likely motor parkinsonism, as well as in reduced \u003cem\u003eBmal1\u003c/em\u003e expression. This latter result offers specific insights into the biology and pathobiology of sleep-wake disturbances in synucleinopathies and supports the role of circadian disruption as a potential biomarker of disease progression and phenoconversion. With a growing recognition of the bidirectional influence of sleep in neurodegeneration [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], such insights may also present opportunities for future symptomatic and disease modifying treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlzheimer\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDementia with Lewy Bodies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpworth Sleepiness Scale\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHealthy Control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHADS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHospital Anxiety and Depression Scale\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHADS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eA\u0026ndash;Hospital Anxiety and Depression Scale\u0026ndash;Anxiety\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHADS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eD\u0026ndash;Hospital Anxiety and Depression Scale\u0026ndash;Depression\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eipRGCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintrinsically photosensitive retinal ganglion cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiRBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIsolated Rapid Eye Movement Sleep Behaviour Disorder\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMorning evening questionnaire\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMovement Disorder Society\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUPDRS\u0026ndash;III\u0026ndash;Movement Disorder Society Unified Parkinson\u0026rsquo;s Disease Rating Scale motor part 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMSE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMini\u0026ndash;Mental State Examination\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMoCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMontreal Cognitive Assessment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBMCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperipheral blood mononuclear cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParkinson's Disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolysomnography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eREM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRapid Eye Movement\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCOPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eS Scales for Outcomes in PD\u0026ndash;Sleep Scale in night\u0026ndash;time sleep (NS) and day\u0026ndash;time sleep (DS)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal sleep time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWASO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewake after sleep onset\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by Royal Prince Alfred Hospital Ethics Review Committee, Sydney, Australia Protocol No X15-0207 \u0026amp; HREC/15/RPAH/272. All participants provided written informed consent prior to commencement of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC, XV, OR and RG declare absence of financial support. Authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. EM is supported by\u0026nbsp;a National Health and Medical Research Council Emerging Leadership Fellowship (2008565) and the US department of Defense Congressionally Directed Medical Research Program Early Investigator Grant (PD220061).\u0026nbsp;SL\u0026nbsp;is supported by a National Health and Medical Research Council Leadership Fellowship (1195830) and has received research funding from\u0026nbsp;the Michael J. Fox Foundation and the Australian Research Council, as well as consulting for Pharmaxis Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC, EM data collection, MC, XV, EM, OR data analysis, MC, EM, RG, SL designed the research project; MC, OR, RG, SL and EM, contributed to writing the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcCann H, Stevens CH, Cartwright H, Halliday GM. \u0026alpha;-Synucleinopathy phenotypes. Parkinsonism \u0026amp; Related Disorders. 2014;20:S62-S7. doi: https://doi.org/10.1016/S1353-8020(13)70017-8.\u003c/li\u003e\n\u003cli\u003eGomperts SN. Lewy Body Dementias: Dementia With Lewy Bodies and Parkinson Disease Dementia. Continuum (Minneap Minn). 2016;22(2 Dementia):435-63. doi: 10.1212/CON.0000000000000309.\u003c/li\u003e\n\u003cli\u003eJellinger KA. Dementia with Lewy bodies and Parkinson\u0026rsquo;s disease-dementia: current concepts and controversies. Journal of Neural Transmission. 2018;125(4):615-50. doi: 10.1007/s00702-017-1821-9.\u003c/li\u003e\n\u003cli\u003eMalhotra RK. Neurodegenerative Disorders and Sleep. 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J Parkinsons Dis. 2019;9(3):603-14. doi: 10.3233/jpd-191627.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dementia with Lewy bodies, Parkinson’s disease, rapid eye movement sleep behaviour disorder, circadian, melatonin, Bmal1, actigraphy, sleep","lastPublishedDoi":"10.21203/rs.3.rs-4717144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4717144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNeurodegenerative synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are strongly associated with sleep disturbances. Furthermore, isolated Rapid Eye Movement Sleep Behaviour Disorder (iRBD) is now established as the strongest risk factor for developing PD or DLB, often preceding the clinical diagnosis by several years.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eWe aimed to investigate sleep/wake cycles and circadian rhythms in patients with early PD and DLB, along with \u0026lsquo;at risk\u0026rsquo; prodromal subjects diagnosed with iRBD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFifteen healthy controls, 20 iRBD, 16 PD and 17 DLB patients within 5 years of diagnosis, underwent assessment. Sleep/wake cycles were evaluated using questionnaires and actigraphy. Salivary and oral mucosa samples were collected every 3 hours to measure melatonin levels and \u003cem\u003eBmal1\u003c/em\u003e clock gene expression over 24-hours.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth subjective and objective measures of sleep/wake cycles demonstrated that the DLB group exhibited the most significant sleep/wake cycle disruption. In the DLB group, no fundamental sine wave could be fitted to the level of melatonin secretion, indicating a severe disruption in the daily rhythm of this hormone. There was a statistically significant pattern of decreasing median \u003cem\u003eBmal1\u003c/em\u003e amplitude from HC, to iRBD, to PD and then to DLB (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis work highlights a differential gradient of objective disruption in the daily circadian rhythms from iRBD to established PD and DLB and is the first to directly demonstrate disruption of clock gene expression and melatonin in DLB. The findings support sleep/wake disruption as a marker of neuropathological severity and potentially a novel therapeutic target across the synucleinopathy spectrum.\u003c/p\u003e","manuscriptTitle":"Alterations in sleep-activity cycles and clock gene expression across the synucleinopathy spectrum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-11 12:18:45","doi":"10.21203/rs.3.rs-4717144/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05ab7a61-cc77-4929-a3ab-8c99b90944fd","owner":[],"postedDate":"August 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-27T07:52:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-11 12:18:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4717144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4717144","identity":"rs-4717144","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}