Sleep Medicine 15 (2014) 116–120
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Sleep Medicine journal homepage: www.elsevier.com/locate/sleep
Original Article
Memory consolidation and inducible nitric oxide synthase expression during different sleep stages in Parkinson disease Dean Wu a,b,c,1, Ing-Jy Tseng d,1, Rey-Yue Yuan e, Chia-Yu Hsieh a,b,e,f, Chaur-Jong Hu a,b,e,f,⇑ a
Department of Neurology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan Sleep Center, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan c Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan d College of Nursing, Taipei Medical University, Taipei, Taiwan e Department of Neurology, Taipei Medical University Hospital, Taipei, Taiwan f Sleep Center, Taipei Medical University Hospital, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 4 September 2013 Accepted 25 September 2013 Available online 18 October 2013 Keywords: Parkinson disease Memory consolidation iNOS NO REM sleep Polysomnography
a b s t r a c t Background: Parkinson disease (PD) is a neurodegenerative disease characterized by motor and nonmotor dysfunctions, which include sleep disturbances. Rapid eye movement (REM) sleep is associated with numerous physiologic changes such as memory consolidation. Compelling evidence suggests that nitric oxide (NO) is crucial to both sleep regulation and memory consolidation. In our study, we explored changes in biologic molecules during various sleep stages and the effects of sleep on memory consolidation in PD. Methods: Ten PD patients and 14 volunteers without PD participated in our study. The gene expression of inducible NO synthase (iNOS) in all sleep stages was measured using realtime polymerase chain reaction (PCR) based on polysomnography (PSG)-guided peripheral blood sampling. In addition, the efficiency of memory consolidation during the sleep of the participants was measured using the Wechsler Memory Scale, third edition (WMS-III). Results: The iNOS expression increased in all sleep stages among the PD patients compared to the control participants, in whom iNOS expression decreased during REM sleep. Regarding memory consolidation, the performance of the controls in logic memory and the patients in visual reproduction tasks improved after sleep. Conclusions: The iNOS synthase expression was different from control participants among PD patients, and the expression was dissimilar in various sleep stages. Sleep might enhance memory consolidation and there are different memory consolidation profiles between PD and control participants demonstrating distinct memory consolidation profiles. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Parkinson disease (PD) is a neurodegenerative disease characterized by an insidious onset and slow progression of rigidity, tremors, bradykinesia, and balance impairment. In addition to motor dysfunction, PD patients may exhibit nonmotor symptoms, such as sensory symptoms, autonomic dysfunction, and sleep disturbances. Several sleep disturbances are particularly common in PD patients, including insomnia, nightmares, sleep apnea, rapid eye movement (REM) sleep behavior disorder, restless legs
⇑ Corresponding author at: Department of Neurology and Sleep Center, Shuang Ho Hospital, Taipei Medical University, 291 Jhongjheng Road, Jhonghe District, New Taipei City, Taiwan. Tel.: +886 2 22490088x8112; fax: +886 2 22490088x2507. E-mail address: [email protected] (C.-J. Hu). 1 These authors contributed equally to this work. 1389-9457/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sleep.2013.09.016
syndrome, periodic limb movement disorder, and excessive daytime sleepiness [1]. All of these symptoms can occur prior to the onset of PD motor symptoms and typically deteriorate during the disease process. The neurotransmitter dopamine plays a role in producing a wakefulness-promoting effect in vertebrates. Dopaminergic deficiency might be attributed to these sleep disturbances. Other systems, such as the serotoninergic, cholingergic, and norepinephrinergic systems, also play roles in sleep, but few studies have focused on PD sleep disorders [2]. All of these neurotransmitters, which play key roles in neurodegenerative diseases, are involved in the initiation and maintenance of sleep. Sleep affects behaviors by altering endocrine, neurotransmitters, and intracellular molecular events [3]. REM sleep, in which PD patients commonly exhibit abnormalities, accompanies numerous physiologic changes, such as a decrease in muscle tone, an increase in brain activities, and irregular cardiopulmonary functioning. These REM-related changes also are associated with
D. Wu et al. / Sleep Medicine 15 (2014) 116–120
dramatic changes in the microenvironment of the brain, including gene-expression patterns and activities of neurotransmitters [4]. In addition, sleep is essential for maintaining neurocognitive functions including memory consolidation [4–6]. Distinct memory is linked to unique sleep-related consolidation mechanisms that work during various sleep stages in REM sleep or slow-wave sleep (SWS) and in different brain regions throughout the night [7]. Increasing evidence suggests that nitric oxide (NO), originally identified as endothelium-derived relaxing factor also is crucial for both sleep regulation and memory consolidation [7]. Four forms of NO synthase are responsive to the generation of NO, which are neuronal (nNOS), endothelial, mitochondrial, and inducible (iNOS). Three processes regulate NO synthesis and NOSs activities, specifically, the L-arginine/arginase substrate-competing system; the citrulline/L-arginine-recycling system; and the asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase NOSs-inhibiting system. Researchers have documented that nitrites and nitrates may back-generate NO under ischemic conditions. This process must be considered regarding patients with neurodegenerative diseases such as PD [8]. A previous study reported that the presence of NO in the brain facilitates sleep, particularly REM sleep, whereas the presence of NO in the periphery may inhibit sleep [8]. In an nNOS/iNOS knockout (KO) young animal study, nNOS and iNOS demonstrated opposite effects on sleep; nNOS KO mice experienced a decrease in REM sleep, and iNOS KO mice exhibited an increase in REM sleep [9]. In contrast to the young animal study, another study indicated that iNOS may be essential to both the initiation and maintenance of REM in aging animals [10]. These studies have suggested that the role of the NO system in sleep regulation is complex. L-NG-nitroarginine methyl ester, an NOS inhibitor, was revealed to induce impairments in immediate, short-, and long-term memories of inhibitory avoidance tasks in animals. The iNOS expression was discovered to substantially increase in brains affected by PD, and this change in the NO system might affect sleep architecture and contribute to the clinically observed sleep and behavioral disturbances in PD patients. We designed our study to explore changes in biologic molecule, iNOS, during various sleep stages in PD patients. In addition, we measured the efficiency of memory consolidation during sleep in PD patients. The central molecules were indirectly examined by sampling peripheral leukocytes, based on the concept of some data supporting the synchronized expression of circadian genes between the central circadian clock and peripheral organs, including the leukocytes [11,12]. Thus we investigated the dissimilarities in iNOS gene expression between PD patients and non-PD control participants in REM sleep, SWS sleep, and wake stages by using polysomnography (PSG)-guided peripheral blood sampling.
2. Methods 2.1. Participants Ten PD patients and 14 non-PD volunteers aged 46–68 years were recruited from Taipei Medical University Hospital (TMUH) and Taipei Medical University Shuang-Ho Hospital. The PD diagnosis was based on the UK Parkinson’s Disease Society Brain Bank criteria. The severity of PD in patients was evaluated using the Hoehn and Yahr Scale. Among the participants, nine patients were in Stage 1 of PD and one patient was in Stage 2. All of the participants provided written informed consent, which was approved by the Joint-Institutional Review Board of Taipei Medical University.
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Participants who possessed a history of sleep disturbance or were regularly taking hypnotic drugs were excluded from our study. 2.2. Study design and memory tests All of the participants arrived at the sleep center of TMUH or Taipei Medical University Shuang-Ho Hospital at approximately 9:00 pm and were asked to provide basic information. They performed the first recall tasks by using the Wechsler Memory Scale, third edition (WMS-III) until 10:00 pm and then underwent PSG after an intravascular catheter was inserted into the cubital vein for further blood sampling. The participants fell asleep in individual rooms at approximately 11:00 pm. They were awoken by light exposure at approximately 6:00 am. Shortly afterward they performed the second recall tasks, which were identical to those performed the previous night (Fig. 1). To evaluate the relationship between sleep architecture and gene expression with memory consolidation in PD patients, the episodic memory tasks, which were designed and defined according to the WMS-III, were included in our study. These tasks comprise a logical memory task, a verbal-paired association task, and a visual reproduction task. All of the tests were conducted by a single member of the medical personnel according to manufacturer instructions. After the participants learned all four of the tasks included in the WMS-III, they were required to perform the first recall prior to sleeping. After approximately 8 h of sleep, they were asked to recall all of the tasks, which were learned the previous day, without being provided any information regarding these tasks; this process was defined as the second recall tasks. The results of recall performance and those of memory consolidation are represented by the percentage of memory retention. 2.3. PSG and blood sampling A PSG examination was conducted on all of the participants by using a Sandman Elite (Tyco Healthcare, Canada) at the sleep center of TMUH, or by using an Embla N7000 (ResMed, Australia) at Shuang-Ho Hospital. Prior to the scheduled sleep at 11:00 pm, venous catheters were inserted into the forearm veins of the participants. During the PSG examination, three samples that each contained 3 cc of peripheral venous blood were collected at three end points: (1) the first REM sleep, (2) non-REM SWS following the REM sleep, and (3) 15 min after the end of the PSG examination when the participants were awoken at 7:00 am by light exposure. The scoring of the PSG examination was performed by qualified PSG technicians based on the Rechtschaffen and Kales rules. Table 1 lists the PSG parameters. 2.4. RNA extraction and realtime polymerase chain reaction After collecting peripheral blood, the blood was immediately transferred to RNAlaterÒ RNA Stabilization Reagent kits (Qiagen; Valencia, CA, USA) to prevent RNA degradation. The total RNA was harvested using an RNA Extraction RiboPure-Blood kit (Ambi-
Fig. 1. Study design. Participants underwent memory tests before and after polysomnography (PSG), and blood samples were collected at three end points based on the PSG findings.
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D. Wu et al. / Sleep Medicine 15 (2014) 116–120 Table 1 Demographic data and polysomnography parameters of patients and control participants. Variable
Control group (n = 14)
Parkinson disease group (n = 10)
P value
Age (y) Gender Men Women BMI TST Sleep efficiency Sleep onset Stage 1 and stage 2 SWS%/TST REM%/TST PLMI/h REM sleep AHI/h Arousal index/h Education (y)
55.500 ± 4.256
59.500 ± 7.576
.113 .8
8 6 25.841 ± 3.311 256.264 ± 51.289 73.243 ± 13.243 26.893 ± 31.519 76.100 ± 13.602 14.893 ± 10.081 9.000 ± 5.956 5.150 ± 10.924 2.714 ± 1.637 10.257 ± 12.277 15.307 ± 10.016 15.143 ± 2.316
4 6 23.304 ± 3.067 295.589 ± 43.904 73.160 ± 9.926 20.972 ± 13.850 83.000 ± 7.963 2.070 ± 4.528 14.380 ± 6.563 15.980 ± 23.964 3.000 ± 0.943 9.470 ± 11.802 19.550 ± 13.015 11.800 ± 3.882
.070 .063 .987 .585 .166 .001 .048 .149 .626 .876 .376 .292
Abbreviations: y, years; BMI, body mass index; TST, total sleep time; SWS, slow-wave sleep; REM, rapid eye movement; PLMI, periodic limb movement index; h, hour; AHI, apnea–hypopnea index.
on; Applied Biosystems; Foster City, CA, USA). The total RNA of single 2-lg samples was included in the cDNA synthesis by using an ImProm-II reverse transcription kit (Promega, MI, USA) according to manufacturer instructions, and the cDNA was stored at 80 °C until use. For determining iNOS gene expression, realtime polymerase chain reaction (PCR) was performed using an ABI-7300 (Applied Biosystems); 1 lL of cDNA was administered in duplicate on eight strip tubes by using an Applied Biosystems Taqman assay (ABI assay ID: Hs00167248_m1), and by using a TaqManÒ Universal PCR Master Mix in a final volume of 20 lL. The threshold cycle changes (DCt) denote the dissimilarity between the Ct of iNOS and the Ct level of the internal control, actin, which is a housekeeping gene (ABI assay ID: Hs99999903_m1). 2.5. Statistical analysis The clinical and PSG variables in these clinical groups were calculated using the Student t test except for sex differences, which were measured using a v2 test. The average DCt of iNOS at REM sleep, SWS, and wake stages among the two groups was calculated. The dissimilarities in DCt between the PD and control groups also were tested using the Student t test. When testing the expression of iNOS, repeated measures were conducted to confirm the differences in paired DCt in REM sleep, SWS sleep, and wake stages among the same individuals. Regarding all analyses, statistical significance was assumed when the P value was less than .05. 3. Results The REM sleep percentage increased and the SWS percentage decreased in the PD group. There was no significant difference between PD and control participants, but there was a trend toward an increased number of REMs and an increased periodic limb movement index in the PD group, as shown in Table 1. Fig. 2 shows the levels of iNOS mRNA expression. Compared with the control group, the PD patients contained significantly higher levels of iNOS expression in REM sleep, SWS, and wake stages. The DCt of iNOS in the PD and control groups were 1.480 ± 0.446 vs 1.67 ± 0.026 (P < .001) during REM sleep, 0.893 ± 0.217 vs 1.681 ± 0.035 (P < .001) during SWS sleep, and 0.618 ± 0.121 vs 1.698 ± 0.031 (P < .001) during the wake stage. Among the PD patients, the DCt of iNOS significantly increased during REM sleep (1.480 ± 0.446 vs 0.893 ± 0.217 and 0.618 ± 0.121; P < .05). These results indicate that the iNOS mRNA expression decreased during REM sleep among the PD patients.
Fig. 2. inducible nitric oxide synthase (iNOS) mRNA levels represented by DCt in the rapid eye movement (REM) sleep, slow-wave sleep (SWS), and wake stages among control and Parkinson disease (PD) patients. iNOS expression is significantly upregulated (lower DCt) in PD patients at the REM sleep, SWS, and wake stages. The iNOS expression during REM sleep is significantly decreased (higher DCt) among PD patients. Abbreviation: NS, nonsignificant. ⁄⁄P < .001; ⁄P < .05.
When using the formula of 2 DDCt, in which DDCt is the difference in mean DCt between the PD group and the control group, the mean mRNA level of the PD group during the REM sleep, SWS, and wake stages were shown to be significantly higher than those of the control group [13,14]. Regarding memory consolidation, the control participants improved in performing logic memory tasks after sleeping and PD patients improved in performing visual reproduction tasks after sleeping (Fig. 3). These results indicate that sleep plays a role in memory consolidation. There are dissimilarities regarding memory consolidation profiles between the PD and non-PD control participants.
4. Discussion In our study, we investigated the expression of the REM sleep and memory-related gene in PD patients in iNOS during various
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Fig. 3. Memory consolidation of sleep measured by the Wechsler Memory Scale, third edition. Logic memory improved in control participants without Parkinson disease (PD), and visual reproduction improved in PD patients. Abbreviations: C, control; LM, logical memory; VPA, verbal paired association; VR, visual reproduction. ⁄P < .05.
sleep stages by using PSG recording. The mRNA of iNOS was significantly increased in PD patients during all of the sleep and wake stages. A previous study demonstrated that oxidative stress played a vital role in the pathophysiology of PD. High levels of neuronal and iNOS were discovered in the substantia nigra of patients and animal models with PD [15]. Another study revealed that iNOS overexpression usually indicated an ongoing inflammation, which is thought to play a role in the development of PD [16]. Neuronal NOS was overexpressed in peripheral blood cells among PD patients, though the blood concentration of NO decreased in PD patients. Previously, there was a lack of information regarding iNOS expression in the peripheral blood of PD patients. Our findings might be compatible with previous studies and support the hypothesis that PD patients demonstrate an increase in oxidative stress, such as iNOS overexpression. However, additional studies of oxidative stress including the measurement of NO are required. In our study, the iNOS mRNA expression significantly decreased during REM sleep compared with the SWS and wake stages among PD patients. Conversely, iNOS expression was unchanged, but it was lower in the non-PD control participants. The role of NO or iNOS in sleep is controversial. NO was revealed both to suppress and to facilitate REM sleep according to various studies. In adult animals, iNOS protein is not expressed, or it only is expressed at extremely low levels. However, there are three principal situations in which iNOS is expressed in neurons and glial cells: sleep rebound associated with sleep deprivation, inflammation, or during aging [17,18]. The increase of iNOS with aging was considered a mechanism for compensating for the neural impairments that develop after many years of adult life [8]. Upregulated iNOS is necessary for producing effective immune defenses. However, the persistent activation of iNOS may lead to the overproduction of NO. In studies on nNOS or iNOS genes that have used transgenic mice, the prominence of NO in REM sleep regulation increased. In iNOS KO animals, REM sleep was increased compared with that of the control mice [9]. In our study, REM sleep significantly increased in the PD patients, suggesting that the downregulation of the iNOS expression might be associated with the increase in REM sleep in PD patients. However, REM sleep increased in PD patients compared with the control group, but the iNOS was still highly expressed. Therefore, the notion that iNOS activation is associated with a decrease in REM sleep can be challenged. Although the relationship between peripheral tissues and the central nervous system regarding iNOS expression is still not clearly defined, we speculate that the suppression of iNOS can serve as a mean for maintaining REM sleep in PD patients.
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Although memory formation is not the only function of sleep, it might be the most crucial function as it assists in establishing the state of consciousness during wakefulness. This idea was formed more than a hundred years ago, dating back to 1885 with the beginning of experimental memory research by Ebbinghaus [19]. Consciousness and the formations of memory possibly are two mutually exclusive processes that cannot simultaneously occur in the neuronal networks of the brain. Without memory, our consciousness might fall apart into pieces of moments. Based on the standard two-stage memory model [20,21], sleep was proposed to be an offline process involving the ‘‘active system consolidation’’ of memory (i.e., the process in which newly encoded memory representations are redistributed to other neuron networks serving as long-term storage) [22]. The occurrence of memory consolidation during sleep has been comprehensively studied [23]. A considerable amount of evidence shows that SWS preferentially supports declarative memory consolidation, whereas REM sleep benefits specifically nondeclarative and emotional aspects of memory. However, the classical classification of sleep stages is crude and cannot adequately reflect on the complexity of sleep-associated processes that contribute to memory consolidation. The specific sleep stages that are responsible for specific memory components still require detailed analysis and investigation. Furthermore, the relationship between NO and memory processing has been previously studied [24–26]. Long-term potentiation and long-term depression (LTD), the predominant mechanisms of learning and memory processes, were believed to be initiated by the NO/cyclic GMP pathway. NO acts as a retrograde messenger of long-term potentiation and LTD in the hippocampus [27]. The expression of LTD-like synaptic plasticity in the hippocampus is related to the formation of several types of motor learning and visual recognition memory [28]. In our study, we observed sleep-enhanced declarative memory consolidation occurring in both the control and PD groups. In addition, logic memory improved in the control participants and visual reproduction improved in the PD patients. Based on the iNOS expression and PSG results, the PD patients contained a higher iNOS expression and demonstrated more REM sleep and less SWS than that of the control participants. Therefore, visual reproduction memory consolidation might be dependent on NO. The other possibility is that logic memory consolidation is dependent on SWS, but visual reproduction memory consolidation is dependent on REM sleep. We also explored the relationship between iNOS expression and various memory modalities, and we subsequently discovered no linear relationship between these two variables (data not shown). Our findings require further confirmation from future studies and research on the mechanisms. Another potential factor that might affect the memory of PD patients is dopamine. Recent studies have indicated that dopamine release implies offline memory reactivation and consolidation [29,30], general working memory capacity [31], and the ability to train working memory [32–34]. A recent study reported that PD patients on dopaminergic medication demonstrated an improved backward digit span performance following a nocturnal sleep interval, compared with PD patients who were not taking dopaminergic medication [35]. In our study, all of the PD patients and none of the control participants were taking dopaminergic medications. Based on the results of our study, if dopamine plays a role in memory consolidation then it is likely that dopamine enhances visual reproduction instead of logic memory. There are several limitations of our study that deserve consideration. The first limitation is the small sample size and the fact that we only recruited early-stage PD patients; it also should be noted that we excluded patients with sleep complaints, as PSG performance at night requires a high level of cooperation. Patients with moderate or severe PD and patients with sleep complaints usually are not able
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to complete the PSG study with enough REM and non-REM sleep periods. The mean apnea–hypopnea indices were high in both the groups; however, they revealed no statistical differences among the groups. The effect of sleep apnea on iNOS expression is uncertain and further investigation is required. Fluctuations in mRNA expression and individual differences might cause the limited significant differences observed in our experiment. However, to our knowledge, our study is the first study to examine biomarker changes in PD patients according to PSG-guided sampling. We encourage additional research on this subject to explore the relationship between biomarkers and sleep events in the neurodegenerative process. In summary, iNOS expression increased in REM sleep, SWS, and wake stages among PD patients in our preliminary study. Our results might reflect the overall increase in the oxidative stress of PD patients. Among all of the PD patients, the iNOS expression decreased during REM sleep. This finding could be associated with the increase in REM sleep, which is suppressed by NO in PD patients. We also showed that sleep might enhance memory consolidation and that the memory consolidation profiles of the PD patients and those of the non-PD control participants are distinct. The relationship between memory consolidation and specific sleep stages of PD patients requires further investigation. Funding sources This study is supported by grants from the National Science Council, Taiwan, NSC 95-2314-B-038-028, Shuang Ho Hospital, 95TMU-TMUH-13. Conflict of interest The ICMJE Uniform Disclosure Form for Potential Conflicts of Interest associated with this article can be viewed by clicking on the following link: http://dx.doi.org/10.1016/j.sleep.2013.09.016.
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Contents lists available at ScienceDirect
Sleep Medicine journal homepage: www.elsevier.com/locate/sleep
Original Article
Memory consolidation and inducible nitric oxide synthase expression during different sleep stages in Parkinson disease Dean Wu a,b,c,1, Ing-Jy Tseng d,1, Rey-Yue Yuan e, Chia-Yu Hsieh a,b,e,f, Chaur-Jong Hu a,b,e,f,⇑ a
Department of Neurology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan Sleep Center, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan c Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan d College of Nursing, Taipei Medical University, Taipei, Taiwan e Department of Neurology, Taipei Medical University Hospital, Taipei, Taiwan f Sleep Center, Taipei Medical University Hospital, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 4 September 2013 Accepted 25 September 2013 Available online 18 October 2013 Keywords: Parkinson disease Memory consolidation iNOS NO REM sleep Polysomnography
a b s t r a c t Background: Parkinson disease (PD) is a neurodegenerative disease characterized by motor and nonmotor dysfunctions, which include sleep disturbances. Rapid eye movement (REM) sleep is associated with numerous physiologic changes such as memory consolidation. Compelling evidence suggests that nitric oxide (NO) is crucial to both sleep regulation and memory consolidation. In our study, we explored changes in biologic molecules during various sleep stages and the effects of sleep on memory consolidation in PD. Methods: Ten PD patients and 14 volunteers without PD participated in our study. The gene expression of inducible NO synthase (iNOS) in all sleep stages was measured using realtime polymerase chain reaction (PCR) based on polysomnography (PSG)-guided peripheral blood sampling. In addition, the efficiency of memory consolidation during the sleep of the participants was measured using the Wechsler Memory Scale, third edition (WMS-III). Results: The iNOS expression increased in all sleep stages among the PD patients compared to the control participants, in whom iNOS expression decreased during REM sleep. Regarding memory consolidation, the performance of the controls in logic memory and the patients in visual reproduction tasks improved after sleep. Conclusions: The iNOS synthase expression was different from control participants among PD patients, and the expression was dissimilar in various sleep stages. Sleep might enhance memory consolidation and there are different memory consolidation profiles between PD and control participants demonstrating distinct memory consolidation profiles. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Parkinson disease (PD) is a neurodegenerative disease characterized by an insidious onset and slow progression of rigidity, tremors, bradykinesia, and balance impairment. In addition to motor dysfunction, PD patients may exhibit nonmotor symptoms, such as sensory symptoms, autonomic dysfunction, and sleep disturbances. Several sleep disturbances are particularly common in PD patients, including insomnia, nightmares, sleep apnea, rapid eye movement (REM) sleep behavior disorder, restless legs
⇑ Corresponding author at: Department of Neurology and Sleep Center, Shuang Ho Hospital, Taipei Medical University, 291 Jhongjheng Road, Jhonghe District, New Taipei City, Taiwan. Tel.: +886 2 22490088x8112; fax: +886 2 22490088x2507. E-mail address: [email protected] (C.-J. Hu). 1 These authors contributed equally to this work. 1389-9457/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sleep.2013.09.016
syndrome, periodic limb movement disorder, and excessive daytime sleepiness [1]. All of these symptoms can occur prior to the onset of PD motor symptoms and typically deteriorate during the disease process. The neurotransmitter dopamine plays a role in producing a wakefulness-promoting effect in vertebrates. Dopaminergic deficiency might be attributed to these sleep disturbances. Other systems, such as the serotoninergic, cholingergic, and norepinephrinergic systems, also play roles in sleep, but few studies have focused on PD sleep disorders [2]. All of these neurotransmitters, which play key roles in neurodegenerative diseases, are involved in the initiation and maintenance of sleep. Sleep affects behaviors by altering endocrine, neurotransmitters, and intracellular molecular events [3]. REM sleep, in which PD patients commonly exhibit abnormalities, accompanies numerous physiologic changes, such as a decrease in muscle tone, an increase in brain activities, and irregular cardiopulmonary functioning. These REM-related changes also are associated with
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dramatic changes in the microenvironment of the brain, including gene-expression patterns and activities of neurotransmitters [4]. In addition, sleep is essential for maintaining neurocognitive functions including memory consolidation [4–6]. Distinct memory is linked to unique sleep-related consolidation mechanisms that work during various sleep stages in REM sleep or slow-wave sleep (SWS) and in different brain regions throughout the night [7]. Increasing evidence suggests that nitric oxide (NO), originally identified as endothelium-derived relaxing factor also is crucial for both sleep regulation and memory consolidation [7]. Four forms of NO synthase are responsive to the generation of NO, which are neuronal (nNOS), endothelial, mitochondrial, and inducible (iNOS). Three processes regulate NO synthesis and NOSs activities, specifically, the L-arginine/arginase substrate-competing system; the citrulline/L-arginine-recycling system; and the asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase NOSs-inhibiting system. Researchers have documented that nitrites and nitrates may back-generate NO under ischemic conditions. This process must be considered regarding patients with neurodegenerative diseases such as PD [8]. A previous study reported that the presence of NO in the brain facilitates sleep, particularly REM sleep, whereas the presence of NO in the periphery may inhibit sleep [8]. In an nNOS/iNOS knockout (KO) young animal study, nNOS and iNOS demonstrated opposite effects on sleep; nNOS KO mice experienced a decrease in REM sleep, and iNOS KO mice exhibited an increase in REM sleep [9]. In contrast to the young animal study, another study indicated that iNOS may be essential to both the initiation and maintenance of REM in aging animals [10]. These studies have suggested that the role of the NO system in sleep regulation is complex. L-NG-nitroarginine methyl ester, an NOS inhibitor, was revealed to induce impairments in immediate, short-, and long-term memories of inhibitory avoidance tasks in animals. The iNOS expression was discovered to substantially increase in brains affected by PD, and this change in the NO system might affect sleep architecture and contribute to the clinically observed sleep and behavioral disturbances in PD patients. We designed our study to explore changes in biologic molecule, iNOS, during various sleep stages in PD patients. In addition, we measured the efficiency of memory consolidation during sleep in PD patients. The central molecules were indirectly examined by sampling peripheral leukocytes, based on the concept of some data supporting the synchronized expression of circadian genes between the central circadian clock and peripheral organs, including the leukocytes [11,12]. Thus we investigated the dissimilarities in iNOS gene expression between PD patients and non-PD control participants in REM sleep, SWS sleep, and wake stages by using polysomnography (PSG)-guided peripheral blood sampling.
2. Methods 2.1. Participants Ten PD patients and 14 non-PD volunteers aged 46–68 years were recruited from Taipei Medical University Hospital (TMUH) and Taipei Medical University Shuang-Ho Hospital. The PD diagnosis was based on the UK Parkinson’s Disease Society Brain Bank criteria. The severity of PD in patients was evaluated using the Hoehn and Yahr Scale. Among the participants, nine patients were in Stage 1 of PD and one patient was in Stage 2. All of the participants provided written informed consent, which was approved by the Joint-Institutional Review Board of Taipei Medical University.
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Participants who possessed a history of sleep disturbance or were regularly taking hypnotic drugs were excluded from our study. 2.2. Study design and memory tests All of the participants arrived at the sleep center of TMUH or Taipei Medical University Shuang-Ho Hospital at approximately 9:00 pm and were asked to provide basic information. They performed the first recall tasks by using the Wechsler Memory Scale, third edition (WMS-III) until 10:00 pm and then underwent PSG after an intravascular catheter was inserted into the cubital vein for further blood sampling. The participants fell asleep in individual rooms at approximately 11:00 pm. They were awoken by light exposure at approximately 6:00 am. Shortly afterward they performed the second recall tasks, which were identical to those performed the previous night (Fig. 1). To evaluate the relationship between sleep architecture and gene expression with memory consolidation in PD patients, the episodic memory tasks, which were designed and defined according to the WMS-III, were included in our study. These tasks comprise a logical memory task, a verbal-paired association task, and a visual reproduction task. All of the tests were conducted by a single member of the medical personnel according to manufacturer instructions. After the participants learned all four of the tasks included in the WMS-III, they were required to perform the first recall prior to sleeping. After approximately 8 h of sleep, they were asked to recall all of the tasks, which were learned the previous day, without being provided any information regarding these tasks; this process was defined as the second recall tasks. The results of recall performance and those of memory consolidation are represented by the percentage of memory retention. 2.3. PSG and blood sampling A PSG examination was conducted on all of the participants by using a Sandman Elite (Tyco Healthcare, Canada) at the sleep center of TMUH, or by using an Embla N7000 (ResMed, Australia) at Shuang-Ho Hospital. Prior to the scheduled sleep at 11:00 pm, venous catheters were inserted into the forearm veins of the participants. During the PSG examination, three samples that each contained 3 cc of peripheral venous blood were collected at three end points: (1) the first REM sleep, (2) non-REM SWS following the REM sleep, and (3) 15 min after the end of the PSG examination when the participants were awoken at 7:00 am by light exposure. The scoring of the PSG examination was performed by qualified PSG technicians based on the Rechtschaffen and Kales rules. Table 1 lists the PSG parameters. 2.4. RNA extraction and realtime polymerase chain reaction After collecting peripheral blood, the blood was immediately transferred to RNAlaterÒ RNA Stabilization Reagent kits (Qiagen; Valencia, CA, USA) to prevent RNA degradation. The total RNA was harvested using an RNA Extraction RiboPure-Blood kit (Ambi-
Fig. 1. Study design. Participants underwent memory tests before and after polysomnography (PSG), and blood samples were collected at three end points based on the PSG findings.
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D. Wu et al. / Sleep Medicine 15 (2014) 116–120 Table 1 Demographic data and polysomnography parameters of patients and control participants. Variable
Control group (n = 14)
Parkinson disease group (n = 10)
P value
Age (y) Gender Men Women BMI TST Sleep efficiency Sleep onset Stage 1 and stage 2 SWS%/TST REM%/TST PLMI/h REM sleep AHI/h Arousal index/h Education (y)
55.500 ± 4.256
59.500 ± 7.576
.113 .8
8 6 25.841 ± 3.311 256.264 ± 51.289 73.243 ± 13.243 26.893 ± 31.519 76.100 ± 13.602 14.893 ± 10.081 9.000 ± 5.956 5.150 ± 10.924 2.714 ± 1.637 10.257 ± 12.277 15.307 ± 10.016 15.143 ± 2.316
4 6 23.304 ± 3.067 295.589 ± 43.904 73.160 ± 9.926 20.972 ± 13.850 83.000 ± 7.963 2.070 ± 4.528 14.380 ± 6.563 15.980 ± 23.964 3.000 ± 0.943 9.470 ± 11.802 19.550 ± 13.015 11.800 ± 3.882
.070 .063 .987 .585 .166 .001 .048 .149 .626 .876 .376 .292
Abbreviations: y, years; BMI, body mass index; TST, total sleep time; SWS, slow-wave sleep; REM, rapid eye movement; PLMI, periodic limb movement index; h, hour; AHI, apnea–hypopnea index.
on; Applied Biosystems; Foster City, CA, USA). The total RNA of single 2-lg samples was included in the cDNA synthesis by using an ImProm-II reverse transcription kit (Promega, MI, USA) according to manufacturer instructions, and the cDNA was stored at 80 °C until use. For determining iNOS gene expression, realtime polymerase chain reaction (PCR) was performed using an ABI-7300 (Applied Biosystems); 1 lL of cDNA was administered in duplicate on eight strip tubes by using an Applied Biosystems Taqman assay (ABI assay ID: Hs00167248_m1), and by using a TaqManÒ Universal PCR Master Mix in a final volume of 20 lL. The threshold cycle changes (DCt) denote the dissimilarity between the Ct of iNOS and the Ct level of the internal control, actin, which is a housekeeping gene (ABI assay ID: Hs99999903_m1). 2.5. Statistical analysis The clinical and PSG variables in these clinical groups were calculated using the Student t test except for sex differences, which were measured using a v2 test. The average DCt of iNOS at REM sleep, SWS, and wake stages among the two groups was calculated. The dissimilarities in DCt between the PD and control groups also were tested using the Student t test. When testing the expression of iNOS, repeated measures were conducted to confirm the differences in paired DCt in REM sleep, SWS sleep, and wake stages among the same individuals. Regarding all analyses, statistical significance was assumed when the P value was less than .05. 3. Results The REM sleep percentage increased and the SWS percentage decreased in the PD group. There was no significant difference between PD and control participants, but there was a trend toward an increased number of REMs and an increased periodic limb movement index in the PD group, as shown in Table 1. Fig. 2 shows the levels of iNOS mRNA expression. Compared with the control group, the PD patients contained significantly higher levels of iNOS expression in REM sleep, SWS, and wake stages. The DCt of iNOS in the PD and control groups were 1.480 ± 0.446 vs 1.67 ± 0.026 (P < .001) during REM sleep, 0.893 ± 0.217 vs 1.681 ± 0.035 (P < .001) during SWS sleep, and 0.618 ± 0.121 vs 1.698 ± 0.031 (P < .001) during the wake stage. Among the PD patients, the DCt of iNOS significantly increased during REM sleep (1.480 ± 0.446 vs 0.893 ± 0.217 and 0.618 ± 0.121; P < .05). These results indicate that the iNOS mRNA expression decreased during REM sleep among the PD patients.
Fig. 2. inducible nitric oxide synthase (iNOS) mRNA levels represented by DCt in the rapid eye movement (REM) sleep, slow-wave sleep (SWS), and wake stages among control and Parkinson disease (PD) patients. iNOS expression is significantly upregulated (lower DCt) in PD patients at the REM sleep, SWS, and wake stages. The iNOS expression during REM sleep is significantly decreased (higher DCt) among PD patients. Abbreviation: NS, nonsignificant. ⁄⁄P < .001; ⁄P < .05.
When using the formula of 2 DDCt, in which DDCt is the difference in mean DCt between the PD group and the control group, the mean mRNA level of the PD group during the REM sleep, SWS, and wake stages were shown to be significantly higher than those of the control group [13,14]. Regarding memory consolidation, the control participants improved in performing logic memory tasks after sleeping and PD patients improved in performing visual reproduction tasks after sleeping (Fig. 3). These results indicate that sleep plays a role in memory consolidation. There are dissimilarities regarding memory consolidation profiles between the PD and non-PD control participants.
4. Discussion In our study, we investigated the expression of the REM sleep and memory-related gene in PD patients in iNOS during various
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Fig. 3. Memory consolidation of sleep measured by the Wechsler Memory Scale, third edition. Logic memory improved in control participants without Parkinson disease (PD), and visual reproduction improved in PD patients. Abbreviations: C, control; LM, logical memory; VPA, verbal paired association; VR, visual reproduction. ⁄P < .05.
sleep stages by using PSG recording. The mRNA of iNOS was significantly increased in PD patients during all of the sleep and wake stages. A previous study demonstrated that oxidative stress played a vital role in the pathophysiology of PD. High levels of neuronal and iNOS were discovered in the substantia nigra of patients and animal models with PD [15]. Another study revealed that iNOS overexpression usually indicated an ongoing inflammation, which is thought to play a role in the development of PD [16]. Neuronal NOS was overexpressed in peripheral blood cells among PD patients, though the blood concentration of NO decreased in PD patients. Previously, there was a lack of information regarding iNOS expression in the peripheral blood of PD patients. Our findings might be compatible with previous studies and support the hypothesis that PD patients demonstrate an increase in oxidative stress, such as iNOS overexpression. However, additional studies of oxidative stress including the measurement of NO are required. In our study, the iNOS mRNA expression significantly decreased during REM sleep compared with the SWS and wake stages among PD patients. Conversely, iNOS expression was unchanged, but it was lower in the non-PD control participants. The role of NO or iNOS in sleep is controversial. NO was revealed both to suppress and to facilitate REM sleep according to various studies. In adult animals, iNOS protein is not expressed, or it only is expressed at extremely low levels. However, there are three principal situations in which iNOS is expressed in neurons and glial cells: sleep rebound associated with sleep deprivation, inflammation, or during aging [17,18]. The increase of iNOS with aging was considered a mechanism for compensating for the neural impairments that develop after many years of adult life [8]. Upregulated iNOS is necessary for producing effective immune defenses. However, the persistent activation of iNOS may lead to the overproduction of NO. In studies on nNOS or iNOS genes that have used transgenic mice, the prominence of NO in REM sleep regulation increased. In iNOS KO animals, REM sleep was increased compared with that of the control mice [9]. In our study, REM sleep significantly increased in the PD patients, suggesting that the downregulation of the iNOS expression might be associated with the increase in REM sleep in PD patients. However, REM sleep increased in PD patients compared with the control group, but the iNOS was still highly expressed. Therefore, the notion that iNOS activation is associated with a decrease in REM sleep can be challenged. Although the relationship between peripheral tissues and the central nervous system regarding iNOS expression is still not clearly defined, we speculate that the suppression of iNOS can serve as a mean for maintaining REM sleep in PD patients.
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Although memory formation is not the only function of sleep, it might be the most crucial function as it assists in establishing the state of consciousness during wakefulness. This idea was formed more than a hundred years ago, dating back to 1885 with the beginning of experimental memory research by Ebbinghaus [19]. Consciousness and the formations of memory possibly are two mutually exclusive processes that cannot simultaneously occur in the neuronal networks of the brain. Without memory, our consciousness might fall apart into pieces of moments. Based on the standard two-stage memory model [20,21], sleep was proposed to be an offline process involving the ‘‘active system consolidation’’ of memory (i.e., the process in which newly encoded memory representations are redistributed to other neuron networks serving as long-term storage) [22]. The occurrence of memory consolidation during sleep has been comprehensively studied [23]. A considerable amount of evidence shows that SWS preferentially supports declarative memory consolidation, whereas REM sleep benefits specifically nondeclarative and emotional aspects of memory. However, the classical classification of sleep stages is crude and cannot adequately reflect on the complexity of sleep-associated processes that contribute to memory consolidation. The specific sleep stages that are responsible for specific memory components still require detailed analysis and investigation. Furthermore, the relationship between NO and memory processing has been previously studied [24–26]. Long-term potentiation and long-term depression (LTD), the predominant mechanisms of learning and memory processes, were believed to be initiated by the NO/cyclic GMP pathway. NO acts as a retrograde messenger of long-term potentiation and LTD in the hippocampus [27]. The expression of LTD-like synaptic plasticity in the hippocampus is related to the formation of several types of motor learning and visual recognition memory [28]. In our study, we observed sleep-enhanced declarative memory consolidation occurring in both the control and PD groups. In addition, logic memory improved in the control participants and visual reproduction improved in the PD patients. Based on the iNOS expression and PSG results, the PD patients contained a higher iNOS expression and demonstrated more REM sleep and less SWS than that of the control participants. Therefore, visual reproduction memory consolidation might be dependent on NO. The other possibility is that logic memory consolidation is dependent on SWS, but visual reproduction memory consolidation is dependent on REM sleep. We also explored the relationship between iNOS expression and various memory modalities, and we subsequently discovered no linear relationship between these two variables (data not shown). Our findings require further confirmation from future studies and research on the mechanisms. Another potential factor that might affect the memory of PD patients is dopamine. Recent studies have indicated that dopamine release implies offline memory reactivation and consolidation [29,30], general working memory capacity [31], and the ability to train working memory [32–34]. A recent study reported that PD patients on dopaminergic medication demonstrated an improved backward digit span performance following a nocturnal sleep interval, compared with PD patients who were not taking dopaminergic medication [35]. In our study, all of the PD patients and none of the control participants were taking dopaminergic medications. Based on the results of our study, if dopamine plays a role in memory consolidation then it is likely that dopamine enhances visual reproduction instead of logic memory. There are several limitations of our study that deserve consideration. The first limitation is the small sample size and the fact that we only recruited early-stage PD patients; it also should be noted that we excluded patients with sleep complaints, as PSG performance at night requires a high level of cooperation. Patients with moderate or severe PD and patients with sleep complaints usually are not able
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to complete the PSG study with enough REM and non-REM sleep periods. The mean apnea–hypopnea indices were high in both the groups; however, they revealed no statistical differences among the groups. The effect of sleep apnea on iNOS expression is uncertain and further investigation is required. Fluctuations in mRNA expression and individual differences might cause the limited significant differences observed in our experiment. However, to our knowledge, our study is the first study to examine biomarker changes in PD patients according to PSG-guided sampling. We encourage additional research on this subject to explore the relationship between biomarkers and sleep events in the neurodegenerative process. In summary, iNOS expression increased in REM sleep, SWS, and wake stages among PD patients in our preliminary study. Our results might reflect the overall increase in the oxidative stress of PD patients. Among all of the PD patients, the iNOS expression decreased during REM sleep. This finding could be associated with the increase in REM sleep, which is suppressed by NO in PD patients. We also showed that sleep might enhance memory consolidation and that the memory consolidation profiles of the PD patients and those of the non-PD control participants are distinct. The relationship between memory consolidation and specific sleep stages of PD patients requires further investigation. Funding sources This study is supported by grants from the National Science Council, Taiwan, NSC 95-2314-B-038-028, Shuang Ho Hospital, 95TMU-TMUH-13. Conflict of interest The ICMJE Uniform Disclosure Form for Potential Conflicts of Interest associated with this article can be viewed by clicking on the following link: http://dx.doi.org/10.1016/j.sleep.2013.09.016.
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