인간이 잠이 부족하면? Sleep increases chromosome dynamics to enable reduction of accumulating DNA damage in single neurons

Sleep increases chromosome dynamics to enable reduction of accumulating DNA damage in single neurons

D. Zada, I. Bronshtein, T. Lerer-Goldshtein, Y. Garini & L. Appelbaum 

 

인간이 잠이 부족하면?    동물이 잠을 자는 건, 후손에 유전자를 전달한다는 '진화적 이익'에 배치된다. 원시시대 자연 생태계에선 포식자에 잡아먹힐 위험이 깨어 있을 때보다 크기 때문이다. 그런데 인간을 포함한 모든 동물은 잠을 자야 살 수 있다. 이 또한 진화의 결과다. 인간은 잠이 부족하면 인지 기능 저하, 면역력 약화 등 여러 가지 신체 이상이 따른다. 계속 잠을 자지 않으면 결국 목숨까지 잃을지 모른다. 하지만 잠을 자야 하는 생물학적 이유는 여전히 규명되지 않았다. 이스라엘 바-일란 대학의 과학자들이 동물실험을 통해 그 비밀의 실마리를 풀었다. 수면이 손상된 뇌 신경세포(뉴런)의 DNA 복구와 직결돼 있다는 것이다. 이 연구결과를 담은 보고서는 과학 저널 '네이처 커뮤니케이션즈(Nature Communications)'에 실렸다. 7일(현지시간) 이스라엘의 유력 일간지 '하레츠(Haaretz)'에 따르면 이 연구를 수행한 건, 바-일란 대학의 리오르 아펠바움 교수팀이다. 연구팀이 고른 실험 대상은 제브라피시다. 이 물고기는 치어일 때 뼛속까지 들여다보일 만큼 투명하고, 잠도 인간처럼 밤에 잔다. 인간과 일부 동물의 적혈구는 예외지만 그 밖의 모든 세포에는 DNA가 있다. DNA는 생기는 시점부터 바로 손상되기 시작한다. 손상의 원인은 각종 산화물, 태양광 등 부지기수며 심지어 뉴런의 활동도 그중 하나다. 이론상 손상된 DNA는 그런 기능을 가진 효소에 의해 복구돼야 한다.   신경세포 이미지/연합뉴스 자료사진 연구팀은 뉴런의 DNA 염색체를 염색하고, 3D 저속촬영으로 움직임을 추적했다. 그랬더니 물고기가 깨어 있을 때 세폭 핵의 염색체 움직임이 줄어들면서 DNA 손상이 증가하는 게 관찰됐다. 수면 부족이 장기화하면 뉴런은 사멸할 수 있다. 하지만 잠 잘 땐 염색체 움직임이 다시 빨라졌다. 이는 손상 부위가 잘 수리되고 있다는 뜻이다. 개별 뉴런의 단위에서 DNA가 정상상태를 유지하려면 충분한 수면이 꼭 필요하다는 것이다. 활발하진 않지만, 낮에도 염색체는 활동하고, 부분적이나마 손상 부위의 복구도 분명히 이뤄진다. 하지만 균형추가 복구 쪽으로 급격히 기우는 건 제브라피시가 잠을 잘 때였다. 아펠바움 교수는 이를 도로 곳곳에 포트홀이 생긴 것에 비유했다. 그는 "특히 주간의 러시아워에 도로의 파손이 늘어나도, 교통량이 줄어드는 야간이 돼야 도로 복구가 가장 편하고 효율적인 것과 마찬가지"라고 말했다. 뉴런 말고 다른 세포의 DNA 복구도 잠잘 때 이뤄지는지는 일단 회의적이라고 한다. 연구팀은 이 부분을 보기 위해 혈관 내벽 세포와 신경아교세포 두 종을 추가로 실험했다. 하지만 주야 사이에 염색체 움직임이나 DNA 손상의 차이는 보이지 않았다. 아펠바움 교수는 "일단 이런 메커니즘은 특별히 뉴런에만 작동하는 것 같다"면서 "향후 과제는 근육과 같은 다른 형태의 세포를 시험해 보는 것"이라고 지적했다. 다음 단계로 연구팀은 뇌의 구석구석을 뒤져 특히 잠잘 때 활성화하는 뉴런을 찾아낼 계획이다. 잠잘 때 작동하는 DNA 복구 시스템이 꿈을 꾸면 어떻게 되는지도 관심거리다. 지난해엔 동물도 꿈을 꿀 수 있다는 연구결과가 발표되기도 했다. 당연히 이번 연구결과는 퇴행성 신경질환의 치료법 개발에도 활용될 수 있다. 만성 수면 부족은 DNA 손상으로 인한 신경세포 사멸의 위험을 높인다. 멀리 갈 거 없이, 많은 뇌 질환 증후 가운데 하나가 불면증이다. 연합뉴스/동아사이언스

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Abstract

Sleep is essential to all animals with a nervous system. Nevertheless, the core cellular function of sleep is unknown, and there is no conserved molecular marker to define sleep across phylogeny. Time-lapse imaging of chromosomal markers in single cells of live zebrafish revealed that sleep increases chromosome dynamics in individual neurons but not in two other cell types. Manipulation of sleep, chromosome dynamics, neuronal activity, and DNA double-strand breaks (DSBs) showed that chromosome dynamics are low and the number of DSBs accumulates during wakefulness. In turn, sleep increases chromosome dynamics, which are necessary to reduce the amount of DSBs. These results establish chromosome dynamics as a potential marker to define single sleeping cells, and propose that the restorative function of sleep is nuclear maintenance.

       

Introduction

Sleep is vital to animal life and is found in all studied animals, ranging from jellyfish to worm, fly, zebrafish, rodents, and humans1,2,3,4,5. Prolonged sleep deprivation can be lethal, and sleep disturbances are associated with various deficiencies in brain performance6. Sleep is regulated by circadian and homeostatic processes7, and is coupled with reduced awareness of the environment and a high risk for survival. Several mechanisms can explain the roles of sleep, ranging from macromolecule biosynthesis, energy conservation, and metabolite clearance, to synaptic plasticity and memory consolidation8,9,10,11,12. However, why sleep has evolved and which fundamental ancestral functions it regulates, remain enigmatic.

Why do animals sleep? Why do humans "waste" a third of their lives sleeping? Researchers now reveal a novel and unexpected function of sleep that could explain how sleep and sleep disturbances affect brain performance, aging and various brain disorders. Using 3D time-lapse imaging techniques in live zebrafish, they were able to define sleep in a single chromosome resolution and show that single neurons require sleep in order to perform nuclear maintenance. Photo: Imaging of chromosome dynamics (green) in single neuron (red, dashed box) in live larva. Credit: David Zada

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In mammals and birds, sleep is defined by behavioral criteria and cycles of electroencephalographic (EEG) patterns, which differentiate between wakefulness and sleep states. In non-mammalian animals, including zebrafish, sleep is solely defined by behavioral criteria, such as periods of immobility associated with a species-specific posture and an increased threshold of arousal to external stimuli13,14,15,16. In all animals, including animals with simple neuronal networks2,3, circuits of sleep- and wake-promoting neurons orchestrate the behavioral states17. Evidence across multiple animals supports the notion that sleep can occur locally in the brain18 or perhaps even in a small number of cells19. Nevertheless, although sleep significantly contributes to the overall temporal organization of the transcriptome20,21, there are no molecular markers that can be reliably used across phylogeny to define sleep in a single cell22.

The nuclear architecture and the dynamic changes in chromatin organization regulate vital cellular processes, including epigenetics, genomic stability, transcription, cell cycle, and DNA replication and repair23,24. Chromatin dynamics, such as chromosome movements and structural genomic arrangements, are regulated by proteins that interact with the nuclear lamina and envelope in dividing cells25,26,27. In mature and non-dividing neurons, the role of chromatin dynamics is less understood28. Accumulating evidence showed that chromatin remodeling is implicated in circadian function. The changes in chromatin organization and epigenetic landscape shape the expression profile of a large number of rhythmic genes29. However, the effect of sleep on chromatin dynamics in neurons is unknown.

Recent works showed that sleep can be induced by cellular stress in Caenorhabditis elegans and mammals30,31. Moreover, sleep has been associated with the faster repair of DNA double-strand breaks (DSBs) in mice and fruit flies32. The causes of DSBs are diverse and include reactive oxygen species (ROS), ionizing radiation, and inadvertent action of nuclear enzymes33. Notably, neuronal activity can also induce DSBs. In specific mouse neurons, DSBs can be generated by physiological brain activity during natural exploration of the environment34. Furthermore, activity-induced DSBs facilitate the expression of immediate early genes in mouse and cell cultures, possibly because they resolve topological constraints in the genome35.

We hypothesized that sleep has evolved in order to enable single neurons to perform nuclear maintenance. To test which nuclear process favors sleep time, real-time imaging of chromosome dynamics, neuronal activity as well as quantification of DSBs and sleep, were coupled with genetic and pharmacological manipulations in live zebrafish. The findings propose a definition for a single sleeping neuron; i.e., increased chromosome dynamics, and suggest a role for sleep; i.e., nuclear maintenance.

Results

Imaging chromosome dynamics in live larvae

Chromatin dynamics constitute a fundamental component of genome regulation and cell function36. In order to visualize and quantify chromosome dynamics in live zebrafish, the zebrafish telomeric repeat binding factor a (terfa) was cloned, and the telomere marker EGFP-Terfa was expressed in zebrafish neurons, resulting in a nucleus-specific punctum pattern (Fig. 1a–e). To verify that EGFP-Terfa marks chromosomes, the human telomere marker uas:dsRED-TRF137 was co-injected with either uas:EGFP-Terfa or the DNA binding-site-deleted construct uas:EGFP-Terfa del into one-cell-stage tg(HuC:Gal4) embryos. While zebrafish and human telomeric markers co-localized (Fig. 1f–i), deletion of the Terfa DNA binding site resulted in non-specific protein aggregates in the nucleus (Fig. 1j–l). To further validate that the puncta mark chromosomes, the zebrafish centromere protein a (cenpa) was cloned, and the EGFP-Cenpa was used as a centromere marker. Two-color imaging showed that EGFP-Cenpa and dsRED-TRF1 puncta were expressed adjacently on the chromosome, but not co-localized, as expected from telomeric and centromeric markers (Fig. 1m–p). To continuously image chromosome dynamics in all neurons of live fish, a stable tg(uas:EGFP-Terfa) transgenic line was generated and crossed with tg(HuC:Gal4) zebrafish (Fig. 1q, r). Neurons in the telencephalon (Te), rhombencephalon (Rh), spinal cord (SC), and habenula (Hb), of 6-day post-fertilization (dpf) larvae were imaged during 9.5 min. Single-particle tracking (SPT) analysis38 was used to detect and quantify the motility of telomere trajectories (Supplementary Movie 1, Fig. 1s, t). While telomeres in the Rh, SC, and Hb neurons showed a similar volume of motion of 0.0069 ± 0.0002, 0.0066 ± 0.0003, and 0.0063 ± 0.0004 µm3, respectively, telomeres in Te neurons showed increased volume of motion (0.01 ± 0.0004 µm3, Supplementary Fig. 1a). Calculation of the mean square displacement (MSD) for single trajectories of all Te and Rh neurons demonstrated anomalous subdiffusion of the puncta (Fig. 1u), which is typical to chromosome diffusion38. Indeed, analysis of both telomeric and centromeric markers that are expressed in the same SC neurons (Fig. 1m–p, Supplementary Movie 2) showed similar dynamics (Supplementary Fig. 1b), verifying that the puncta mark chromosomes. Altogether, these results transfer in vitro chromatin experiments to whole organisms, and demonstrate the capability of monitoring chromosome dynamics in live larvae.

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https://www.nature.com/articles/s41467-019-08806-w

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