Periodic Reporting for period 1 - CIRCautophAGING (INTER-REGULATION OF CIRCADIAN AND AUTOPHAGIC PROCESSES IN STEM CELLS: IMPACT ON TISSUE REGENERATION AND AGING)
Période du rapport: 2021-01-04 au 2023-01-03
The CIRCautophAGING project aims to elucidate circadian rhythms in muscle stem cells (MuSCs), also called satellite cells. Organisms developed adaptive mechanisms, termed circadian rhythms to align physiologic functions to 24 hours (h) light/dark cycle on Earth. The mechanisms underlying their control are complex, and studies led by Dr. Hall, Dr. Rosbash and Dr. Young, which were awarded the Nobel Prize in 2017, represented a remarkable advance in the chronobiology field for their discoveries about circadian rhythms molecular regulation and generation at a cellular level. This mechanism is physiologically relevant since many basic and essential functions in mammals happen at a certain time of the day, they are adaptive and entrainable mechanisms that respond to external environmental cues. Among the most basic circadian-regulated behavior it is worth mentioning the sleep-wake cycle, hormone release, and food-fasting rhythms. Perturbation of circadian rhythms leads to gastrointestinal, metabolic, reproductive, and cardiovascular diseases as observed in studies on night shift workers whose circadian rhythms are deregulated. Furthermore, circadian rhythm deregulation is considered to be one of the hallmarks of aging. During aging the MuSCs number and function drop, causing a delay in muscle regeneration, this has been associated with impaired autophagy and senescence in old MuSCs. Other studies underlined that the circadian rhythms of these cells are rewired as an organism ages. To elucidate the contribution of circadian rhythms to the homeostasis and functions of these cells is key to understanding the changes that occur in aging and can be modulated in order to delay their biological decay or rejuvenate them.
Circadian rhythms are influenced and can be trained by external cues like light or habits such as exercise and diet. In virtually all the cells of the body, between 10-30% of genes follows an oscillating pattern of transcription/translation throughout the 24 h. The transcription factors responsible for this are known, and the generation of transgenic models based on their modulation represented a turning point in this field. At the molecular level, the transcription factor BMAL1 (brain and muscle ARNT-like 1) is considered the master regulator of circadian genes. Overall, this regulation depends on complex feedback loops controlled by other transcription factors and their regulators. Briefly, when BMAL1 is activated it dimerizes with CLOCK (circadian locomotor output cycles kaput) and binds to E-box elements to activate the transcription of controlled clock genes (CCGs). Period and (Per) cryptochrome (Cry) are among the CCGs regulated by the BMAL1/CLOCK complex, their encoded proteins also dimerize and act as inhibitors of BMAL1/CLOCK transcriptional activity. PER and CRY are eventually degraded by the proteasome, which releases the repression of BMAL1/CLOCK. Additionally, there are auxiliary loops that involve nuclear receptor subfamily 1 group D member 1 (Nr1d1 or REV-ERB-α) and member 2 (Nr1d2 or REV-ERBβ), which inhibit BMAL1 transcription. On the other hand, the retinoic acid receptor (RAR)-related orphan receptor (ROR) activates BMAL1 transcription.
The light-synchronized central clock has classically been thought to be dominant over clocks in peripheral tissues, however, recent reports have highlighted a role for local peripheral clocks themselves in governing their own rhythmic programs. It is increasingly established that oscillations are the result of integrated signals that comes from external cues as well as inter- intra-organ communication signals. How this regulation impinges on stem cells is unknown as in part due to technical challenges associated with working with these cells, as well as the previous lack of suitable animal models. To address this, we dissected the contribution of the local intrinsic clock 1) in a system where only MuSCs lack the clock regulator Bmal1, and 2) in a system in which the clock is reconstituted in MuScs in an otherwise Bmal1-null context. Overall, this ambitious project aimed to elucidate: (i) the impact of circadian rhythms on MuSCs by investigating the contribution of the cell-intrinsic (local) clock to their homeostasis, (ii) the crosstalk between the central and local clocks by specifically restoring the clocks in these two types of cell/tissue in an otherwise arrhythmic model (iii) the contribution of autophagy in MuSCs circadian regulation.
Circadian rhythms are influenced and can be trained by external cues like light or habits such as exercise and diet. In virtually all the cells of the body, between 10-30% of genes follows an oscillating pattern of transcription/translation throughout the 24 h. The transcription factors responsible for this are known, and the generation of transgenic models based on their modulation represented a turning point in this field. At the molecular level, the transcription factor BMAL1 (brain and muscle ARNT-like 1) is considered the master regulator of circadian genes. Overall, this regulation depends on complex feedback loops controlled by other transcription factors and their regulators. Briefly, when BMAL1 is activated it dimerizes with CLOCK (circadian locomotor output cycles kaput) and binds to E-box elements to activate the transcription of controlled clock genes (CCGs). Period and (Per) cryptochrome (Cry) are among the CCGs regulated by the BMAL1/CLOCK complex, their encoded proteins also dimerize and act as inhibitors of BMAL1/CLOCK transcriptional activity. PER and CRY are eventually degraded by the proteasome, which releases the repression of BMAL1/CLOCK. Additionally, there are auxiliary loops that involve nuclear receptor subfamily 1 group D member 1 (Nr1d1 or REV-ERB-α) and member 2 (Nr1d2 or REV-ERBβ), which inhibit BMAL1 transcription. On the other hand, the retinoic acid receptor (RAR)-related orphan receptor (ROR) activates BMAL1 transcription.
The light-synchronized central clock has classically been thought to be dominant over clocks in peripheral tissues, however, recent reports have highlighted a role for local peripheral clocks themselves in governing their own rhythmic programs. It is increasingly established that oscillations are the result of integrated signals that comes from external cues as well as inter- intra-organ communication signals. How this regulation impinges on stem cells is unknown as in part due to technical challenges associated with working with these cells, as well as the previous lack of suitable animal models. To address this, we dissected the contribution of the local intrinsic clock 1) in a system where only MuSCs lack the clock regulator Bmal1, and 2) in a system in which the clock is reconstituted in MuScs in an otherwise Bmal1-null context. Overall, this ambitious project aimed to elucidate: (i) the impact of circadian rhythms on MuSCs by investigating the contribution of the cell-intrinsic (local) clock to their homeostasis, (ii) the crosstalk between the central and local clocks by specifically restoring the clocks in these two types of cell/tissue in an otherwise arrhythmic model (iii) the contribution of autophagy in MuSCs circadian regulation.
We used several transgenic models to dissect the role of circadian clocks and autophagy in transcriptional rhythms within MuSCs. In the first, the essential circadian clock gene Bmal1 was deleted only in MuSCs. In the second, we used a model where the essential autophagy regulator Atg7 was deleted in MuSCs. To study if the MuSCs’ circadian regulation is autonomous, a murine model of total body Bmal1-KO with reconstitution of BMAL1 only in MuSCs. In the third, to study the crosstalk between the MuSCs clock and the central regulator in the brain, the suprachiasmatic nucleus (SCN), we used full BMAL1-KO mice and then used Cre-dependent stop cassette excision to reconstitute BMAL1 in the SCN and MuSCs either alone, or simultaneously. A circadian transcriptome has been performed for every mouse line mentioned, plus wild-type (WT) mice, at least 4 replicates (males and females) have been collected every 4 h over the 24 h. MuSCs represent 5-7% of all nuclei within the fiber basal lamina and they are FACS-isolated from a muscle cell suspension obtained by muscle enzymatic digestion, thanks to specific surface markers. From the transcriptomic data, the circadian GO categories mainly dependent on the local clock are related to response to hypoxia, NfKb and Wnt regulation, inhibition of apoptosis, and cell proliferation while the metabolism of lipids follows a circadian pattern that does not depend on BMAL1, suggesting that this function is dependent on external input. Regarding the contribution of autophagy to the circadian output, we found that genetic impairment of autophagy induced 148 transcripts to oscillate, these transcripts were not found to oscillate in the WT, and they account for functions related to protein stabilization, muscle development, senescence, skeletal muscle tissue regeneration. Part of these results have been disseminated during international conferences such as the “EMBO Workshop on Muscle formation, maintenance, regeneration and pathology”, the “Senestherapy-III: Cell senescence: mechanisms and therapies” and the “European Biological Rhythms Society (EBRS) 2022 conference” by presenting posters and a short talk.
This work defined the existence of a communication axis between the SCN and muscle stem cells for the first time. More broadly, this work contributes to dissecting the mechanisms through which different tissues in an organism can coordinate to sense the time of the day which is an open question in the field of chronobiology, in addition, to the impact of circadian clocks on stem cells. We expect to confirm the MuSC-specific circadian processes that are important for stem cell homeostasis. This acts as the basis of regenerative therapies that can be developed for devastating disorders affecting muscle such as muscular dystrophies and sarcopenia, which affect millions worldwide.
No website has been developed for the project.
No website has been developed for the project.