Circadian regulation of endoplasmic reticulum calcium response in mouse cultured astrocytes
Curation statements for this article:-
Curated by eLife
eLife assessment
This is a useful study that identifies circadian changes in the gene expression profile of cultured mouse astrocytes. Mechanistic details linking circadian rhythmicity in HERP, a regulator of calcium signals in the endoplasmic reticulum, to altered phosphorylation of Connexin 43 remain currently incomplete. With improved manuscript clarity and statistical analysis, this work could be of interest to the field of astrocyte and circadian biology.
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Abstract
The circadian clock, an internal time-keeping system orchestrates 24-hour rhythms in physiology and behavior by governing rhythmic transcription within cells. Astrocyte, the most abundant glial cell type, play crucial roles in central nervous system functions. However, a detailed understanding of how the circadian clock impacts functions of astrocyte remains largely unexplored. In this study, utilizing circadian clock-synchronized mouse cultured cortical astrocytes and RNA sequencing, we identified 412 circadian rhythmic transcripts with a distinct astrocyte-specific expression pattern. A Gene Ontology analysis of these rhythmic transcripts highlighted genes implicated in Ca 2+ homeostasis as being under circadian control. Notably, Herpud1 (Herp) exhibited robust circadian rhythmicity at both mRNA and protein levels, a rhythm disrupted in astrocytes lacking the circadian transcription factor, BMAL1. HERP regulated endoplasmic reticulum (ER) Ca 2+ release by modulating the degradation of inositol 1,4,5-trisphosphate receptors (ITPRs). Intriguingly, ATP-stimulated ER Ca 2+ release varied with the circadian cycle, being more pronounced at subjective night, likely owing to the rhythmic expression of ITPR2. Furthermore, this rhythmic ER Ca 2+ response led to day/night variations in the phosphorylation of Cx43 (Ser368) and the gap junctional communication. Given the role of gap junction channel (GJC) in propagating Ca 2+ signals, we suggest that this circadian regulation of ER Ca 2+ responses could markedly affect astrocytic modulation of synaptic activity according to the time of day. Overall, our study enhances the understanding of how circadian clock influences astrocyte function in the CNS, shedding light on their potential role in daily variations of brain activity and health.
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eLife assessment
This is a useful study that identifies circadian changes in the gene expression profile of cultured mouse astrocytes. Mechanistic details linking circadian rhythmicity in HERP, a regulator of calcium signals in the endoplasmic reticulum, to altered phosphorylation of Connexin 43 remain currently incomplete. With improved manuscript clarity and statistical analysis, this work could be of interest to the field of astrocyte and circadian biology.
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Reviewer #1 (Public Review):
Summary:
In Ryu et al., the authors use a cortical mouse astrocyte culture system to address the functional contribution of astrocytes to circadian rhythms in the brain. The authors' starting point is transcriptional output from serum-shocked culture, comparative informatics with existing tools and existing datasets. After fairly routine pathway analyses, they focus on the calcium homeostasis machinery and one gene, Herp, in particular. They argue that Herp is rhythmic at both mRNA and protein levels in astrocytes. They then use a calcium reporter targeted to the ER, mitochondria, or cytosol and show that Herp modulates calcium signaling as a function of circadian time. They argue that this occurs through the regulation of inositol receptors. They claim that the signaling pathway is clock-controlled by a …
Reviewer #1 (Public Review):
Summary:
In Ryu et al., the authors use a cortical mouse astrocyte culture system to address the functional contribution of astrocytes to circadian rhythms in the brain. The authors' starting point is transcriptional output from serum-shocked culture, comparative informatics with existing tools and existing datasets. After fairly routine pathway analyses, they focus on the calcium homeostasis machinery and one gene, Herp, in particular. They argue that Herp is rhythmic at both mRNA and protein levels in astrocytes. They then use a calcium reporter targeted to the ER, mitochondria, or cytosol and show that Herp modulates calcium signaling as a function of circadian time. They argue that this occurs through the regulation of inositol receptors. They claim that the signaling pathway is clock-controlled by a limited examination of Bmal1 knockout astrocytes. Finally, they switch to calcium-mediated phosphorylation of the gap junction protein Connexin 43 but do not directly connect HERP-mediated circadian signaling to these observations. While these experiments address very important questions related to the critical role of astrocytes in regulating circadian signaling, the mechanistic arguments for HERP function, its role in circadian signaling through inositol receptors, the connection to gap junctions, and ultimately, the functional relevance of these findings is only partially substantiated by experimental evidence.
Strengths:
- The paper provides useful datasets of astrocyte gene expression in circadian time.
- Identifies HERP as a rhythmic output of the circadian clock.
- Demonstrates the circadian-specific sensitivity of ATP -> calcium signaling.
- Identifies possible rhythms in both Connexin 43 phosphorylation and rhythmic movement of calcium between cells.
Weaknesses:
- It is not immediately clear why the authors chose to focus on Ca2+ homeostasis or Herp from their initial screens as neither were the "most rhythmic" pathways in their primary analyses.
- It would have been interesting (and potentially important) to know whether various methods of cellular synchronization would also render HERP rhythmic (e.g., temperature, forskolin, etc). If Herp is indeed relatively astrocyte-specific and rhythmic, it should be easy to assess its rhythmicity in vivo.
- The authors show that Herp suppression reduces ATP-mediated suppression of calcium whereas it initially increases Ca2+ in the cytosol and mitochondria and then suppresses it. The dynamics of the mitochondrial and cytosolic responses are not discussed in any detail and it is unclear what their direct relationship is to Herp-mediated ER signaling. What is the explanation for Herp (which is thought to be ER-specific) to calcium signaling in other organelles?
- What is the functional significance of promoting ATP-mediated suppression of calcium in ER?
- The authors then nicely show that the effect of ATP is dependent on intrinsic circadian timing but do not explain why these effects are antiphase in cytosol or mitochondria. Moreover, the ∆F/F for calcium in mitochondria and cytosol both rise, cross the abscissa, and then diminish - strongly suggesting a biphasic signaling event. Therefore, one wonders whether measuring the area under the curve is the most functionally relevant measurement of the change.
- Why are mitochondrial and cytosolic calcium not also demonstrated for Bmal1 KO astrocytes?
- The authors claim that Herp acts by regulating the degradation of ITPRs but this hypothesis - rather central to the mechanisms proposed in this study - is not experimentally substantiated.
- There is no clear demonstration of the functional relevance of the circadian rhythms of ATP-mediated calcium signaling.
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Reviewer #2 (Public Review):
Summary:
The article entitled "Circadian regulation of endoplasmic reticulum calcium response in mouse cultured astrocytes" submitted by Ryu and colleagues describes the circadian control of astrocytic intracellular calcium levels in vitro.
Strengths:
The authors used a variety of technical approaches that are appropriate
Weaknesses:
Statistical analysis is poor and could lead to a misinterpretation of the data
Several conceptual issues have been identified.
Overinterpretation of the data should be avoided. This is a mechanistic paper done completely in vitro, all references to the in vivo situation are speculative and should be avoided.
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Reviewer #3 (Public Review):
Astrocyte biology is an active area of research and this study is timely and adds to a growing body of literature in the field. The RNA-seq, Herp expression, and Ca2+ release data across wild-type, Bmal1 knockout, and Herp knockdown cellular models are robust and lend considerable support to the study's conclusions, highlighting their importance. Despite these strengths, the manuscript presents a gap in elucidating the dynamics of HERP and the involvement of ITPR1/2 in modulating Ca2+ release patterns and their circadian variations, which remains insufficiently supported and characterized. While the Connexin data underscore the importance of rhythmic Ca2+ release triggered by ATP, the relationship here appears correlational and the role of HERP and ITPR in Cx function remains to be characterized. Moreover, …
Reviewer #3 (Public Review):
Astrocyte biology is an active area of research and this study is timely and adds to a growing body of literature in the field. The RNA-seq, Herp expression, and Ca2+ release data across wild-type, Bmal1 knockout, and Herp knockdown cellular models are robust and lend considerable support to the study's conclusions, highlighting their importance. Despite these strengths, the manuscript presents a gap in elucidating the dynamics of HERP and the involvement of ITPR1/2 in modulating Ca2+ release patterns and their circadian variations, which remains insufficiently supported and characterized. While the Connexin data underscore the importance of rhythmic Ca2+ release triggered by ATP, the relationship here appears correlational and the role of HERP and ITPR in Cx function remains to be characterized. Moreover, enhancing the manuscript's clarity and readability could significantly benefit the presentation and comprehension of the findings.
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