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. 2020 Jul 29:8:622.
doi: 10.3389/fcell.2020.00622. eCollection 2020.

Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development

Ankur Sheel  1   2 , Rong Shao  1   2   3 , Christine Brown  1 , Joanne Johnson  1 , Alexandra Hamilton  1 , Danhui Sun  1   2 , Julia Oppenheimer  4 , Wendy Smith  5 , Pablo E Visconti  2   6 , Michele Markstein  1   2 , Carol Bigelow  7 , Lawrence M Schwartz  1   2
Affiliations

Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development

Ankur Sheel et al. Front Cell Dev Biol. .

Abstract

The term programmed cell death (PCD) was coined in 1965 to describe the loss of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. While it was subsequently demonstrated that this hormonally controlled death requires de novo gene expression, the signal transduction pathway that couples hormone action to cell death is largely unknown. Using the ISMs from the tobacco hawkmoth Manduca sexta, we have found that Acheron/LARP6 mRNA is induced ∼1,000-fold on the day the muscles become committed to die. Acheron functions as a survival protein that protects cells until cell death is initiated at eclosion (emergence), at which point it becomes phosphorylated and degraded in response to the peptide Eclosion Hormone (EH). Acheron binds to a novel BH3-only protein that we have named BBH1 (BAD/BNIP3 homology 1). BBH1 accumulates on the day the ISMs become committed to die and is presumably liberated when Acheron is degraded. This is correlated with the release and rapid degradation of cytochrome c and the subsequent demise of the cell. RNAi experiments in the fruit fly Drosophila confirmed that loss of Acheron results in precocious ecdysial muscle death while targeting BBH1 prevents death altogether. Acheron is highly expressed in neurons and muscles in humans and drives metastatic processes in some cancers, suggesting that it may represent a novel survival protein that protects terminally differentiated cells and some cancers from death.

Keywords: BAD; BBH1; Drosophila; Manduca sexta; apoptosis; autophagy; cytochrome c; intersegmental muscle.

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Figures

FIGURE 1
FIGURE 1
Acheron expression during ISM development. (A) RNA-seq analysis of Acheron mRNA in the ISMs from day 13 until day 18, the day of adult eclosion. “20E” represents animals that were injected on day 17 to delay the time of cell death and then assayed on day 18. Acheron expression changed during development and was induced on day 18 (p = 0.002) but blocked by pre-treatment with 20E (p = 0.025, n = 3 independent replicates for each stage of development). (B) qPCR quantification of Acheron mRNA late in development and following 20E treatment. Only the day 18 sample was significantly different from day 13 (p = 0.02). (It should be noted that these data agree well with our independent validations of this RNA-seq dataset using qPCR; Tsuji et al., 2020). (C) (Left) Western blot of Acheron expression at carefully timed stages between day 15 of pupal/adult development and 2 h post-eclosion (PE). Acheron expression was prevented by treatment with 20E. The blots were also probed for tubulin (bottom), which served as a loading control. (D) Detailed examination of Acheron expression on late day 18 starting at 4:30 p.m. Animals in the colony eclose at about 5:30 p.m. (E,F) Immunohistochemical staining of Acheron before (E) and after (F) adult eclosion.
FIGURE 2
FIGURE 2
Phosphorylation and degradation of Acheron. (A) ISM Western blot with an anti-phospho-serine/threonine antibody detects a protein doublet the same size as Acheron at the time of eclosion. Comparable bands were not detected before or after this time point. (B) In vitro phosphorylation and degradation of Acheron. Endogenous Acheron is present in day 18 ISM extracts but not in extracts from post-eclosion (PE) muscles. Mixing the two extracts failed to induce Acheron degradation. The addition of cGMP and IBMX resulted in a higher molecular weight Acheron protein that was then susceptible to degradation when subsequently exposed to PE ISM extracts. (C) This experiment was repeated in the presence of 32P and then analyzed by film autoradiography, which demonstrated that Acheron is phosphorylated prior to degradation. These experiments were performed four times.
FIGURE 3
FIGURE 3
Quantification of signal transduction genes during ISM development. (A) RNA-seq analysis of Eclosion Hormone Receptor (EHR) and Ecdysis Triggering Hormone Receptors (ETHR) A and B across ISM development. RNA-seq analysis of EHR (blue), ETHR-A (red), and ETHR-B (green). EHR was significantly induced on day 18 (p < 0.001, n = 3 independent replicates for each stage of development). (B) Expression of cGMP-dependent protein kinase (PKG) (blue) and cAMP-dependent protein kinase (red) across the ISMs across development. PKG was induced during development (p = 0.002, n = 3 independent replicates for each stage of development).
FIGURE 4
FIGURE 4
Cloning and analysis of BBH1. (A) Co-immunoprecipitation (co-IP) assays were used to identify binding partners in mouse C2C12 myoblasts. Acheron binds to CASK, a previously identified partner, but not the pro-survival protein Bcl-2. It does bind to the BH3-only protein BAD, but not the related protein BAX. (B) Co-IP assays with day 18 ISM extracts demonstrate that Acheron binds to a protein that cross-reacts with an anti-mouse BAD antibody. (C) Expression of BBH1 protein across ISM development. Tubulin was used as a loading control. (D) RNA-seq quantification of BBH1 expression during development. There was no significant change (p = 0.28, n = 3 independent replicates for each stage of development). (E) The anti-BAD antibody recognizes a bacterially expressed portion of Manduca BBH1 protein in the induced (“I”) sample but not the uninduced sample (“U”).
FIGURE 5
FIGURE 5
The role of Acheron and BBH1 on ecdysial muscle death in Drosophila. (A) Data from Flybase modENCODE Tissue Expression Data based on RNA-seq analysis (CG17386). Acheron is transiently induced ∼600-fold just before adult eclosion. (B) Percentage of Drosophila that eclosed successfully in control and Acheron RNAi expressing animals. Acheron RNAi blocked eclosion (p < 0.0001, mean ± SE, n = 28 and 202, respectively). (C) Pre-eclosion pharate adult transgenic flies expressing red fluorescent protein (RFP) in the skeletal muscles of control (top) and Acheron (CG17386) RNAi flies. (D) Fluorescent imaging of the same animals showing that Acheron RNAi knockdown led to the precocious death of the ptilinal muscles. (E) Percentage of flies that eclosed successfully in control and BBH1 RNAi animals (p = 1.0, mean ± SE, n = 28 and 154, respectively). (F) Fluorescent image of a control fly head 36 h post-eclosion showing the normal loss of the ptilinal muscles. (G) Fluorescent image of a BBH1 RNAi fly head 36 h post-eclosion showing the retention of the ptilinal muscles. Note that the muscles in the proboscis (bottom front of the heads) persist in the adult. wt, wild type. Scale bars = 100 μm.
FIGURE 6
FIGURE 6
Expression of cytochrome c in the ISMs. Cytochrome c immunohistochemical staining of ISMs demonstrates abundant punctate staining in the ISMs prior to adult eclosion (A), which was lost throughout the tissue following eclosion (B). The nuclei are stained blue. Scale bar = ∼280 μm. (C) Western blot analysis of Acheron and cytochrome c in the ISMs before and after adult eclosion.
FIGURE 7
FIGURE 7
Model for ISM cell death. On day 17 of pupal–adult development, the circulating levels of 20E decline below a threshold that triggers the expression of both the Eclosion Hormone Receptor (EHR) and Acheron. Acheron binds to and stabilizes the pro-death protein BBH1, which then accumulates. A further decline of 20E on day 18 triggers the release of eclosion hormone, which binds to the EHR and drives the production of cGMP, the conversion of inactive protein kinase G (PKGi) into PKG active (PKGa), and Acheron phosphorylation, which leads to its degradation. This liberates BBH1, which then induces the release and degradation of cytochrome c from mitochondria and the subsequent non-apoptotic death of the muscles. (Solid lines denote events that have been demonstrated experimentally, while dashed lines have not been formally tested).
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