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Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Blohberger J, Kunz L, Einwang D, Berg U, Berg D, Ojeda SR, Dissen GA, Fröhlich T, Arnold GJ, Soreq H, Lara H, Mayerhofer A - Cell Death Dis (2015)

Bottom Line: AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo.The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death.Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis.

View Article: PubMed Central - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig-Maximilian-University (LMU), Munich, Germany.

ABSTRACT
Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

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Related in: MedlinePlus

Nec-1 and NSA block ARP-induced increase in cell death, while Z-VAD-FMK does not. (a) LDH cytotoxicity assay performed with cultured human GCs. ARP (50 ng/ml) significantly increases cytotoxicity compared with control groups (Scr 50 ng/ml; ARPin 50 ng/ml; P<0.05; analysis of variance (ANOVA)). (b) Z-VAD-FMK (ZVF; 20 μM) does not block ARP-regulated increase in cytotoxicity. Treatment with ZVF only significantly decreases cytotoxicity compared with control (DMSO 1‰ P<0.05; ANOVA). (c) No increased activity of caspase 3/7 was detected in ARP-treated cells compared with control groups (P<0.05; ANOVA). Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. (d) Nec-1 (20 μM) significantly blocks ARP-regulated increase in cytotoxitcity. Ethanol (EtOH; 0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (e) Treatment with Nec-1 causes significant lower cytotoxicity compared with control group (P<0.05, t-test). (f) NSA (0.5 μM) is able to block ARP-dependent increase in cytotoxicity (P<0.05; t-test). DMSO (0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (g) Treatment with NSA causes significant lower cytotoxicity compared with control group (P<0.05, t-test). All values of LDH-assays are shown as mean±S.E.M. of n=10 (except Z-VAD-FMK stimulation, panel b, n=7) independent preparations of cells from two to five patients each. (h) Identification of RIPK1 and RIPK3 protein in cultured human GCs by western blotting. Arrows indicate the expected mass of protein (RIPK1: 76 kDa; RIPK3: 57 kDa; p-MLKL: 54 kDa). Serum control was negative. (i) Identification of MLKL and p-MLKL in cultured human GCs by western blotting. ARP treatment for 5 h increased the levels of p-MLKL compared with control group, which was treated with the control peptide (Scr). Arrows point to the expected mass of the proteins (MLKL 37 kDa; p-MLKL: 54 kDa). Different letters in a–g indicate statistically significant differences between the treatment groups
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fig5: Nec-1 and NSA block ARP-induced increase in cell death, while Z-VAD-FMK does not. (a) LDH cytotoxicity assay performed with cultured human GCs. ARP (50 ng/ml) significantly increases cytotoxicity compared with control groups (Scr 50 ng/ml; ARPin 50 ng/ml; P<0.05; analysis of variance (ANOVA)). (b) Z-VAD-FMK (ZVF; 20 μM) does not block ARP-regulated increase in cytotoxicity. Treatment with ZVF only significantly decreases cytotoxicity compared with control (DMSO 1‰ P<0.05; ANOVA). (c) No increased activity of caspase 3/7 was detected in ARP-treated cells compared with control groups (P<0.05; ANOVA). Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. (d) Nec-1 (20 μM) significantly blocks ARP-regulated increase in cytotoxitcity. Ethanol (EtOH; 0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (e) Treatment with Nec-1 causes significant lower cytotoxicity compared with control group (P<0.05, t-test). (f) NSA (0.5 μM) is able to block ARP-dependent increase in cytotoxicity (P<0.05; t-test). DMSO (0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (g) Treatment with NSA causes significant lower cytotoxicity compared with control group (P<0.05, t-test). All values of LDH-assays are shown as mean±S.E.M. of n=10 (except Z-VAD-FMK stimulation, panel b, n=7) independent preparations of cells from two to five patients each. (h) Identification of RIPK1 and RIPK3 protein in cultured human GCs by western blotting. Arrows indicate the expected mass of protein (RIPK1: 76 kDa; RIPK3: 57 kDa; p-MLKL: 54 kDa). Serum control was negative. (i) Identification of MLKL and p-MLKL in cultured human GCs by western blotting. ARP treatment for 5 h increased the levels of p-MLKL compared with control group, which was treated with the control peptide (Scr). Arrows point to the expected mass of the proteins (MLKL 37 kDa; p-MLKL: 54 kDa). Different letters in a–g indicate statistically significant differences between the treatment groups

Mentions: In contrast to the AChE-S and AChE-E, the AChE-R is a soluble monomer and its specific C-terminal peptide ARP has been shown to possess additional non-enzymatic functions.41 To explore assumed non-enzymatic effects in human GCs, we used a synthetic ARP peptide (Figure 4). Live cell imaging performed over a 24-h time period revealed massive cell death events in the ARP-treated cells (50 ng/ml) compared with the untreated control group (Figure 4a; Supplementary Data). A scrambled control peptide (Scr; 50 ng/ml) and heat-inactivated ARP (ARPin; 50 ng/ml; 10 min, 95 °C) exhibited no bioactivity. Confluence measurements furthermore underpinned this observation (Figure 4b). Cell death events were first observed after approximately 2–3 h upon the addition of ARP and continued throughout a 24-h period. Many of the dying cells showed a characteristic morphology upon ARP treatment. It involved cytoplasmic ballooning (Figure 4c), which albeit at much lower frequency could be found in control cells as well. Lactate dehydrogenase (LDH) assays with 10 independent preparations of cultured GCs were performed to detect LDH release as measure for plasma membrane damage. The results showed a significant increase after 5 h, indicating cytotoxicity of ARP treatment compared with the control, Scr and ARPin (Figure 5a). The pan-caspase inhibitor Z-VAD-FMK (20 μM) did not prevent the ARP-dependent increase in cytotoxicity seen in LDH measurements. Addition of Z-VAD-FMK to GCs, however, blocked basally occurring cell death, presumably apoptosis, which was observed in the control group, in which the solvent dimethyl sulfoxide (DMSO; 1‰) was tested (Figure 5b; seven independent GC preparations). ARP stimulation did not change the activities of caspase 3/7 over control groups (Figure 5c; three independent GC preparations). The results indicate that the type of cell death that is involved is not typical caspase-dependent apoptosis. The RIPK1 inhibitor Nec-1 (20 μM) significantly blocked the ARP-dependent increase in LDH release when added to ARP-exposed GCs (Figure 5d; 10 independent GC preparations). This effect points to necroptosis as cause for the increased number of cell deaths by ARP stimulation. In the GC group treated with Nec-1 alone, a significantly reduced cytotoxicity became apparent, indicating a basal level of necroptosis in GCs (Figure 5e; 10 independent GC preparations). Addition of NSA (0.5 μM), a blocker of MLKL, effectively reduced necroptotic cell death, indicated by its ability to inhibit ARP-induced cytotoxicity (Figure 5f; 10 independent GC preparations). As in the case of Nec-1, NSA significantly reduced cytotoxicity in GCs compared with untreated control (Figure 5g), and this further indicates that necroptosis is a form of cell death in GCs. As Nec-1 is dissolved in ethanol and NSA in DMSO, we excluded non-specific possible cytotoxic actions of the corresponding solvents (ethanol (0.1‰) and DMSO (0.1‰)) using the LDH assay, (Figures 5d and f). Furthermore, RIPK1, RIPK3 and MLKL, key proteins in the necroptosis pathway, were identified in three GC preparations by using western blotting (Figures 5h and i; for RT-PCR data, see Supplementary Data). ARP, but not the control peptide, increased the levels of p-MLKL after 5 h, which corresponds to the time, when the cytotoxicity of ARP was confirmed by LDH measurements (Figure 5i). This experiment was repeated using three independent GC preparations (Supplementary Data).


Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Blohberger J, Kunz L, Einwang D, Berg U, Berg D, Ojeda SR, Dissen GA, Fröhlich T, Arnold GJ, Soreq H, Lara H, Mayerhofer A - Cell Death Dis (2015)

Nec-1 and NSA block ARP-induced increase in cell death, while Z-VAD-FMK does not. (a) LDH cytotoxicity assay performed with cultured human GCs. ARP (50 ng/ml) significantly increases cytotoxicity compared with control groups (Scr 50 ng/ml; ARPin 50 ng/ml; P<0.05; analysis of variance (ANOVA)). (b) Z-VAD-FMK (ZVF; 20 μM) does not block ARP-regulated increase in cytotoxicity. Treatment with ZVF only significantly decreases cytotoxicity compared with control (DMSO 1‰ P<0.05; ANOVA). (c) No increased activity of caspase 3/7 was detected in ARP-treated cells compared with control groups (P<0.05; ANOVA). Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. (d) Nec-1 (20 μM) significantly blocks ARP-regulated increase in cytotoxitcity. Ethanol (EtOH; 0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (e) Treatment with Nec-1 causes significant lower cytotoxicity compared with control group (P<0.05, t-test). (f) NSA (0.5 μM) is able to block ARP-dependent increase in cytotoxicity (P<0.05; t-test). DMSO (0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (g) Treatment with NSA causes significant lower cytotoxicity compared with control group (P<0.05, t-test). All values of LDH-assays are shown as mean±S.E.M. of n=10 (except Z-VAD-FMK stimulation, panel b, n=7) independent preparations of cells from two to five patients each. (h) Identification of RIPK1 and RIPK3 protein in cultured human GCs by western blotting. Arrows indicate the expected mass of protein (RIPK1: 76 kDa; RIPK3: 57 kDa; p-MLKL: 54 kDa). Serum control was negative. (i) Identification of MLKL and p-MLKL in cultured human GCs by western blotting. ARP treatment for 5 h increased the levels of p-MLKL compared with control group, which was treated with the control peptide (Scr). Arrows point to the expected mass of the proteins (MLKL 37 kDa; p-MLKL: 54 kDa). Different letters in a–g indicate statistically significant differences between the treatment groups
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fig5: Nec-1 and NSA block ARP-induced increase in cell death, while Z-VAD-FMK does not. (a) LDH cytotoxicity assay performed with cultured human GCs. ARP (50 ng/ml) significantly increases cytotoxicity compared with control groups (Scr 50 ng/ml; ARPin 50 ng/ml; P<0.05; analysis of variance (ANOVA)). (b) Z-VAD-FMK (ZVF; 20 μM) does not block ARP-regulated increase in cytotoxicity. Treatment with ZVF only significantly decreases cytotoxicity compared with control (DMSO 1‰ P<0.05; ANOVA). (c) No increased activity of caspase 3/7 was detected in ARP-treated cells compared with control groups (P<0.05; ANOVA). Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. (d) Nec-1 (20 μM) significantly blocks ARP-regulated increase in cytotoxitcity. Ethanol (EtOH; 0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (e) Treatment with Nec-1 causes significant lower cytotoxicity compared with control group (P<0.05, t-test). (f) NSA (0.5 μM) is able to block ARP-dependent increase in cytotoxicity (P<0.05; t-test). DMSO (0.1‰) has no effect on ARP-dependent increase in cytotoxicity. (g) Treatment with NSA causes significant lower cytotoxicity compared with control group (P<0.05, t-test). All values of LDH-assays are shown as mean±S.E.M. of n=10 (except Z-VAD-FMK stimulation, panel b, n=7) independent preparations of cells from two to five patients each. (h) Identification of RIPK1 and RIPK3 protein in cultured human GCs by western blotting. Arrows indicate the expected mass of protein (RIPK1: 76 kDa; RIPK3: 57 kDa; p-MLKL: 54 kDa). Serum control was negative. (i) Identification of MLKL and p-MLKL in cultured human GCs by western blotting. ARP treatment for 5 h increased the levels of p-MLKL compared with control group, which was treated with the control peptide (Scr). Arrows point to the expected mass of the proteins (MLKL 37 kDa; p-MLKL: 54 kDa). Different letters in a–g indicate statistically significant differences between the treatment groups
Mentions: In contrast to the AChE-S and AChE-E, the AChE-R is a soluble monomer and its specific C-terminal peptide ARP has been shown to possess additional non-enzymatic functions.41 To explore assumed non-enzymatic effects in human GCs, we used a synthetic ARP peptide (Figure 4). Live cell imaging performed over a 24-h time period revealed massive cell death events in the ARP-treated cells (50 ng/ml) compared with the untreated control group (Figure 4a; Supplementary Data). A scrambled control peptide (Scr; 50 ng/ml) and heat-inactivated ARP (ARPin; 50 ng/ml; 10 min, 95 °C) exhibited no bioactivity. Confluence measurements furthermore underpinned this observation (Figure 4b). Cell death events were first observed after approximately 2–3 h upon the addition of ARP and continued throughout a 24-h period. Many of the dying cells showed a characteristic morphology upon ARP treatment. It involved cytoplasmic ballooning (Figure 4c), which albeit at much lower frequency could be found in control cells as well. Lactate dehydrogenase (LDH) assays with 10 independent preparations of cultured GCs were performed to detect LDH release as measure for plasma membrane damage. The results showed a significant increase after 5 h, indicating cytotoxicity of ARP treatment compared with the control, Scr and ARPin (Figure 5a). The pan-caspase inhibitor Z-VAD-FMK (20 μM) did not prevent the ARP-dependent increase in cytotoxicity seen in LDH measurements. Addition of Z-VAD-FMK to GCs, however, blocked basally occurring cell death, presumably apoptosis, which was observed in the control group, in which the solvent dimethyl sulfoxide (DMSO; 1‰) was tested (Figure 5b; seven independent GC preparations). ARP stimulation did not change the activities of caspase 3/7 over control groups (Figure 5c; three independent GC preparations). The results indicate that the type of cell death that is involved is not typical caspase-dependent apoptosis. The RIPK1 inhibitor Nec-1 (20 μM) significantly blocked the ARP-dependent increase in LDH release when added to ARP-exposed GCs (Figure 5d; 10 independent GC preparations). This effect points to necroptosis as cause for the increased number of cell deaths by ARP stimulation. In the GC group treated with Nec-1 alone, a significantly reduced cytotoxicity became apparent, indicating a basal level of necroptosis in GCs (Figure 5e; 10 independent GC preparations). Addition of NSA (0.5 μM), a blocker of MLKL, effectively reduced necroptotic cell death, indicated by its ability to inhibit ARP-induced cytotoxicity (Figure 5f; 10 independent GC preparations). As in the case of Nec-1, NSA significantly reduced cytotoxicity in GCs compared with untreated control (Figure 5g), and this further indicates that necroptosis is a form of cell death in GCs. As Nec-1 is dissolved in ethanol and NSA in DMSO, we excluded non-specific possible cytotoxic actions of the corresponding solvents (ethanol (0.1‰) and DMSO (0.1‰)) using the LDH assay, (Figures 5d and f). Furthermore, RIPK1, RIPK3 and MLKL, key proteins in the necroptosis pathway, were identified in three GC preparations by using western blotting (Figures 5h and i; for RT-PCR data, see Supplementary Data). ARP, but not the control peptide, increased the levels of p-MLKL after 5 h, which corresponds to the time, when the cytotoxicity of ARP was confirmed by LDH measurements (Figure 5i). This experiment was repeated using three independent GC preparations (Supplementary Data).

Bottom Line: AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo.The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death.Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis.

View Article: PubMed Central - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig-Maximilian-University (LMU), Munich, Germany.

ABSTRACT
Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Show MeSH
Related in: MedlinePlus