Limits...
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.

Show MeSH

Related in: MedlinePlus

p-MLKL in primate ovarian tissue. (a and b) Cells in human corpus luteum (CL) are positive for p-MLKL. (c) IgG control lacks staining. (d) GCs in antral follicles of rhesus monkeys show positive staining for p-MLKL. (e) IgG control lacks staining. Bars indicate 100 μm (a) and 40 μm (b–e)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4385929&req=5

fig6: p-MLKL in primate ovarian tissue. (a and b) Cells in human corpus luteum (CL) are positive for p-MLKL. (c) IgG control lacks staining. (d) GCs in antral follicles of rhesus monkeys show positive staining for p-MLKL. (e) IgG control lacks staining. Bars indicate 100 μm (a) and 40 μm (b–e)

Mentions: We used a monoclonal antibody, which was recently described11 and allows in human tissue immunohistochemical identification of p-MLKL, that is, a marker for necroptosis. The human corpus luteum showed specific staining for p-MLKL (Figures 6a and b), while the immunoglobulin G (IgG) control was negative (Figure 6c). In rhesus monkey follicles, the GCs were immunoreactive for p-MLKL (Figure 6d). The IgG control was devoid of staining (Figure 6e).


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)

p-MLKL in primate ovarian tissue. (a and b) Cells in human corpus luteum (CL) are positive for p-MLKL. (c) IgG control lacks staining. (d) GCs in antral follicles of rhesus monkeys show positive staining for p-MLKL. (e) IgG control lacks staining. Bars indicate 100 μm (a) and 40 μm (b–e)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385929&req=5

fig6: p-MLKL in primate ovarian tissue. (a and b) Cells in human corpus luteum (CL) are positive for p-MLKL. (c) IgG control lacks staining. (d) GCs in antral follicles of rhesus monkeys show positive staining for p-MLKL. (e) IgG control lacks staining. Bars indicate 100 μm (a) and 40 μm (b–e)
Mentions: We used a monoclonal antibody, which was recently described11 and allows in human tissue immunohistochemical identification of p-MLKL, that is, a marker for necroptosis. The human corpus luteum showed specific staining for p-MLKL (Figures 6a and b), while the immunoglobulin G (IgG) control was negative (Figure 6c). In rhesus monkey follicles, the GCs were immunoreactive for p-MLKL (Figure 6d). The IgG control was devoid of staining (Figure 6e).

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