Long-term incubation with mifepristone (MLTI) increases the spine density in developing Purkinje cells: new insights into progesterone receptor mechanisms

Abstract

Cerebellar Purkinje cells (PC) physiologically reveal an age-dependent expression of progesterone with high endogenous concentrations during the neonatal period. Even if progesterone has been previously shown to induce spinogenesis, dendritogenesis and synaptogenesis in immature PC, data about the effects of progesterone on mature PC are missing, even though they could be of significant therapeutic interest. The current study demonstrates for the first time a progesterone effect, depending on the developmental age of PC. Comparable with the physiological course of the progesterone concentration, experimental treatment with progesterone for 24 h achieves the highest effects on the dendritic tree during the early neonate, inducing an highly significant increase in dendritic length, spine number and spine area, while spine density in mature PC could not be further stimulated by progesterone incubation. Observed progesterone effects are certainly mediated by classical progesterone receptors, as spine area and number were comparable to controls when progesterone incubation was combined with mifepristone (incubation for 24 h), an antagonist of progesterone receptors A and B (PR-A/PR-B). In contrast, an increase in the spine number and area of both immature and mature PC was detected when slice cultures were incubated with mifepristone for more than 72 h (mifepristone long-time incubation, MLTI). By including time-lapse microscopy, electron microscopic techniques, PCR, western blot, and MALDI IMS receptor analysis, as well as specific antagonists like trilostane and AG 205, we were able to detect the underlying mechanism of this diverging mifepristone effect. Thus, our results provide new insights into the function and signaling mechanisms of the recently described progesterone receptor membrane component 1 (PGRMC1) in PC. It is highly suitable that progesterone does not just induce effects by the well-known genomic mechanisms of the classical progesterone receptors but also acts through PGRMC1 mediated non-genomic mechanisms. Thus, our results provide first proofs for a previously discussed progesterone-dependent induction of neurosteroidogenesis in PC by interaction with PGRMC1. But while genomic progesterone effects mediated through classical PR-A and PR-B seem to be restricted to the neonatal period of PC, PGRMC1 also transmits signals by non-genomic mechanisms like regulation of the neurosteroidogenesis in mature PC. Thus, PGRMC1 might be an interesting target for future clinical studies and therapeutic interventions.

New Aspects of Progesterone Interactions with the Actin Cytoskeleton and Neurosteroidogenesis in the Cerebellum and the Neuronal Growth Cone

“…Its exact mechanism of action is still unclear, but various studies have indicated an interaction with steroidogenesis (Hughes et al 2007;Brinton et al 2008;Rohe et al 2009). Indeed, current results strongly suggest that PGRMC1 directly interacts with the production of neurosteroids in Purkinje cells (Wessel et al 2014). Other research in different neuronal tissues revealed that increased PRGMC1 activation in response to progesterone leads to an increase in receptor expression.…”

“…Progesterone-induced up-regulation of PGRMC1 mRNA was observed after a spinal cord injury in rats (Labombarda et al 2003), whereas PGRMC1 expression was significantly lowered in the hypothalamic tissue of mice harboring a double knockout of progesterone receptor A and B (Krebs et al 2000). In the hippocampus, an increase in PGRMC1 was induced by cyclic progesterone exposure (Zhao et al 2012), and we, too, described a similar progesterone-dependent up-regulation of PGRMC1 in Purkinje cells (Wessel et al 2014). Not only did we observe an increase in PRGMC1 protein but also an increase in dendritic spine density when classical progesterone receptors were antagonized by mifepristone for more than 72 hours (mifepristone long-time incubation, MLTI) (Wessel et al 2014).…”

“…In the hippocampus, an increase in PGRMC1 was induced by cyclic progesterone exposure (Zhao et al 2012), and we, too, described a similar progesterone-dependent up-regulation of PGRMC1 in Purkinje cells (Wessel et al 2014). Not only did we observe an increase in PRGMC1 protein but also an increase in dendritic spine density when classical progesterone receptors were antagonized by mifepristone for more than 72 hours (mifepristone long-time incubation, MLTI) (Wessel et al 2014). Even though the accompanied increase in the amount of PGRMC1 strongly indicates that the increase in dendritic spine density after MLTI is mediated by endogenous progesterone via PGRMC1, it is important to note that mifepristone itself can have a protective effect on different neuronal cells in experimental studies (Behl et al 1997;McCullers et al 2002;Ghoumari et al 2003;Rakotomamonjy et al 2011).…”

“…Thus, a recent investigation of changes in the dendritic spine density in neonatal [7 days in vitro (div)], juvenile (15 div) and matured (30 div) Purkinje cells after incubation with progesterone confirm that progesterone indeed induces an increase in the number of dendritic spines in immature and young Purkinje cells. But, interestingly, these new data strongly indicate that, in mature Purkinje cells, the effects of progesterone are no longer detectable (Wessel et al 2014). Furthermore, incubation with mifepristone or mifepristone plus progesterone failed to show measurable effects on dendritic length or dendritic spine density in mature Purkinje cells (Wessel et al 2014).…”

“…Nevertheless, none of these studies have demonstrated an influence of mifepristone on Purkinje cell morphology and dendritic spine density; but they have demonstrated an increase in primary Purkinje cell survival during the critical postnatal week (Ghoumari et al 2003(Ghoumari et al , 2006Rakotomamonjy et al 2011). In contrast, MLTI was shown to increase the dendritic spine density and area without influencing Purkinje cell survival (Wessel et al 2014); the slice cultures used were from 10-day pups in order to avoid apoptosis in cerebellar slice cultures during the first postnatal days. Given that (i) mifepristone incubation started after the critical period of apoptosis; (ii) the PGRMC1 amount was shown to increase under MLTI; (iii) Purkinje cell morphology was affected; and (iv) these effects were absent when PGRMC1 was blocked, it is well-justified to consider that endogenous progesterone and PGRMC1 are critical for the increase in dendritic spine density and area induced by MLTI.…”