| Alzheimer’s disease(AD) is the most common type of age-associated dementia. The pathology of the disease is characterized by senile plaques,intracellular neurofibrillary tangles, and extensive neuronal loss. Beta-amyloid peptide(Aβ) is the major component of senile plaques, which is considered extremely critical for AD development. Although the mechanisms underlying Aβ-induced neurotoxicity are not fully understood, alteration in mitochondrial homeostasis has been reported to play a central role in Aβ-induced cell apoptosis. It is likely that mitochondrial abnormalities may be a common pathway leading to neuronal dysfunction in many neurodegenerative diseases,including AD. There is accumulating evidence that Aβ alters the expression of genes from Bcl-2 family, which are considered to be critical for the regulation of mitochondrial apoptotic pathways. It has been suggested that a high Bax to Bcl-2 ratio can cause mitochondrial membrane potential(MMP) collapse,release of cytochrome c, and subsequent apoptosis. Jun N-terminal kinase(JNK) is a putative upstream signaling component of mitochondrial apoptotic pathway. Interestingly, JNK activation is observed in Aβ-induced neurons, and its inhibition remarkably attenuates Aβ toxicity. So, there is a possibility that inhibiting mitochondrial-related apoptotic pathway or regulating its upstream signaling(e.g. JNK signaling) may attenuate Aβ-induced neurotoxicity.Steroid hormones and their metabolites within the central nervous system are commonly defined as neuroactive steroids or neurosteroids.Neuroprotective properties of neurosteroids have been reported in numerous studies. Progesterone(PROG), beyond helping maintaining pregnancy, has also been a neuroactive steroid. Over the past two decades, the neuroprotective effects of progesterone have been documented in experimental models of neurodegeneration, spinal cord injury, brain ischemia,stroke and traumatic brain injury(TBI). Based on its safety and effectiveness demonstrated in a variety of TBI models, progesterone has been tested in human clinical trials for traumatic brain injury. Evidence suggests it exerts its neuroprotective effects through several pathways, including improving neuronal survival, reducing swelling, and inhibiting apoptosis and inflammatory reaction. Some study show that progesterone play a role mainly through its metabolite allopregnanolone in nervous system.The exact mechanism by which progesterone exerts protective effects in the central nervous system remains unclear. Like most steroids, progesterone exerts its effects by binding to specific cellular receptors. It is shown that classic progesterone receptors(classic PR), which are widely expressed in the central nervous system, have extensive physiological functions. Recently,there is growing evidence that progesterone achieves neuroprotective actions by non-classic progesterone receptors mechanisms. Progesterone receptor membrane component 1, a prominent candidate mediating the nongenomic signaling events of progesterone, was suggested to be involved in antiapoptotic action.Part I The protection of progesterone in Aβ induced neuron injuryObjective: To observe the protective effect of progesterone in Aβinduced primary cultured rat cortical neuron injury and investigate whether the protective effects of progesterone was mediated by its metabolites allopregnanolone.Methods: issociated cortical neurons were prepared from postnatal day0(P0) Sprague Dawley rat, and determined the neurons purity withβⅢ-Tubulin(TUJ1) antibody and Hoechst 33258 stain. AD cell model was established based the Aβ25-35 induced primary cultured rat cortical neuron injury. The viability of neurons was determined by MTT assay and the apoptosis was indentified by Hoechest 33258 dying after the neurons treated with different concentration of progesterone, allopregnanolone or finasteride.Results:1 Neuronal cultures are more than 90% pure.2 The effect of progesterone on neuronal viability after Aβ25–35 treatment MTT assays revealed that neurons exposed to 25 μM of Aβ35–25(the control reverse sequence of the peptide Aβ25-35), showed no difference in cell viability with control cultures. However, similar concentration of Aβ25–35exhibited a significant decrease in cell viability(59.93 ± 5.97) %(P<0.01).Co-incubation with progesterone significantly improved neuronal survival in a concentration-dependent and time-dependent manner. 1 μM progesterone for48 h yielded maximal rescue(86.71 ± 3.24) %(P<0.01).3 The effect of progesterone on neuronal apoptosis after Aβ25–35treatmentApoptotic cells were characterized by the fragmented or intensely stained nuclei. Only a few apoptotic cells were observed in the control group(11.7 ±1.96) %; Compared with control group, apoptotic rate did not show significant difference with the addition of progesterone under basal condition(10.27 ±2.27) %. The amount of apoptotic cells markedly increased after exposure to25 μM Aβ25–35 for 48 h(49.56 ± 7.29) %. Treatment with progesterone attenuated this increase(26.82 ± 2.15) %.4 The effect of adding finasteride on the neuroprotective role of progesteroneMTT assay show that, compared with control group, cell viability did not show significant difference with the addition of finasteride under Aβ25–35incubation, indicating that pretreating with finasteride did not affect the neuroprotective role of progesterone.5 The effect of allopregnanolone on neuronal viability after Aβ25–35treatmentCompared with control group, neurons exposed to 25 μM of Aβ25–35exhibited a significant decrease in cell viability(57.9±4.8) %; Compared with Aβ group, at the concentration from 0.001 μM to 1 μM, cell viability did not show significant difference with the addition of allopregnanolone(P>0.05);on the contrary, at the concentration of 10 μM, allopregnanolone decrease the cell viability.Conclusions:1 Progesterone, which can increase cell viability and decrease apoptosis rate, protects against Aβ-induced neuronal toxicity in a concentration-dependent and time-dependent manner.2 Progesterone protects against Aβ-induced neuronal toxicity in vitro not through its metabolites allopregnanolone.Part II Progesterone treatment inhibits mitochondrial apoptotic pathwayObjective: To observe the protective effect of progesterone on neuronal mitochondrial apoptotic pathway under Aβ incubation and explore the potential molecular mechanism.Methods: Under Aβ25–35 induced AD cell model, the mitochondrial membrane potential was detected by using the fluorescent dye rhodamine 123 after progesterone incubation; we examined the expression of Bcl-2, Bax and the active form of caspase-3 by Western blotting.Results:1 The effect of progesterone on neuronal mitochondrial membrane potential after Aβ25–35 treatmentUnder a fluorescence microscope, after being exposed to 25 μM Aβ25–35for 48 h, the cells revealed a significant decrease of MMP to 65% of the control level(P<0.01). The co-incubation of Aβ25–35-insulted cells with progesterone resulted in the preservation of the mitochondrial membrane potential, with significant differences(about 80% of control group), relatively to Aβ25–35-only exposed cells.2 The effect of progesterone on expression level of Bax/Bcl-2 under Aβ25–35 induced AD neuronal model.Western blots results show that Bax/Bcl-2 proteins ratio under Aβchallenge was about 8 times, with significant differences(P<0.01), relatively to normal neuron cells. Compared with Aβ modle group, progesterone attenuates about 50% of Aβ-induced Bax/Bcl-2 proteins ratio(P<0.01).Compared with control group, Bax/Bcl-2 proteins ratio did not showsignificant difference with the addition of progesterone under basal condition(P<0.01).3 The effect of progesterone on expression level of cleaved-caspase 3under Aβ25–35 induced AD neuronal model.Western blots results show that cleaved-caspase 3 expression level under Aβ challenge was about 5 times, with significant differences(P<0.01),relatively to normal neuron cells. Compared with Aβ modle group,progesterone attenuates about 45% of Aβ-induced cleaved-caspase 3expression level(P<0.01). Compared with control group, cleaved-caspase 3expression level did not show significant difference with the addition of progesterone under basal condition(P<0.01).Conclusions:Progesterone reversed the Aβ-induced alternations of Bax/Bcl-2 proteins ratio and inhibited the production of active caspase-3 induced by Aβ, rescuing mitochondrial membrane potential. These indicate that progesterone attenuates Aβ25–35-induced neuronal toxicity by inhibiting mitochondria-associated apoptotic pathway.Part Ⅲ The protective effect of progesterone was partially through PGRMC1Objective: To explore whether progesterone exhibits neuroprotective effects against Aβ25–35-induced neuronal toxicity through classic PR or PGRMC1.Methods: We used Westem blot to examine the PGRMC1 and classic PR expression in neurons exposed to Aβ25–35. Then We used MTT, Hoechest33258 to detect the affection of RU486 and AG205 on progesterone protection under Aβ25–35 induced AD cell model. In the end, the Westem blot was carried out to explore whether the mitochondrial protection of progesterone is mediated by classic PR or PGRMC1 after Aβ treatment.Results:1 PGRMC1 and classical PR were confirmed expressed in primary rat cerebral cortical neurons using immunocytochemical stainingResults show that Pgrmc1 are rich in in neuron body and Synaptic processes, few expressed in nucleus. While classic PR mainly expressed in nucleus, few in cytoplasm.2 Western blots results show that the effect of Aβ on PGRMC1 and classic PR expression in primary rat cortical neurons.Western blots results show that neurons treated by Aβ have a modest PGRMC1 expression upregulation than control culture with the vehicles(P<0.05). However, compared with control neurons, classic PR expression in Aβ treated group had no significant increase.3 Western blots results show that the effect RU486 or AG205 on progesterone protection against Aβ-induced neurotoxicity.Neuron cultures were pretreated with AG205 or RU486 for 60 min followed by exposure to 25 μM Aβ25–35 with or without 1 μM progesterone for48 h. The addition of RU486 did not affect the neuroprotective role of progesterone. However, at concentration of 5 μM, AG205 incompletely blocked progesterone-mediated neuroprotection against Aβ toxicity. Hoechst33258 staining results also show that 5 μM AG205 partly inhibited neuroprotective effect of progesterone under Aβ incubation.4 Involvement of PGRMC1 in progesterone inhibiting Aβ-induced mitochondrial apoptotic pathwayPtreating with AG205 significantly weakened the protective effects of progesterone against Aβ25–35-induced MMP dissipation. To gain insights into the PGRMC1-regulated signaling pathway, the mitochondrial dependent apoptosis-related proteins and caspase cascade were investigated. The progesterone inhibitory effects on proteins ratio of Bax/Bcl-2 and increased actived caspase-3 were partly blocked by AG205. These data showed that progesterone protective effect against Aβ25–35-induced cells injury might involve inhibition of mitochondrial apoptotic pathway in a PGRMC1-dependent manner.Conclusions:1 Aβ induces PGRMC1 expression upregulation but not classic PR inprimary rat cortical neurons2 Progesterone protective effect against Aβ25-35-induced cells injury involve inhibition of mitochondrial apoptotic pathway partly though PGRMC1.Part Ⅳ Progesterone exerts neuroprotection by suppressing JNK activationObjective: The contribution of JNK signaling to the neuroprotective effect of progesterone was investigated.Methods: Western blot was used to detect JNK signaling after Neurons exposed to Aβ were pretreated with JNK inhibitor SP600125 or treated with progesterone for 48 h, alone or in combination. MTT and Hoechst 33258 staining was used to detect cell viability and apoptosis rate after Neurons exposed to Aβ were pretreated with JNK inhibitor SP600125 or treated with progesterone for 48 h, alone or in combination. Western blot was used to detect JNK signaling after Neurons exposed to Aβ were pretreated with AG205 or treated with progesterone for 48 h, alone or in combination.Results:1 Progesterone can functionally mimick SP600125 to partly inhibit Aβ-induced phosphorylation of the JNK Western blot results confirmed that the JNK inhibitor SP600125 can block Aβ-induced JNK phosphorylation. Progesterone can functionally mimick SP600125 to partly inhibit Aβ-induced phosphorylation of the JNK.2 Progesterone can functionally mimick SP600125 to partly inhibit Aβ-induced neuronal toxicity MTT and Hoechst 33258 staining results showed that pretreatment with SP600125 partly prevented Aβ-induced neuronal toxicity, indicating that inhibition of JNK signaling provides protection against Aβ25–35-induced neurotocity.3 Progesterone inhibited Aβ-induced phosphorylation of the JNK independent of PGRMC1Although progesterone neuroprotection against Aβ toxicity dependent in part on inhibiting JNK activation, pretreatment of cells with AG205 showed no significant effects on the progesterone inhibition of Aβ-induced p-JNK expression. These data indicated that progesterone inhibited Aβ-induced phosphorylation of the JNK independent of PGRMC1.Conclusions:1 Progesterone can functionally mimick SP600125 to partly inhibit Aβ-induced phosphorylation of the JNK.2 Progesterone can functionally mimick SP600125 to partly inhibit Aβ-induced neuronal toxicity.3 Progesterone inhibited Aβ-induced phosphorylation of the JNK independent of PGRMC1.In conclusion, the present studies use Aβ25-35 treated cortical neurons as a cellular model of AD. We examined the effects of progesterone on Aβ25-35-induced neurotoxicity and the underlying mechanisms by MTT,Hoechst33258 stain, immunocytochemistry and Western blot analysis respectively. Using cellular model, we found that progesterone significantly improved neuronal survival in a concentration-dependent and time-dependent manner. 1μM progesterone for 48 h yielded maximal rescue. Thus, these concentration and time were used in subsequent mechanism study. Firstly results showed that progesterone exerts neuroprotection not by its metabolites allopregnanolone. Furthermore, we observed that progesterone alleviated mitochondrial dysfunction by rescuing mitochondrial membrane potential under Aβ challenge. Moreover, progesterone could also attenuate Bax/Bcl-2proteins ratio upregulation and inhibit the activation of caspase-3 in Aβ-treated neurons. These indicate that progesterone attenuates Aβ25–35-induced neuronal toxicity by inhibiting mitochondria-associated apoptotic pathway. Then we further found that the protective effect of progesterone was partially abolished by PGRMC1 inhibitor AG205 rather than classic PR antagonist RU486 in this study. Additionally, progesterone protected neurons by inhibiting Aβ-induced activation of JNK, which was anupstream signaling component in Aβ-induced mitochondria-associated apoptotic pathway. But this process was independent of PGRMC1. Taken together, these results suggest that progesterone exerts a protective effect against Aβ25–35-induced insults at least in part by two complementary pathways:(1) progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway, and(2) blocking Aβ-induced JNK activation. The present study provides new insights into the mechanism by which progesterone brings neuroprotection. |
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