Protective Effect Of Raloxifene On ALS Cell Model And Its Mechanism

Amyotrophic lateral sclerosis?ALS?is a devastating adult-onset neurodegenerative disease characterized by selective loss of both upper and lower motor neurons,resulting in generalized muscular atrophy,paralysis,and muscle weakness.The underlying mechanisms of ALS are not completely understood.In recent years,the 43-kDa TAR DNA-binding protein?TDP-43?has been identified as the major component of ubiquitin-positive cytoplasmic inclusions in the central nervous system?CNS?of TDP-43 proteinopathies,including ALS.Immunocytochemical studies have demonstrated that cytoplasmic inclusions in the CNS of ALS contain phosphorylated and ubiquitinated TDP-43 C-terminal fragments?CTFs?.Moreover,recent studies have shown that TDP-43 mutations account for 5%of familial ALS cases and up to 4%of sporadic cases.TDP-43 is believed to play an important role in the pathogenesis of ALS.TDP-25,the 25-kDa CTF of TDP-43,is cleaved by activated caspase-3.Interestingly,the C-terminal fragment-25?CTF25?of TDP-43 is prone to misfolding and thus to form aggregates,and the overexpression of TDP-25 induces mitochondrial autophagy and leads to ubiquitin-positive inclusions,similar to the mutant TDP-43 model.Previous studies have found that TDP-25 forms compact cytoplasmic inclusions and significantly enhances apoptosis.Furthermore,TDP-25-transgenic rats reportedly suffer from selective forelimb impairments similar to those observed in ALS due to increased levels of TDP-25.In our laboratory,we established a cellular model in which NSC-34 cells stably express the CTF25of TDP-43,which we term TDP-25 cells.The current therapeutic options for ALS are currently very limited given that the pathogenesis of ALS is unclear.ALS is usually fatal within 3 to 5years after diagnosis.Riluzole and edaravone have been approved for treatment of ALS in the United States and have shown a modest survival benefit.Therefore,the identification of effective new therapeutic agents for ALS is urgently needed.Epidemiological studies have revealed that pre-menopausal women are less susceptible to ALS and other neurodegenerative diseases than males or post-menopausal women.In addition,the lifespan of ovariectomized female mice is reportedly shorter than that of intact female mice in an ALS animal model.These data suggest that estrogen might play a neuroprotective role in ALS.The use of estrogen as a therapeutic agent for neurodegenerative diseases has been investigated over the last decade.E2?17?-estradiol?has been shown to be neuroprotective in a variety of neurodegenerative disorders,such as stroke,Parkinson's disease,and Alzheimer's disease.The effects of estrogen are primarily mediated by three different receptors.The two classical nuclear estrogen receptors,ER?and ER?,function traditionally as nuclear transcription factors,and the third is a membrane receptor termed GPR30 that was identified as a novel estrogen receptor that mediates a rapid,non-genomic response to estrogens.Studies have demonstrated that classic ERs play an indispensable role in estrogenic neuroprotection.However,GPR30 knockout attenuates estrogen-induced neuroprotection.Studies have shown that GPR30 plays a role in the nervous systems.The precise expression pattern of GPR30 remains controversial,and the estrogen signaling pathway through GPR30 has not been characterized in TDP-25 cells.Estrogen is also known to exert undesirable stimulatory effects on the breast and uterus,which might potentially increase the risk of developing breast and uterine cancers or stroke.The results from studies showing the positive effects of E2 have occasionally been overshadowed by its undesirable side-effects and poor compliance.These potential limitations have prompted interest in the development and therapeutic use of neuroprotective nonsteroidal selective estrogen receptor modulators?SERMs?as alternative treatments that mimic the beneficial effects of estrogen but minimize the adverse outcomes.Among these SERMS is raloxifene?Ral?,a widely used drug.Several in vitro and in vivo experimental studies have demonstrated that Ral has neuroprotective effects in various animal models of neurodegenerative disorders.Ral acts by binding the classical receptors ER?and ER?but is also reported to mediate an effect through GPR30.The neuroprotective effect of Ral has been suggested to be mediated through a cooperation between GPR30and ER?/ER?in certain tissues?e.g.,the brain,endocrine organs and various cancer cells?.Ral is a non-steroidal benzothiophene SERM approved by the FDA for the treatment and prevention of osteoporosis in menopausal women and invasive breast cancer due to its agonistic effect on bone and its antagonistic effect on breast tissue.Ral displays agonist and antagonist estrogenic responses depending on the target tissue.Specifically,it acts as an antagonist on estrogen receptors?ERs?in the breast and uterus and exerts estrogenic effects on bone,cholesterol metabolism,and the nervous system.Because Ral does not increase the risk of cancer in reproductive organs and has non-feminizing effects in men,Ral could be a potential alternative to E2for neuroprotection.Nonetheless,the protective efficacy of Ral on TDP-25cells has not been reported,and the neuroprotective effect of Ral in the context of ALS remains to be studied.Emerging evidence supports the hypothesis that the activation of autophagy might be protective in some neurodegenerative diseases by enhancing the removal of toxic protein aggregates.Autophagy has been shown to be involved in the degradation of CTF25 overexpression,and its activation is a potentially useful strategy for the treatment of neurodegenerative diseases associated with TDP-43 proteinopathies.Estrogen reportedly augments the level of autophagy in calcified arteries,and estrogen-induced autophagy plays a protective role in osteoblast survival.Previous data show that apoptosis might play a role in the pathomechanism of ALS and the degeneration of motor neurons in this disease,and neuronal apoptosis is associated with the onset and mortality of ALS.Cells pretreated with physiological E2 concentrations exhibit decreased death and reduced activation of the pro-apoptotic cascade.Therefore,we postulated that Ral,similar to estrogen,would enhance the protective autophagy pathway,suppress apoptosis and consequently limit motoneuron degeneration.In the present study,we evaluated the neuroprotective effects of Ral on TDP-25 cells compared E2.We also assessed the underlying mechanisms involved in Ral-and E2-induced neuroprotection via ER?/ER?or GPR30 signaling.Part One Expression of ER?/ER?and GPR30 in TDP-25 cellsObjective:To identify expression of ER?/ER?and GPR30 in TDP-25cells.Methods:In our laboratory,a new cellular model of ALS that stably expresses the CTF25 of TDP-43 has been established.These cells,termed TDP-25 cells.To investigate the localization of ER?,ER?and GPR30 in TDP-25 cells,we performed immunofluorescence staining and confocal microscopy analysis.Results:1.Immunofluorescence staining confirmed that ER?/ER?and GPR30were expressed in all cultured TDP-25 cells.2.ER?was extensively distributed throughout the cell and was significantly concentrated in the nuclear region.However,very little ER?expression was observed in the periphery of the cell.Compared with control E cells,TDP-25 cells showed a higher nucleus:cytoplasm ratio of ER?expression.3.In comparison,ER?expression was more extensively distributed throughout the cell,including in extrusions of the cellular membrane.No significant difference was observed between nuclear and cytoplasmic staining.The expression of ER?in TDP-25 cells showed no major differences from E cells.4.Notably,in TDP-25 cells,GPR30 showed a more restricted,punctate staining pattern in the nuclear and perinuclear areas,with rare staining in other parts of the cytoplasm.Additionally,no staining was observed on the borders of the cells.GPR30 expression in E cells showed a more restricted expression,except for the punctate staining pattern in perinuclear areas.Part Two E2 and Ral promote cell viability through ER?/ER?and GPR30Objective:To observe the effect of E2 and Ral on the proliferation of TDP-25 cells and its mechanism through ER?/ER?and GPR30.Methods:For experiments investigating the E2 and Ral responses,the cells were seeded into 6,24 or 96-well plates in DMEM medium with 10%FBS and allowed to attach overnight.The next day,the medium was replaced with serum-free DMEM for 24 h of hormone starvation.The cells were then exposed to DMEM medium supplemented with 10%v/v charcoal-stripped FBS.The different treatments were applied simultaneously,and the cells were incubated for 24 h.The presence of ER?,ER?and GPR30 in TDP-25 cells was observed by western blot analyses.To study the efficacy of E2 and Ral on TDP-25 cells proliferation,we examined cell viability using CCK-8 assays,determine the treatment concentration of E2 or Ral in the next experiment.E2?10^-77 M?or Ral?10^-77 M?were applied to TDP-25 cells to investigate their effects on the localization of ER?,ER?and GPR30 using immuno-fluorescence.To determine whether ER?/ER?or GPR30 signaling are required for E2 or ral-induced cell proliferation,we stimulated TDP-25 cell cultures with 10^-77 M E2 or ral with or without the specific antagonists G15?for GPR30?and ICI 182,780?for ER?/ER??or the agonist G-1?for GPR30?.Results:1.The protein levels of ER?and GPR30 in TDP-25 cells treated with several concentrations of E2(10^-9,10^-8 and 10^-7 M)were significantly higher than those of control TDP-25 cells without E2 treatment.Ral treatment at 10^-8M and 10^-77 M increased the ER?protein levels as well as GPR30 expression.The highest expression of these proteins was obtained with 10^-7 M Ral.However,in contrast to E2,ER?showed no apparent increase with any Ral concentration.2.The results showed that TDP-25 cell viability was significantly stimulated by E2 or Ral at a dose of 10^-77 M.3.In the control group,GPR30 protein failed to show a punctate staining pattern after sequential starvation?serum-free DMEM?and estrogen deprivation?charcoal-stripped DMEM?for 24 h.The GPR30 staining appeared light and showed restricted distributions in the nuclear and perinuclear areas,with no detected expression in other parts of the cytoplasm.Moreover,no expression was observed on the cell border.Following treatment with 10^-77 M E2 for 24 h,GPR30 expression showed an apparent increase in the cytoplasm compared with the controls,with a higher cytoplasm:nucleus expression ratio compared with control cells.Notably,after treatment with10^-77 M Ral,GPR30 expression showed further increases in the cytoplasm.As observed by immunofluorescence,there were no significant changes in ER?and ER?localization after E2?10^-77 M?or Ral?10^-77 M?treatment.4.Ral and E2 enhanced TDP-25 cell proliferation at a dose of 10^-77 M.The addition of either G15 or ICI182,780 completely inhibited E2-stimulated TDP-25 cell viability.For Ral,the elevated TDP-25 cell viability was completely blocked by G15 but not ICI 182,780.TDP-25 cell viability increased after 24 h of exposure to G-1 compared with the control and G15groups.Part Three Ral and E2 enhance autophagy and suppress apoptosis through ER?/ER?and GPR30.Objective:To investigate the effect of Ral and E2 on autophagy and apoptosis through ER and GPR30.Methods:TDP-25 cells were seeded into 6-well plates in DMEM medium with 10%FBS and allowed to attach overnight.The next day,the medium was replaced with serum-free DMEM for 24 h of hormone starvation.The cells were then exposed to DMEM medium supplemented with 10%v/v charcoal-stripped FBS.E2(10^-7M)or Ral(10^-7M)were applied simultaneously,and the cells were incubated for 24 h.To investigate changes in autophagic activity,we performed western blot analyses to determine the expression levels of the autophagic markers LC3-II and P62 in TDP-25 cells.We examined the expression levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins Bax and cleaved Caspase-9 by western blot.To determine whether ER?/ER?or GPR30 signaling are required for effects of E2 and Ral on autophagy or apoptosis,we stimulated TDP-25 cell cultures with E2(10^-7 M)or Ral(10^-7 M)with or without the specific antagonists G15?for GPR30?and ICI 182,780?for ER?/ER??or the agonist G-1?for GPR30?.Results:1.The p62 levels decreased after treatment with E2,and this effect was blocked by ICI 182,780 and G15.The expression of LC3-II increased in the E2 group,and this change was blocked by ICI 182,780 but not G15 treatment.2.Ral treatment significantly decreased both the p62 and LC3-II levels,and this decrease in the p62 levels was reversed by ICI 182,780 and G15treatment.The decrease in LC3-II was reversed by ICI 182,780 and attenuated by G15,although this latter difference was not significant.3.The protein levels of Bcl-2,Bax and cleaved Caspase-9 following treatment with E2 were examined by immunoblotting.A significant increase in Bcl-2 and a decrease in Bax and Caspase-9 were observed,and these effects were reversed by ICI 182,780 treatment.The decrease in cleaved Caspase-9expression was also reversed by G15.In addition,G-1 decreased the expression of cleaved Caspase-9 compared with that found in the E2+G15 and G15 groups.The changes in protein levels of Bcl-2 and Bax were not reversed by G15 or affected by G-1.4.Ral treatment significantly decreased the cleaved Caspase-9 and Bax levels,whereas the protein levels of Bcl-2 showed no significant difference between the Ral-treated and control groups.The decrease in cleaved Caspase-9 levels was reversed by ICI 182,780 and G15 treatment.Moreover,significant decreases in the Bax levels were observed following Ral treatment,and these effects were reversed by ICI 182,780 but not G15.In addition,G-1treatment did not significantly decrease the Caspase-9 or Bax levels.Conclusion:Overall,our results show that Ral,similar to E2,provides neuroprotection in TDP-25 cells through ER?/ER?and GPR30.These effects are mediated by increasing autophagy,decreasing apoptosis and enhancing cell viability.Ral mimics the beneficial effects of estrogen but minimizes the adverse outcomes and might thus be a promising therapeutic strategy for ALS.This study highlights a new mechanism of Ral neuroprotection for ALS and provides support for the consideration of ER?/ER?and GPR30 in the development of therapeutic neuroprotective agents.