Inhibition of abnormal C/EBPβ/α‐Syn signaling pathway through activation of Nrf2 ameliorates Parkinson's disease‐like pathology

Abstract Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs) in the brain. These LBs are primarily composed of α‐Synuclein (α‐Syn), which has aggregated. A recent report proposes that CCAAT/enhancer‐binding proteins β (C/EBPβ) may act as an age‐dependent transcription factor for α‐Syn, thereby initiating PD pathologies by regulating its transcription. Potential therapeutic approaches to address PD could involve targeting the regulation of α‐Syn by C/EBPβ. This study has revealed that Nrf2, also known as nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2), suppresses the transcription of C/EBPβ in SH‐SY5Y cells when treated with MPP+. To activate Nrf2, sulforaphane, an Nrf2 activator, was administered. Additionally, C/EBPβ was silenced using C/EBPβ‐DNA/RNA heteroduplex oligonucleotide (HDO). Both approaches successfully reduced abnormal α‐Syn expression in primary neurons treated with MPP+. Furthermore, sustained activation of Nrf2 via its activator or inhibition of C/EBPβ using C/EBPβ‐HDO resulted in a reduction of aberrant α‐Syn expression, thus leading to an improvement in the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) in mouse models induced by 1‐methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP) and those treated with preformed fibrils (PFFs). The data presented in this study illustrate that the activation of Nrf2 may provide a potential therapeutic strategy for PD by inhibiting the abnormal C/EBPβ/α‐Syn signaling pathway.


| INTRODUC TI ON
Neurodegeneration in Parkinson's disease (PD) is marked by the progressive deterioration of dopaminergic neurons in the substantia nigra pars compacta (SNc).This results in motor symptoms as a consequence of the simultaneous loss of nigrostriatal dopaminergic termini.PD is the second most prevalent neurodegenerative disorder that occurs with age (Kang, Zhang, Liu, Manfredsson, He, et al., 2017).The etiology of PD is presently unclear.However, it is noteworthy that both sporadic and familial PD share identical pathological characteristics.Specifically, degeneration of dopaminergic neurons occurs in the SNc, with the remaining dopaminergic neurons containing intraneuronal proteinaceous cytoplasmic inclusions referred to as Lewy bodies (LBs).These pathological features are commonly observed in both sporadic and familial cases of PD (Dauer & Przedborski, 2003;Yang et al., 2020).The main component of LBs is alpha-synuclein (α-Syn), and the aggregation of α-Syn is considered a critical factor in the development of PD (Volpicelli-Daley et al., 2011).Phosphorylation of the α-Syn protein at the serine 129 site is a critically important post-translational modification that enhances the generation of pathogenic α-Syn aggregates (Zhang et al., 2019).The pathogenesis of both sporadic and familial PD has been identified as the mutation or multiplication of α-Syn (Ellis et al., 2001;Jiang et al., 2013;Okochi et al., 2000).Mutations in the α-Syn (SNCA) gene, including A53T, A30P, E46K, H50Q, Q51D, and A53E, lead to autosomal-dominant PD (Toffoli et al., 2020).Soluble monomers, toxic oligomers, and insoluble fibrils of α-Syn have been observed in the brains of patients diagnosed with PD (Lashuel et al., 2013).These findings suggest that the abnormal accumulation of α-Syn contributes to the accelerated death of dopaminergic neurons in both familial and sporadic cases of PD.
CCAAT/enhancer-binding proteins (C/EBP) belong to the class of transcription factors known as basic-leucine zipper (bZIP).The family consists of six members, with C/EBPβ being particularly abundant in the brain.In this context, it plays a crucial role in memory formation and synaptic plasticity (Pulido-Salgado et al., 2015).C/EBPβ can be activated by pro-inflammatory cytokines such as IL-1β, IL-6, and TNFα.It regulates the pro-inflammatory program in glial cells, playing a crucial role in inducing neurotoxic effects through microglia activation (Magalini et al., 1995;Poli, 1998;Pulido-Salgado et al., 2015;Straccia et al., 2011;Wedel & Ziegler-Heitbrock, 1995).
It has been reported that the expression of C/EBPβ in human neuroblastoma cells increases with dopamine treatment in a dosedependent manner.This increase in expression is correlated with the sequential escalation of α-Syn, indicating that C/EBPβ may be involved in the development of PD (Gomez-Santos et al., 2005).
Recent studies have confirmed the link between C/EBPβ and the onset of neurodegenerative disorders.C/EBPβ acts as a key transcription factor for δ-secretase and regulates its mRNA levels in both aging and Alzheimer's disease (AD) brains (Wang et al., 2018).
Moreover, C/EBPβ serves as the primary transcription factor for α-Syn and regulates their mRNA expressions during aging and in the brains of individuals affected by PD (Wu et al., 2021).As a result, inhibiting abnormal C/EBPβ expression could be a potential therapeutic approach to counteract the advancement of PD.
Nrf2 (nuclear factor (erythroid-derived 2)-like 2; NFE2L2) is a transcription factor that plays a critical role in mediating the expression of Phase II detoxification and antioxidant genes.Upon activation, Nrf2 disassociates from Keap1 and translocates to the nucleus where it dimerizes with other bZIP proteins, such as small Maf proteins.This process forms a transactivation complex that binds to antioxidant response elements (AREs), enabling the transactivation of target genes involved in cellular defense against oxidative stress (Kobayashi et al., 2013;Kometsi et al., 2020;Ma, 2013;Suzuki et al., 2013;Suzuki & Yamamoto, 2015;Yamamoto et al., 2018).Sulforaphane (SFN), an isothiocyanate compound naturally occurring in broccoli, has been discovered as a potent activator of the Nrf2 signaling pathway.Activation of this pathway by SFN results in enhanced antioxidant and anti-inflammatory effects (Cheung & Kong, 2010;Fahey et al., 1997;Lin et al., 2008;Zhang et al., 1994).A recent study has demonstrated the critical role of C/EBPβ as a transcription factor for α-Syn, highlighting its involvement in regulating α-Syn expression in both aging and PD brains (Wu et al., 2021).In this study, our objective was to examine the potential of Nrf2 activator SFN and Nrf2 overexpression in inhibiting the transcription of the C/EBPβ gene in SH-SY5Y cells treated with MPP + .Initially, we utilized quantitative PCR (qPCR) and western blot assays to assess the impact of MPP + on mRNA and protein expression of C/EBPβ in SH-SY5Y cells.The results revealed that MPP + significantly induced an elevation in both C/EBPβ mRNA and protein levels.However, this effect was effectively abrogated by treatment with SFN and overexpression of Nrf2 (Figure 1a,b).Secondly, it is of interest to investigate the expression of pro-inflammatory cytokines IL-1β and IL-6 in MPP + -treated SH-SY5Y cells, as these cytokines are known to activate C/EBPβ and induce neurotoxic effects (Magalini et al., 1995;Poli, 1998;Pulido-Salgado et al., 2015;Straccia et al., 2011;Wedel & Ziegler-Heitbrock, 1995).Enzyme-linked immunosorbent assay (ELISA) revealed that the levels of IL-1β and IL-6 were increased in SH-SY5Y cells treated with MPP + , but were subsequently reduced following treatment with SFN and Nrf2 overexpression (Figure 1c,d).

Since
Finally, to further clarify the impact of SFN and Nrf2 overexpression on inhibiting the expression of C/EBPβ, we conducted luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay.The luciferase reporter assays revealed that MPP + activated the C/EBPβ promoter in SH-SY5Y cells; however, this activation was inhibited by SFN and Nrf2 overexpression (Figure 1e).2a).However, the administration of SFN reversed these effects (Figure 2a).Immunofluorescence staining further demonstrated that MPP + caused a redistribution of both C/ EBPβ and α-Syn within primary neurons, resulting in elevated immunoreactivity (Figure 2b).Again, SFN treatment effectively reversed this effect (Figure 2b).Additionally, nuclear/cytosol fractionation immunoblot analysis indicated that C/EBPβ was predominantly expressed in the nuclei of primary neurons.MPP + treatment increased C/EBPβ expression, which was mitigated by SFN (Figure S1).Subsequently, we developed C/EBPβ-HDO, a gene silencing technology designed to suppress the expression of C/EBPβ (Asada et al., 2021;Nishina et al., 2015;Yoshioka et al., 2019).The effectiveness of C/EBPβ-HDO in suppressing C/EBPβ mRNA and protein levels in SH-SY5Y cells was confirmed through qPCR and western blot experiments (Figure S2A,B).These results indicate that C/ EBPβ-HDO holds promise as a tool for inhibiting C/EBPβ expression.
To further explore the potential of C/EBPβ-HDO, we investigated whether inhibiting C/EBPβ expression could alleviate abnormal α-Syn expression in primary neurons treated with MPP + .Western blot analysis revealed a significant increase in the expression of C/EBPβ and α-Syn in MPP + -treated primary neurons (Figure 2c).However, this effect was reversed with the use of C/EBPβ-HDO (Figure 2c).
Immunofluorescence staining also showed an increase in the immunoreactivity of C/EBPβ and α-Syn in MPP + -treated primary neurons, which was again attenuated by C/EBPβ-HDO (Figure 2d).Furthermore, nuclear/cytosol fractionation immunoblot analysis F I G U R E 2 Treating primary neurons with SFN to activate Nrf2 or using C/EBPβ-HDO to silence C/EBPβ expression can effectively mitigate the upregulation of abnormal α-Syn expression triggered by MPP + .(a) SFN significantly prevents the abnormalities caused by MPP + in primary neurons, including the downregulation of Nrf2 and the upregulation of C/EBPβ, p-α-Syn, and α-Syn expression levels.This attenuation was confirmed using western blot analysis (mean ± SEM, n = 6 per group, one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001).(b) Immunofluorescence staining analysis shows that treatment with SFN can effectively reduce the levels of C/EBPβ and α-Syn expression in primary neurons treated with MPP + .Scale bar = 50 μm.(c) Schematic illustration of the construction of C/EBPβ-HDO.C/EBPβ-HDO administration is an effective approach to reducing the upregulation of both C/EBPβ, pα-Syn, and α-Syn expression levels in MPP + -treated primary neurons.This was confirmed using western blot analysis (mean ± SEM, n = 6 per group, one-way ANOVA, *p < 0.05, **p < 0.01).(d) C/EBPβ-HDO treatment leads to a significant decrease in both C/EBPβ and α-Syn immunoreactivity levels in primary neurons that have been treated with MPP + .Scale bar = 50 μm.indicated that C/EBPβ-HDO reduced the elevated C/EBPβ expression in the nucleus of MPP + -treated primary neurons (Figure S3).The results demonstrated that the activation of Nrf2 or the silencing of C/EBPβ expression was effective in decreasing aberrant α-Syn expression in primary neurons treated with MPP + .

| Activation of Nrf2 by glucoraphanin has
been demonstrated to decrease the degeneration of dopaminergic neurons in A53T mice treated with 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) MPTP is a well-known neurotoxin that is responsible for inducing pathology similar to PD.Previous studies have demonstrated that injecting MPTP in SNCA mice can lead to the progression of PD (Kang, Zhang, Liu, Manfredsson, Benskey, et al., 2017;Luo et al., 2019).Our in vitro findings suggest that activation of Nrf2 by SFN inhibits the transcription of C/EBPβ, thus preventing abnormal expression of α-Syn.In this study, we aimed to determine whether Nrf2 activation can alleviate MPTP-induced PD-like symptoms in mice by suppressing C/EBPβ expression.To test this hypothesis, we used glucoraphanin, an activator of Nrf2 and a biosynthetic precursor of SFN (Yao et al., 2016).In a 30-day oral administration of 0.1% glucoraphanin, we observed a significant increase in the duration time of the rotarod test for A53T mice treated with MPTP (Figure 3a,b).Additionally, the ELISA assay showed that glucoraphanin treatment effectively improved the reduced dopamine concentration in the striatum and SNc of A53T mice treated with MPTP (Figure 3c,d).Furthermore, immunofluorescence staining revealed that glucoraphanin attenuated the decreased TH immunoreactivity in both the striatum and SNc of MPTP-treated A53T mice (Figure 3e,f,j,k).Co-staining of TH with C/EBPβ, pα-Syn, and α-Syn proteins suggested that the loss of TH neurons in the SNc of MPTP-treated A53T mice is associated with an increase in C/EBPβ, pα-Syn, and α-Syn levels, but these effects were markedly reduced after glucoraphanin treatment (Figure 3g-i, l-n).Immunofluorescent staining for IBA1 and GFAP also found that glucoraphanin significantly alleviated the elevated IBA1 and GFAP immunoreactivity in the SNc of MPTP-treated A53T mice (Figure S4).Western blot analysis further confirmed the beneficial effects of glucoraphanin on TH and Nrf2 protein expression, while decreasing the levels of C/EBPβ, pα-Syn, and α-Syn protein expression in the SNc of MPTP-treated A53T mice (Figure 3o).According to the results,

| C/EBPβ -HDO-mediated inhibition of aberrant C/EBPβ expression results in a marked decrease in degeneration of dopaminergic neurons in PFFs-treated A53T mouse models
Injection of human α-Syn PFFs into the rat SNs, in combination with adeno-associated virus (AAV)-mediated overexpression of human α-Syn, triggers Lewy-like α-Syn pathology in DA neurons and impairs motor behavior (Thakur et al., 2017) Overall, our findings suggest that targeting the abnormal C/EBPβ/α-Syn signaling pathway through Nrf2 activation can be an effective therapeutic strategy for alleviating PD-like pathological features.
Nrf2 acts as a crucial transcriptional mediator for genes related to antioxidant and Phase II detoxification.The administration of SFN, a potent Nrf2 activator, activates the Nrf2 signaling pathway, leading to the production of antioxidant and anti-inflammatory effects (Cheung & Kong, 2010;Fahey et al., 1997;Lin et al., 2008;Zhang et al., 1994).Studies suggest that Nrf2 activity declines with age, but pharmacological restoration can enhance its transcriptional activity in older subjects.Furthermore, though Nrf2 is typically found in the cytosol, it is also present in the nucleus of age-matched patients with PD.In the nucleus, Nrf2 helps mitigate oxidative stress by promoting Nrf2-dependent transcription of antioxidant enzymes in the SNc region of dopaminergic neurons (Jazwa et al., 2011).Therefore, it is evident that Nrf2 has a role in the pathogenesis of PD.
C/EBPβ functions as a critical transcription factor involved in neuroinflammation and has emerged as a potential target for therapeutic interventions in various neurodegenerative diseases (Pulido-Salgado et al., 2015).Pro-inflammatory cytokines, such as IL-1α, IL-6, and TNFα, can activate C/EBPβ, leading to the activation of microglia and exerting detrimental effects on the nervous system (Magalini et al., 1995;Poli, 1998;Pulido-Salgado et al., 2015;Straccia et al., 2011;Wedel & Ziegler-Heitbrock, 1995).These findings suggest that suppressing the inflammatory response might confer neuroprotective benefits by attenuating abnormal C/EBPβ expression.
Recent studies have identified C/EBPβ as the primary transcription factor responsible for the mRNA and protein expressions of α-Syn during aging and in PD-affected brains (Wu et al., 2021).Research conducted on transgenic mice with human wild-type α-Syn revealed that overexpression of C/EBPβ led to increased levels of α-Syn, resulting in augmented PD pathologies and motor disorders.Since pro-inflammatory cytokines have been shown to promote C/ EBPβ expression (Magalini et al., 1995;Poli, 1998;Pulido-Salgado et al., 2015;Straccia et al., 2011;Wedel & Ziegler-Heitbrock, 1995), we also examined their levels in MPP + -treated SH-SY5Y cells and found that both SFN treatment and Nrf2 overexpression attenuated pro-inflammatory cytokine expression.Therefore, it is possible that Nrf2's ability to bind to the C/EBPβ promoter and inhibit inflammatory reactions contributes to its ability to reduce C/EBPβ transcription in MPP + -treated SH-SY5Y cells.However, the exact mechanism through which Nrf2 inhibits C/EBPβ transcription remains unclear and warrants further investigation.
Numerous genetic and biochemical studies have demonstrated that α-Syn is the primary constituent of LBs and plays a central role in the development of PD (Luk et al., 2012;Shahmoradian et al., 2019).Therefore, potential therapeutic approaches for PD may involve strategies aimed at reducing the abnormal expression of α-Syn.
Interestingly, recent investigations have revealed that C/EBPβ acts as a critical transcription factor for α-Syn in both aging and PDaffected brains (Wu et al., 2021) Chronic neuroinflammation is a crucial pathogenic factor implicated in various neurodegenerative disorders.It primarily acts by increasing the levels of glia-derived cytokines, which trigger neurotoxic effects on vulnerable dopaminergic neurons (Barcia et al., 2003;Halliday & Stevens, 2011;Taylor et al., 2013).Reactive microglia/ astrocytes expressing IBA1/GFAP have been observed in the SNc region of both mouse models and patients with PD, suggesting a potential involvement of gliosis-derived inflammatory processes in the development of PD pathology (Costa et al., 2013).Consistent with this observation, inhibiting the glial activation-mediated inflammatory response can provide protection to dopaminergic neurons both in vivo and in vitro (Wi et al., 2020).In PD mouse models, oral administration of 0.1% glucoraphanin and repeated intrathecal injection Primary rat neurons were cultured as previously described (Kim et al., 2017;Wu et al., 2021).All rats were purchased from the Guangdong Experimental Animal Center, China.The protocol was reviewed and approved by the Jinan University Institutional Animal Care and Use Committee.During embryonic Day 15, we dissected the brains of fetal rats and exposed it to a cold Hank's equilibrium salt solution that was free of Ca 2+ /Mg 2+ (HBSS).We used 4 mL of HBSS per embryo.The tissue was dissociated using 0.05% trypsin Nrf2 plays a crucial role as a transcriptional mediator of antioxidants, it is worth investigating its potential to inhibit C/EBPβ transcription.Pro-inflammatory cytokines are known to activate C/EBPβ, which in turn regulates α-Syn expression in both aging and PD brains.Therefore, exploring the effects of Nrf2 activation on the abnormal C/EBPβ/α-Syn signaling pathway in a PD mouse model could provide valuable insights into mitigating PD-like pathology.F I G U R E 1 Activating Nrf2 using SFN or overexpressing Nrf2 can effectively suppress C/EBPβ transcription.(a,b) Treatment with SFN or Nrf2 overexpression effectively mitigates the induction of C/EBPβ mRNA and protein levels in SH-SY5Y cells stimulated with MPP + .Both qPCR and western blot analyses provide compelling evidence showing significant decreases in C/EBPβ mRNA and protein levels following SFN or Nrf2 treatments, as compared to MPP + alone-treated cells (mean ± SEM, n = 6 or 7 per group for mRNA, n = 5 per group for protein, one-way ANOVA, **p < 0.01, ***p < 0.001).(c, d) Treatments with SFN or Nrf2 overexpression are highly effective in suppressing the induction of pro-inflammatory cytokine expressions triggered by MPP + in SH-SY5Y cells.The levels of IL-1β and IL-6 noticeably reduced following treatment with either SFN or Nrf2, as compared to the MPP + treated group, as demonstrated by ELISA assay results (mean ± SEM, n = 7 or 8 per group, one-way ANOVA, **p < 0.01, ***p < 0.001).(e) Treatment with SFN or Nrf2 overexpression effectively suppresses MPP + -induced C/EBPβ promoter activation.The luciferase reporter assay results show remarkable reductions in C/EBPβ promoter activity levels following treatment with either SFN or Nrf2, as compared to MPP + treated cells (mean ± SEM, n = 6 per group, one-way ANOVA, ***p < 0.001).(f) Treatment with SFN or Nrf2 overexpression can effectively mitigate the MPP + -induced dissociative effects of Nrf2 with the C/EBPβ promoter in SH-SY5Y cells.ChIP assay results demonstrate an increase in the dissociation of Nrf2 with the C/EBPβ promoter following treatment with MPP + , which is blocked upon treatment with either SFN or Nrf2 overexpression.
Furthermore, as depicted in Figure1f, our ChIP assay indicated that MPP + increased the dissociation effects of Nrf2 from the C/ EBPβ promoter in SH-SY5Y cells; nevertheless, this effect was relieved by SFN and Nrf2 overexpression.The results of this study suggest that both SFN and Nrf2 overexpression exert a protective effect against MPP + -induced damage in SH-SY5Y cells by reducing the transcription of C/EBPβ.

2. 2 |
The activation of Nrf2 by SFN or the silencing of C/EBPβ expression by C/EBPβ -DNA/ RNA heteroduplex oligonucleotide (HDO) can reduce abnormal α-Syn expression in primary neurons treated with MPP + SFN-induced activation of Nrf2 reduces transcription of C/EBPβ in vitro.C/EBPβ, the primary transcription factor controlling α-Syn expression in PD, can potentially be regulated through activation of Nrf2.Therefore, we investigated whether SFN-induced activation of Nrf2 could decrease abnormal α-Syn expression by inhibiting the expression of C/EBPβ in vitro.To investigate this hypothesis, primary neurons were treated with MPP + to establish a model.The results revealed a significant reduction in Nrf2 expression and an increase in C/EBPβ, pα-Syn, and α-Syn expression in primary neurons following MPP + treatment (Figure glucoraphanin has been shown to improve PD-like symptoms in A53T mice treated with MPTP.This suggests that the activation of Nrf2 through glucoraphanin leads to the inhibition of abnormal expression of C/EBPβ and α-Syn in the SNc of MPTP-treated A53T mice, resulting in a reduction in the degeneration of dopaminergic neurons.2.4 | Silencing the expression of C/EBPβ using C/EBPβ -HDO has been demonstrated to mitigate the degeneration of dopaminergic neurons in A53T mice when treated with MPTP The activation of Nrf2 by its activator inhibits the abnormal expression of both C/EBPβ and α-Syn in the SNc of A53T mice treated with MPTP.This study employed a C/EBPβ-HDO construct to investigate the direct silencing effect on C/EBPβ expression in A53T mice treated with MPTP, with the aim of analyzing its impact on abnormal α-Syn expression.The mice received a total of four intrathecal injections of C/EBPβ-HDO at a concentration of 100 nM, with a volume of 2 μL per week.In the SNc of wild-type (WT) mice, C/EBPβ-HDO significantly decreased both C/EBPβ mRNA and protein expression levels (Figure S5A,B).Subsequently, in A53T mice treated with MPTP, two repeated intrathecal administrations of C/EBPβ-HDO (100 nM per 2 μL) on Day 1 and Day 5 significantly improved the duration time in the rotarod test (Figure 4a,b).ELISA analysis revealed that C/EBPβ-HDO led to a significant enhancement of dopamine concentration in both the striatum and SNc regions of MPTP-treated A53T mice (Figure 4c,d).Immunofluorescence studies demonstrated that C/EBPβ-HDO effectively restored diminished TH immunoreactivity in both the striatum and SNc regions of MPTP-treated A53T mice (Figure 4e,f,j,k).Additionally, using immunofluorescence duallabeling assays for TH and C/EBPβ, TH and pα-Syn, TH and α-Syn, F I G U R E 3 Activating Nrf2 can significantly mitigate the degeneration of dopaminergic neurons in A53T mice treated with MPTP.(a) Schedule of treatment.(b) Rotarod test.The duration time for the rotarod test were recorded and compared (mean ± SEM, n = 14 per group, one-way ANOVA, **p < 0.01 and ***p < 0.001).(c, d) Glucoraphanin attenuated the decreased concentration of dopamine in the striatum and SNc of MPTP-treated A53T mice.ELISA assay showed that the decreased concentration of dopamine in the striatum and SNc of MPTP-treated A53T mice were significantly ameliorated by glucoraphanin (mean ± SEM, n = 6 or 7 per group, one-way ANOVA, *p < 0.05, ***p < 0.001).(e,f) Immunofluorescence staining showed that the TH neuron reduction in both striatum and SNc regions with the treatment of MPTP, which was attenuated by treatment with glucoraphanin.Scale bar = 200 μm.(g) Immunofluorescent double staining of TH and C/EBPβ demonstrates that the loss of TH neurons is coupled with an increase of C/EBPβ.Scale bar = 50 μm.(h) Immunofluorescent double staining of TH and pα-Syn demonstrates that the loss of TH neurons is coupled with an increase of pα-Syn.Scale bar = 50 μm.(i) Immunofluorescent double staining of TH and α-Syn demonstrates that the loss of TH neurons is coupled with an increase of α-Syn.Scale bar = 50 μm.(j-n) The immunostaining intensities of TH, C/EBPβ, pα-Syn, and α-Syn were measured and compared among (mean ± SEM, n = 5 per group for TH, C/EBPβ, pα-Syn, and α-Syn, one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001).(o) Glucoraphanin attenuates the downregulation of TH and Nrf2 and the upregulation of C/EBPβ, pα-Syn, and α-Syn expression in MPTP-treated A53T mice.Western blot suggested that administration of glucoraphanin significantly ameliorated the downregulation of Nrf2 and TH and the upregulation of C/EBPβ, pα-Syn, and α-Syn protein expression in the SNc of MPTP-treated A53T mice (mean ± SEM, n = 5 per group, one-way ANOVA, *p < 0.05, **p < 0.01).we observed a simultaneous increase in the expression of C/EBPβ, pα-Syn, and α-Syn, along with a reduction in TH neuron numbers in the SNc area of MPTP-exposed A53T mice; this phenomenon was alleviated after treatment with C/EBPβ-HDO (Figure 4g-i, l-n).Immunofluorescence analyses of IBA1 and GFAP showed that C/ EBPβ-HDO significantly decreased the elevated immunoreactivity of IBA1 and GFAP in the SNc region of MPTP-treated A53T mice (Figure S6).Western blot analyses confirmed the increase in TH protein expression and the decrease in C/EBPβ, pα-Syn, and α-Syn protein expression in the SNc of MPTP-treated A53T mice following intervention with C/EBPβ-HDO (Figure 4o).The findings indicate that by directly suppressing the expression of C/EBPβ, C/EBPβ-HDO therapy can inhibit abnormal α-Syn production in the SNc area of A53T mice exposed to MPTP.This inhibition ultimately leads to a reduction in the degeneration of dopaminergic neurons.2.5 | The activation of Nrf2 through the use of SFN and the suppression of C/EBPβ expression via C/EBPβ -HDO treatment were found to hinder the aggregation of abnormal α-Syn in preformed fibrils (PFFs)-treated HEK293α-Syn-YFP cells To investigate the influence of SFN on PFFs-induced α-Syn aggregation in HEK293α-Syn-YFP cells, we activated Nrf2 and evaluated its effects.Our observations revealed the formation of intracellular fluorescence spots in HEK293α-Syn-YFP cells following exposure to PFFs, indicating the aggregation of α-Syn.Remarkably, SFN intervention significantly suppressed this aggregative activity (Figure S7A).Moreover, western blot analysis demonstrated that PFFs decreased the levels of Nrf2 expression while increasing those of C/EBPβ, pα-Syn, α-Syn, insoluble pα-Syn, and insoluble α-Syn expression.However, when SFN was used as an intervention agent, Nrf2 expression was significantly increased, and the levels of C/EBPβ, pα-Syn, α-Syn, insoluble pα-Syn, and insoluble α-Syn were significantly reduced in PFFs-treated HEK293α-Syn-YFP cells (Figure S7B).Therefore, our data indicate that SFN-mediated activation of Nrf2 has the potential to serve as a therapeutic tool against abnormal C/EBPβ expression and α-Syn aggregation in PFFstreated HEK293α-Syn-YFP cells.In addition, we conducted experiments to evaluate the impact of C/EBPβ-HDO administration on α-Syn aggregation in PFFsstimulated HEK293α-Syn-YFP cells.After treatment with PFFs, YFPα-Syn aggregated into small fluorescent clusters located within intracellular spaces.However, upon therapy with C/EBPβ-HDO, a corresponding decrease in aggregation levels was observed (Figure S7C).Furthermore, western blotting revealed that C/EBPβ-HDO administration significantly reduced the increased expression levels of C/EBPβ, pα-Syn, α-Syn, insoluble pα-Syn, and insoluble α-Syn in PFFs-treated HEK293α-Syn-YFP cells (Figure S7D).Overall, our data demonstrate that the suppression of C/EBPβ expression mediated by C/EBPβ-HDO leads to a remarkable reduction in abnormal α-Syn aggregation in PFFs-exposed HEK293α-Syn-YFP cells.
. Conversely, our in vitro findings suggest that targeted suppression of abnormal C/EBPβ expression can hinder the pathological aggregation of α-Syn in HEK293α-Syn-YFP cells treated with PFFs.Therefore, this study aimed to investigate the potential of C/EBPβ-HDO therapy in attenuating the degradation of dopaminergic neurons in the SNc region of A53T mice exposed to PFFs by inhibiting the expression of C/EBPβ.In the PFFs-treated α-Syn-A53T mice, administration of repeated intrathecal injections of C/EBPβ-HDO at a concentration of 100 nM per 2 μL per week for a total of four times demonstrated a significant enhancement in the duration of the rotarod test (Figure 5a,b).An F I G U R E 4 Silencing the expression of C/EBPβ can significantly reduce the degeneration of dopaminergic neurons in A53T mice treated with MPTP.(a) Schedule of treatment.(b) Rotarod test.The duration time for the rotarod test were recorded and compared (mean ± SEM, n = 14 per group, one-way ANOVA, **p < 0.01 and ***p < 0.001).(c, d) C/EBPβ-HDO attenuated the decreased concentration of dopamine in the striatum and SNc of MPTP-treated A53T mice.ELISA assay showed that the decreased concentration of dopamine in the striatum and SNc of MPTP-treated A53T mice were significantly ameliorated by C/EBPβ-HDO (mean ± SEM, n = 8 per group, one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001).(e,f) Immunofluorescence staining showed the TH neuron reduction in both striatum and SNc regions with the treatment of MPTP, which was attenuated by treatment with C/EBPβ-HDO.Scale bar = 200 μm.(g) Immunofluorescent double staining of TH and C/EBPβ demonstrates that the loss of TH neurons is coupled with an increase of C/EBPβ.Scale bar = 50 μm.(h) Immunofluorescent double staining of TH and pα-Syn demonstrates that the loss of TH neurons is coupled with an increase of pα-Syn.Scale bar = 50 μm.(i) Immunofluorescent double staining of TH and α-Syn demonstrates that the loss of TH neurons is coupled with an increase of α-Syn.Scale bar = 50 μm.(j-n) The immunostaining intensities of TH, C/EBPβ, pα-Syn, and α-Syn were measured and compared among (mean ± SEM, n = 5 per group for TH, C/EBPβ, pα-Syn, and α-Syn, one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001).(o) C/EBPβ-HDO attenuates the downregulation of TH and Nrf2 and the upregulation of C/EBPβ, pα-Syn, and α-Syn expression in MPTP-treated A53T mice.Western blot suggested that administration of C/EBPβ-HDO significantly ameliorated the downregulation of Nrf2 and TH and the upregulation of C/EBPβ, pα-Syn, and α-Syn protein expression in the SNc of MPTP-treated A53T mice (mean ± SEM, n = 6 per group, one-way ANOVA, **p < 0.01 and ***p < 0.001).ELISA study revealed that infusion of C/EBPβ-HDO led to a significant increase in dopamine levels in both the striatum and SNc regions of PFFs-treated A53T mice (Figure 5c,d).Additionally, administration of C/EBPβ-HDO resulted in substantial restoration of TH immunoreactivity within the striatum and SNc regions of PFFsexposed mice (Figure 5e,f,j,k).The findings from immunofluorescent co-staining showed that an increase in C/EBPβ, pα-Syn, and α-Syn levels accompanied the loss of TH neurons in the SNc region of PFFsstimulated A53T mice.However, the impact of these changes was notably lessened with the intervention of C/EBPβ-HDO (Figure 5gi,l-n).Immunofluorescence results for IBA1 and GFAP indicated that C/EBPβ-HDO treatment greatly reduced the heightened IBA1 and GFAP immunoreactivity observed in the SNc of PFFs-exposed A53T mice (Figure S8).Additionally, western blot assays provided further validation for our findings, showing that TH expression levels were upregulated, while C/EBPβ, pα-Syn, α-Syn, insoluble pα-Syn, and insoluble α-Syn expression levels were downregulated in the SNc region of PFFs-treated A53T mice following C/EBPβ-therapy (Figure 5o and Figure S9).Our findings indicate the downregulation of C/EBPβ expression can mitigate dopaminergic neuron death in the SNc region of PFFs-treated A53T mice.This effect is likely attributed to the subsequent inhibition of abnormal α-Syn expression and its associated pathological effects on cells.3 | DISCUSS ION Our study has revealed that Nrf2 inhibits abnormal C/EBPβ transcription in MPP + -treated SH-SY5Y cells.Administration of SFN, an activator of Nrf2, and silencing of C/EBPβ through C/EBPβ-HDO significantly reduce levels of abnormal α-Syn expression in MPP +stimulated primary neurons.Additionally, administering glucoraphanin to activate Nrf2 results in a significant reduction in abnormal C/ EBPβ and α-Syn expression in the SNc of MPTP-treated A53T mice, thereby mitigating dopaminergic neuronal degradation.Moreover, our results demonstrate that directly silencing abnormal C/EBPβ expression using C/EBPβ-HDO can reduce α-Syn expression levels in the SNc of MPTP-treated A53T mice, ultimately attenuating dopaminergic neuron degeneration.Importantly, we discovered that inhibiting abnormal α-Syn expression by suppressing C/EBPβ expression with C/EBPβ-HDO significantly mitigates dopaminergic neuron degeneration in the SNc region of PFFs-treated A53T mice.

F
The therapeutic agent C/EBPβ-HDO can significantly mitigate PD pathology induced by PFFs in vivo.(a) The schedule of treatment.PFFs (3.75 μg/μL, total 5 μg protein) were injected into the right site of substantia nigra.C/EBPβ -HDO (100 nM, 2 μL) was myelin injection on Day 7, Day 14, Day 21, and Day 28.(b) Rotarod test.The duration time for the rotarod test were recorded and compared (mean ± SEM, n = 14 per group, one-way ANOVA, ***p < 0.001).(c,d) C/EBPβ-HDO attenuated the decreased concentration of dopamine in the striatum and SNc of PFFs-treated A53T mice.ELISA assay showed that the decreased concentration of dopamine in the striatum and SNc of PFFs-treated A53T mice were significantly ameliorated by C/EBPβ-HDO (mean ± SEM, n = 6 or 7 per group, one-way ANOVA, *p < 0.05, **p < 0.01).(e,f) Immunofluorescence staining showed that the TH neuron reduction in both striatum and SNc regions with the treatment of PFFs, which was attenuated by treatment with C/EBPβ-HDO.Scale bar = 200 μm.(g) Immunofluorescent double staining of TH and C/EBPβ demonstrates that the loss of TH neurons is coupled with an increase of C/EBPβ.Scale bar = 50 μm.(h) Immunofluorescent double staining of TH and pα-Syn demonstrates that the loss of TH neurons is coupled with an increase of pα-Syn.Scale bar = 50 μm.(i) Immunofluorescent double staining of TH and α-Syn demonstrates that the loss of TH neurons is coupled with an increase of α-Syn.Scale bar = 50 μm.(j-n): The immunostaining intensities of TH, C/EBPβ, pα-Syn, and α-Syn were measured and compared among (mean ± SEM, n = 5 per group for TH, C/EBPβ, pα-Syn, and α-Syn, one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001).(o) C/EBPβ-HDO attenuates the downregulation of TH and Nrf2 and the upregulation of C/EBPβ, pα-Syn, and α-Syn expression in PFFs-treated A53T mice.Western blot suggested that administration of C/EBPβ-HDO significantly ameliorated the downregulation of Nrf2 and TH and the upregulation of C/ EBPβ, pα-Syn, and α-Syn protein expression in the SNc of PFFs-treated A53T mice (mean ± SEM, n = 6 per group, one-way ANOVA, *p < 0.05 and **p < 0.01).Conversely, depletion of C/EBPβ in similar transgenic mice with human α-Syn resulted in reduced expression of α-Syn, leading to mitigated PD pathologies and motor impairments (Wu et al., 2021).Thus, targeting the inhibition of abnormal C/EBPβ transcription may hold potential in preventing PD pathologies.Our research aimed to investigate whether activation of Nrf2 SFN treatment or Nrf2 overexpression could reduce C/EBPβ transcription in MPP + -treated SH-SY5Y cells.We used a combination of qPCR, western blotting, luciferase reporter assays, and ChIP analysis to examine the effects.Our findings revealed that both SFN treatment and Nrf2 overexpression attenuated the increased expression of C/EBPβ mRNA and protein, as well as decreased the activation of the C/EBPβ promoter and the dissociation of Nrf2 with the C/EBPβ promoter in MPP + -treated SH-SY5Y cells.These results provide evidence that activating Nrf2 through SFN treatment or overexpression can inhibit C/EBPβ transcription in MPP + -treated SH-SY5Y cells.
. Our in vitro study demonstrated that the activation of Nrf2 effectively inhibited the abnormal transcription of C/EBPβ.To further investigate the mechanisms underlying this observation, we treated primary neurons exposed to MPP + with SFN or C/EBPβ-HDO to activate Nrf2 or silence C/EBPβ.We observed that the activation of Nrf2 via SFN treatment or the silencing of C/EBPβ through C/EBPβ-HDO attenuated abnormal α-Syn expression levels in the MPP + -treated primary neurons.Treatment with glucoraphanin, a precursor of SFN, resulted in the downregulation of downstream targets such as C/EBPβ and α-Syn, consequently promoting extensive survival of dopaminergic neurons.This was accompanied by evident motor improvements in MPTP-treated A53T mice.Additionally, the activation of Nrf2 through SFN treatment or the silencing of C/EBPβ through C/EBPβ-HDO efficiently suppressed Lewy-like pathology in HEK293α-Syn-YFP cells treated with PFFs.Silencing the abnormal expression of C/EBPβ through the inhibition of abnormal α-Syn also facilitated the survival of dopaminergic neurons and ameliorated motor deficits in A53T mice treated with PFFs.Our findings suggest that the activation of Nrf2 or the silencing of abnormal C/EBPβ expression can lead to reduced aberrant α-Syn levels, thereby alleviating α-Syn-induced pathological changes.
of C/EBPβ-HDO successfully prevented glial activation and reduced degeneration of TH neurons in the SNc.These findings strongly suggest that the neuroprotective effects of glucoraphanin and C/EBPβ-HDO may arise from their ability to inhibit glial activation in the SNc region of PD mouse models.In conclusion, our data support the effectiveness of Nrf2 activation in suppressing abnormal C/EBPβ transcription.The activation of Nrf2 through its activator or the inhibition of C/EBPβ using C/ EBPβ-HDO can effectively reduce the degeneration of dopaminergic neurons in mouse models of PD by decreasing abnormal α-Syn expression levels.Based on these findings, it can be inferred that blocking the aberrant C/EBPβ/α-Syn signaling pathway through Nrf2 activation could serve as a promising therapeutic strategy for PD.Thus, Nrf2 activators like SFN and glucoraphanin, along with C/ EBPβ-HDO, show great potential as therapeutic agents for combating PD.4 | MATERIAL S AND ME THODS4.1 | Mice and cell linesMale transgenic mice expressing A53T human α-Syn and wild type mice (12 weeks old, 25-30 g) were used for the experiments.The animals were housed under controlled temperature and kept in a 12-h light/dark cycle with ad libitum access to food and water.The animal protocol was approved by the Jinan University Institutional Animal Care and Use Committee, and all experiments were performed following the Guide for Animal Experimentation of Jinan University.