https://orcid.org/0000-0002-2997-7478
Abstract
This study aimed to characterize the functional relevance and mechanistic basis of the histone demethylase Jumonji domain-containing protein-3 (JMJD3) in preserving dopaminergic neuron survival in Parkinson’s disease (PD). Mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lesions and MN9D dopaminergic neuronal cell lines exposed to 6-OHDA, respectively, were used to simulate in vivo and in vitro PD-like environments. PD-related genes with differential expressions were identified using RNA sequencing of hippocampal tissues collected from MPTP-lesioned mice. A specific lentiviral shRNA vector was used to investigate the effects of JMJD3 on neuron activities in vitro and PD-like phenotypes in vivo. JMJD3 was found to up-regulate the expression of Snail family transcriptional repressor 2 (SNAI2) through the inhibition of H3 on lysine 27 (H3K27me3) enrichment in the SNAI2 promoter region. As a result, the viability of 6-OHDA-exposed MN9D cells was stimulated, and cell apoptosis was diminished. Knockdown of SNAI2 decreased the expression of yes-associated protein (YAP) and HIF1α while also reducing the viability of 6-OHDA-exposed MN9D cells and increasing cell apoptosis. The in vivo experiments demonstrated that JMJD3 activated the SNAI2/YAP/HIF1α signaling pathway, inhibiting PD-like phenotypes in MPTP-lesioned mice. Thus, the findings provide evidence that JMJD3 inhibits the enrichment of H3K27me3 at the SNAI2 promoter, leading to the upregulation of SNAI2 expression and activation of the YAP/HIF1α signaling pathway, ultimately exerting a protective effect on PD mice. This finding suggests that targeting the JMJD3-SNAI2 pathway could be a promising therapeutic strategy for PD. Further in-depth studies are needed to elucidate the underlying mechanisms and identify potential downstream targets of this pathway.
Introduction
Parkinson’s disease (PD) is a degenerative neurological condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (1–4). Despite its prevalence, the etiopathogenesis and the cause of nigrostriatal dopaminergic neuron loss remain elusive (5,6). Thus, a mechanistic understanding of dopaminergic neuron survival is imperative to develop potential therapeutic targets.
One potential target is Jumonji domain-containing protein-3 (JMJD3), a histone demethylase that can regulate the trimethylation of histone H3 on lysine 27 (H3K27me3) (7). JMJD3 has been found to enhance dopaminergic neuron differentiation in the fetal midbrain. In addition, it leads to M2 microglia polarization by modifying the histone H3K27me3, which could potentially contribute to the immune pathogenesis of PD (8). JMJD3’s increased activity has also been linked to Snail family transcriptional repressor 2 (SNAI2) induction in hepatocellular carcinoma cells through H3K27me3 reduction in the SNAI2 gene promoter (9,10).
SNAI2 is a transcription factor that plays an essential role in tissue development and tumorigenesis (11). After three months of nerve injury, the distal nerve can maintain high SNAI2 expression (12). SNAI2 can form binary complexes with yes-associated protein (YAP) to regulate its transcriptional activity and function (13). YAP is associated with neuron survival and differentiation, and its deficiency can lead to dopaminergic neuron death and apoptosis in PD (14). Inhibition of YAP can also suppress the upregulation of HIF1α induced by cyclic mechanical stress in rat cartilage chondrocytes (15,16). High expression of HIF1α is associated with the neuroprotection in PD provided by lactoferrin, a multi-functional iron-binding globular glycoprotein (17).
Therefore, it is reasonable to hypothesize that JMJD3 controls dopaminergic neuron survival and plays a critical role in PD by regulating the SNAI2/YAP/HIF1α axis. To test this hypothesis, we generated a PD mouse model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) since the degeneration of midbrain dopaminergic neurons is a hallmark of PD, and MPTP triggers this degeneration selectively when administered systemically (18–20). We conducted transcriptome sequencing technology and in vitro experiments to validate our hypothesis, revealing the specific mechanisms of the JMJD3/SNAI2/YAP/HIF1α signaling axis for the first time. Our study illuminates a novel therapeutic target and reveals a potential treatment strategy for PD through developing mechanistic understanding and epigenetic regulation theories.
Results
RNA sequencing and bioinformatics analysis for key genes involved in the pathogenesis of PD
Three pairs of hippocampal tissues from normal mice and MPTP-lesioned mice were used for mRNA sequencing. After quality control and mRNA identification, the mRNAs were subjected to differential analysis using the R ‘edgeR’ package. The results yielded 454 significantly up-regulated genes and 210 downregulated genes (Fig. 1A, Supplementary Material, Table S3, S4), among which SNAI2 was the most downregulated (Fig. 1B). JMJD3 is a histone demethylase that regulates target gene expression by modulating the level of H3K27me3 in the promoter region of target genes (21). Meanwhile, JMJD3 (also known as KDM6B) was poorly expressed in the hippocampal tissues of MPTP-lesioned mice (Fig. 1C and D). Therefore, we hypothesized that JMJD3 may regulate SNAI2 expression activation by affecting the histone methylation in the SNAI2 promoter region.
Bioinformatics analysis for differentially expressed genes involved in the pathogenesis of PD. (A) A heat map of the expression of differentially expressed genes in normal and PD samples by mRNA sequencing. (B) A histogram of SNAI2 expression in normal and PD samples following mRNA sequencing. (C) A histogram of JMJD3 expression in normal and PD samples following mRNA sequencing. (D) Predicted binding sites in the SNAI2 promoter.
JMJD3 inhibits H3K27me3 enrichment in the promoter region of SNAI2 and up-regulates SNAI2 expression in vivo and in vitro
PD was mimicked in MPTP-lesioned mice, which was used to explore whether the low expression of SNAI2 in PD was associated with the regulation of histone methylation in the SNAI2 promoter region by JMJD3. The Nissl staining results (Fig. 2A) showed that the number of Nissl bodies in normal mice was higher than that of MPTP-induced mice, demonstrating the successful establishment of the PD mouse model.
JMJD3 up-regulates SNAI2 expression via inhibition of H3K27me3 enrichment in the promoter region of SNAI2 in vivo and in vitro. (A) The number of Nissl bodies in the brain tissue of normal mice and MPTP-lesioned mice observed by Nissl staining. (B) Western blot of JMJD3 and SNAI2 proteins in normal and MPTP-lesioned mice brain tissue. (C) Immunohistochemistry of JMJD3 and SNAI2 proteins in normal and MPTP-lesioned mice brain tissue. (D) SNAI2 promoter pulled down by anti-JMJD3 or anti-H3K27me3 antibodies determined by ChIP assay in normal and MPTP-lesioned mice’s brain tissue. SNAI2 promoter expression was measured by qPCR. (E) Western blot of JMJD3, SNAI2 and HIF1α proteins in the control and 6-OHDA-treated MN9D cells. (F) Western blot of JMJD3 and SNAI2 proteins in the 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing oe-JMJD3 or sh-JMJD3. (G) Western blot of JMJD3 and SNAI2 proteins in the 6-OHDA-treated MN9D cells treated with GSKJ4. (H) SNAI2 promoter pulled down by anti-JMJD3 or anti-H3K27me3 antibodies determined by ChIP assay in the 6-OHDA-treated MN9D cells treated with GSKJ4, with SNAI2 promoter expression measured by qPCR. Data are presented as mean ± standard deviation. The significance of differences in data between the two groups was analyzed using an unpaired t-test. The cell experiment was repeated three times independently. n = 9 mice for each treatment. P-values are provided in the histogram and indicate the significance of differences compared to normal, control, oe-NC or sh-NC.
Based on western blot and immunohistochemistry (Fig. 2B and C), expression of JMJD3 and SNAI2 was reduced in the brain tissues of MPTP-lesioned mice compared with the normal mice. ChIP-qPCR results (Fig. 2D) showed that the SNAI2 promoter pulled down by the anti-JMJD3 antibody was reduced in the brain tissue of MPTP-lesioned mice, while an anti-H3K27me3 antibody amplified it. The above results showed that the enrichment of JMJD3 in the SNAI2 promoter region was decreased, while that of H3K27me3 was increased in MPTP-lesioned mice.
In the MN9D cells induced by 6-OHDA, JMJD3 and SNAI2, protein expression was lower than in the control cells (Fig. 2E). To further verify whether JMJD3 regulates SNAI2 expression, we performed group treatments in MN9D cells. First, a western blot was used to confirm the successful transfection of the shRNA lentivirus targeting JMJD3 and SNAI2, respectively. The results revealed that sh-JMJD3-1 and sh-JMJD3-2 significantly inhibited the expression of JMJD3, with the highest silencing efficiency seen in sh-JMJD3-2 (Supplementary Material, Fig. S1A). Similarly, sh-SNAI2-1 and sh-SNAI2-2 effectively suppressed SNAI2 expression, with the best inhibitory effect observed using sh-SNAI2-2 for subsequent experiments (Supplementary Material, Fig. S1B). Subsequently, we examined the fluorescence expression of EGFP protein in cells infected with lentivirus using fluorescence microscopy (Supplementary Material, Fig. S1C). The results showed that cells overexpressing or silencing the target genes had obvious EGFP protein expression. Finally, western blot analysis demonstrated that in comparison to the oe-NC group, both JMJD3 and SNAI2 expressions were significantly up-regulated in the oe-JMJD3 group (Fig. 2F). Moreover, compared to the sh-NC group, both JMJD3 and SNAI2 expressions were remarkably downregulated in the sh-JMJD3 group, supporting the notion that JMJD3 represses SNAI2 expression.
Meanwhile, the results shown in Figure 2G revealed decreased JMJD3 and SNAI2 protein expression in the 6-OHDA-treated MN9D cells treated with GSKJ4. ChIP-qPCR data (Fig. 2H) exhibited reduced SNAI2 promoter pulled down by anti-JMJD3 antibody in the 6-OHDA-treated MN9D cells treated with GSKJ4, but the anti-H3K27me3 antibody elevated it. The above results showed that the enrichment of JMJD3 was decreased in the SNAI2 promoter region, and that of H3K27me3 was increased in the in vitro PD model.
JMJD3 silencing down-regulates SNAI2 expression, thereby reducing the viability of 6-OHDA-exposed MN9D cells and promoting cell apoptosis
Then this study moved to verify whether JMJD3 affected SNAI2 expression activation by regulating H3K27me3 levels in the SNAI2 promoter region. Western blot results (Fig. 3A) displayed downregulated JMJD3 and SNAI2 expression in 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing sh-JMJD3. No alteration was noted in JMJD3 expression, but SNAI2 expression was up-regulated in response to sh-JMJD3 + oe-SNAI2. Additionally, ChIP-qPCR results (Fig. 3B) unveiled decreased SNAI2 promoter pulled down by anti-JMJD3 antibody in the 6-OHDA-treated MN9D cells treated with sh-JMJD3, but the anti-H3K27me3 antibody elevated it. Upon treatment with sh-JMJD3 + oe-SNAI2, there was no difference in the SNAI2 promoter pulled down by the anti-JMJD3 antibody or anti-H3K27me3 antibody in the 6-OHDA-treated MN9D cells.
JMJD3 silencing down-regulates SNAI2 expression, inhibiting the viability of 6-OHDA-exposed MN9D cells and promoting cell apoptosis. 6-OHDA-treated MN9D cells were transduced with lentiviral vectors expressing sh-JMJD3 or in combination with oe-SNAI2. (A) Western blot of JMJD3 and SNAI2 proteins in 6-OHDA-exposed MN9D cells. (B) SNAI2 promoter pulled down by anti-JMJD3 or anti-H3K27me3 antibodies determined by ChIP assay in the 6-OHDA-exposed MN9D cells. SNAI2 promoter expression was measured by qPCR. (C) The viability of 6-OHDA-exposed MN9D cells measured by CCK-8 assay. (D) Western blot of Ki67 and PCNA proteins in 6-OHDA-exposed MN9D cells. (E) Apoptosis of 6-OHDA-exposed MN9D cells measured by flow cytometry. Data are presented as mean ± standard deviation. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. Bonferroni-corrected two-way ANOVA was applied to compare data among multiple groups at varied time points. The cell experiment was repeated three times independently. P-values are provided in the histogram and indicate the significance of differences as compared with sh-NC + oe-NC or sh-JMJD3 + oe-NC.
According to the results of the CCK-8 assay and western blot (Fig. 3C and D), JMJD3 silencing repressed cell viability and diminished Ki67 and PCNA expression; conversely, the effect of JMJD3 silencing was reversed by SNAI2 overexpression. Furthermore, flow cytometric data (Fig. 3E) presented enhanced cell apoptosis without JMJD3. Opposite results were found upon SNAI2 overexpression. The results demonstrate that JMJD3 silencing could decrease SNAI2 expression by increasing H3K27me3 levels in the SNAI2 promoter region, thus repressing the viability of 6-OHDA-exposed MN9D cells and inducing apoptosis. However, overexpression of SNAI2 attenuated the inhibitory effect of JMJD3 silencing on 6-OHDA-exposed MN9D cells.
Downregulation of SNAI2 leads to downregulation of YAP in the MPTP-lesioned mice and 6-OHDA-treated MN9D cells
Evidence has shown that SNAI2 up-regulates YAP expression (22), and YAP is associated with neuron survival and differentiation (14). Western blot and immunohistochemistry results revealed reduced YAP expression in the brain tissues of MPTP-lesioned mice versus the normal mice (Fig. 4A and B). Furthermore, YAP exhibited poor expression in the 6-OHDA-treated MN9D cells relative to control cells (Fig. 4C). In addition, the western blot results showed that SNAI2 and YAP expression was elevated in the 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing oe-SNAI2 (Fig. 4D). The above results indicate that low expression of SNAI2 can decrease the expression of YAP in vivo and in vitro.
Downregulated expression of SNAI2 contributes to inhibition of YAP expression in vivo and in vitro. (A) Western blot of YAP protein in normal and MPTP-lesioned mice brain tissue. (B) Immunohistochemistry of YAP protein in normal and MPTP-lesioned mice brain tissue. (C) Western blot of YAP protein in the control and 6-OHDA-treated MN9D cells. (D) Western blot of YAP1 and SNAI2 proteins in 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing oe-SNAI2. Data are presented as mean ± standard deviation. The significance of differences in data between the two groups was analyzed using an unpaired t-test. The cell experiment was repeated three times independently. n = 9 mice for each treatment. P-values are provided in the histogram and indicate the significance of differences as compared with normal, control or oe-NC.
SNAI2 silencing down-regulates YAP expression, thereby inhibiting the viability of 6-OHDA-exposed MN9D cells and promoting cell apoptosis
Next, the study centered on validating the effect of SNAI2 on the viability and apoptosis of 6-OHDA-exposed MN9D cells by regulating YAP expression. Western blot results (Fig. 5A) suggested downregulated SNAI2 and YAP expression in 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing sh-SNAI2; conversely, sh-SNAI2 + oe-YAP resulted in elevated YAP expression while failing to change SNAI2 expression.
SNAI2 silencing reduces the viability of 6-OHDA-exposed MN9D cells and induces apoptosis by down-regulating YAP expression. 6-OHDA-treated MN9D cells were transduced with lentiviral vectors expressing sh-SNAI2 or in combination with or-YAP. (A) Western blot of SNAI2 and YAP proteins in 6-OHDA-exposed MN9D cells. (B) The viability of 6-OHDA-exposed MN9D cells measured by CCK-8 assay. (C) Western blot of Ki67 and PCNA proteins in 6-OHDA-exposed MN9D cells. (D) Apoptosis of 6-OHDA-exposed MN9D cells measured by flow cytometry. Data are presented as mean ± standard deviation. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. Bonferroni-corrected two-way ANOVA was applied to compare data among multiple groups at varied time points. The cell experiment was repeated three times independently. P-values are provided in the histogram and indicate the significance of differences as compared with sh-NC + oe-NC or sh-SNAI2 + oe-NC.
Based on the results of the CCK-8 assay and western blot (Fig. 5B and C), knockdown of SNAI2 decreased cell viability and diminished Ki67 and PCNA expression; these results were reversed by further YAP overexpression. Moreover, flow cytometric data (Fig. 5D) presented that cell apoptosis was stimulated without SNAI2. Opposite results were found upon YAP overexpression. The above results indicate that SNAI2 silencing could inhibit the viability of 6-OHDA-exposed MN9D cells and promote apoptosis by down-regulating YAP expression. YAP overexpression could reverse these effects.
HIF1α, downregulated in PD models, can be up-regulated by YAP
YAP has been reported to stabilize HIF1α and promote its expression (23). Meanwhile, HIF1α expression is downregulated in PD (24). Therefore, in this study, we analyzed the effect of YAP on HIF1α expression in PD. Western blot and immunohistochemistry (Fig. 6A and B) demonstrated that the expression of HIF1α was reduced in the brain tissue of MPTP-lesioned mice.
YAP increases HIF1α expression in the mouse and cell PD models. (A) Western blot of HIF1α protein in the brain tissue of normal mice and MPTP-lesioned mice. (B) Immunohistochemistry of HIF1α protein in normal and MPTP-lesioned mice brain tissue. (C) Western blot of HIF1α protein in the control and 6-OHDA-treated MN9D cells. (D) Western blot of YAP and HIF1α proteins in the 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing or-YAP. Data are presented as mean ± standard deviation. The significance of differences in data between the two groups was analyzed using an unpaired t-test. The cell experiment was repeated three times independently. n = 9 mice for each treatment. P-values are provided in the histogram and indicate the significance of differences as compared with normal, control or oe-NC.
As depicted in Figure 6C 6-OHDA-treated MN9D cells also declined HIF1α expression compared with control cells. In addition, treatment with oe-YAP in 6-OHDA-treated MN9D cells increased YAP and HIF1α expression. The above results suggest that HIF1α is downregulated in the PD model, and YAP could up-regulate HIF1α expression (Fig. 6D).
YAP silencing inhibits the viability of 6-OHDA-exposed MN9D cells and induces cell apoptosis by down-regulating HIF1α expression
The effect of YAP on the viability and apoptosis of 6-OHDA-exposed MN9D cells by regulating HIF1α expression was the subsequent focus of the current study. Western blot results (Fig. 7A) showed reduced YAP and HIF1α expression in 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing sh-YAP. In contrast, HIF1α expression was amplified following treatment with sh-YAP + oe-HIF1α in addition to unaltered YAP expression.
YAP silencing decreases HIF1α expression to impair the viability of 6-OHDA-exposed MN9D cells and stimulate cell apoptosis. 6-OHDA-treated MN9D cells were transduced with lentiviral vectors expressing sh-YAP or in combination with one-HIF1α. (A) Western blot of YAP and HIF1α proteins in 6-OHDA-exposed MN9D cells. (B) The viability of 6-OHDA-exposed MN9D cells measured by CCK-8 assay. (C) Western blot of Ki67 and PCNA proteins in 6-OHDA-exposed MN9D cells. (D) Apoptosis of 6-OHDA-exposed MN9D cells measured by flow cytometry. Data are presented as mean ± standard deviation. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. Bonferroni-corrected two-way ANOVA was applied to compare data among multiple groups at varied time points. The cell experiment was repeated three times independently. P-values are provided in the histogram and indicate the significance of differences as compared with sh-NC + oe-NC or sh-YAP + oe-NC.
According to the results of the CCK-8 assay and western blot (Fig. 7B and C), cell viability was repressed, and Ki67 and PCNA expression was diminished upon YAP silencing, the effect of which was abolished by robustly induced HIF1α. Furthermore, flow cytometric analysis results (Fig. 7D) revealed that cell apoptosis was stimulated without YAP, but opposite results were found upon HIF1α overexpression. Overall, the above results show that YAP silencing could down-regulate HIF1α expression, inhibit the viability of 6-OHDA-exposed MN9D cells, and augment cell apoptosis. Conversely, overexpression of HIF1α could reverse the pro-apoptotic effect of YAP silencing on 6-OHDA-exposed MN9D cells and protect MN9D cells against 6-OHDA-induced cell death.
JMJD3 silencing inactivates the SNAI2/YAP/HIF1α signaling axis, thus inhibiting viability of 6-OHDA-exposed MN9D cells and stimulating cell apoptosis
The aforementioned results encouraged us to validate the effect of JMJD3 on the viability and apoptosis of 6-OHDA-exposed MN9D cells by regulating the SNAI2/YAP/HIF1α signaling axis. A decline was found in the expression of JMJD3, SNAI2, YAP and HIF1α in 6-OHDA-treated MN9D cells transduced with lentiviral vectors expressing sh-JMJD3; however, further treatment with or-HIF1α up-regulated HIF1α expression but did not affect JMJD3, SNAI2 and YAP expression, as revealed by western blot (Fig. 8A). Moreover, the results of the CCK-8 assay and western blot (Fig. 8B and C) showed descending cell viability trend. The result diminished Ki67 and PCNA expression in the absence of JMJD3, while contrary results were found upon further HIF1α overexpression.
JMJD3 silencing downregulates the SNAI2/YAP/HIF1α signaling axis to repress the viability of 6-OHDA-exposed MN9D cells and stimulate cell apoptosis. 6-OHDA-treated MN9D cells were transduced with lentiviral vectors expressing sh-JMJD3 or in combination with oe-HIF1α. (A) Western blot of JMJD3, SNAI2, YAP and HIF1α proteins in 6-OHDA-exposed MN9D cells. (B) The viability of 6-OHDA-exposed MN9D cells measured by CCK-8 assay. (C) Western blot of Ki67 and PCNA proteins in 6-OHDA-exposed MN9D cells. (D) Apoptosis of 6-OHDA-exposed MN9D cells measured by flow cytometry. Data are presented as mean ± standard deviation. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. Bonferroni-corrected two-way ANOVA was applied to compare data among multiple groups at varied time points. The cell experiment was repeated three times independently. P-values are provided in the histogram and indicate the significance of differences as compared with sh-NC + oe-NC or sh-JMJD3 + oe-NC.
According to the flow cytometric data (Fig. 8D), JMJD3 knockdown augmented cell apoptosis, which was negated by HIF1α overexpression. These lines of evidence demonstrate that JMJD3 silencing could decrease HIF1α expression, inhibiting the viability of 6-OHDA-exposed MN9D cells and stimulating cell apoptosis; however, overexpression of HIF1α could reverse the pro-apoptotic effect of JMJD3 silencing on 6-OHDA-exposed MN9D cells and protect MN9D cells against 6-OHDA-induced cell death.
JMJD3 silencing inactivates the SNAI2/YAP/HIF1α signaling axis, thereby alleviating PD-like phenotypes in MPTP-lesioned mice
Finally, we aimed to determine the mechanism of JMJD3 on the MPTP-lesioned mice by regulating the SNAI2/YAP/HIF1α signaling axis in vivo. Regarding the issue of transfection efficiency in vivo, we also used fluorescence microscopy to detect EGFP protein expression within mouse brain tissue (Supplementary Material, Fig. S1D). The EGFP protein expression experiment showed significant EGFP protein expression within mouse brain tissue infected with lentivirus or treated with silencing vectors for overexpression. As shown in Figure 9A, JMJD3, SNAI2, YAP and HIF1α protein expression were downregulated in the brain tissue of sh-JMJD3-treated mice. In response to sh-JMJD3 + oe-HIF1α, JMJD3, SNAI2 and YAP protein expression showed no difference, while HIF1α protein expression was up-regulated. Nissl staining results (Fig. 9B) showed a decreased number of Nissl bodies in the absence of JMJD3. Further overexpression of HIF1α increased the number of Nissl bodies. Thus, it can be concluded that JMJD3 silencing reduces Nissl bodies and promotes PD development by inhibiting the SNAI2/YAP/HIF1α signaling axis activation, which was reversed by overexpression of HIF1α.
JMJD3 silencing disrupts the SNAI2/YAP/HIF1α signaling axis to retard PD-like phenotypes in MPTP-lesioned mice. MPTP-lesioned mice were treated with sh-JMJD3 or combined with one-HIF1α. (A) Western blot of JMJD3, SNAI2, YAP, and HIF1α proteins in the brain tissue of MPTP-lesioned mice. (B) The number of Nissl bodies in the brain tissue of MPTP-lesioned mice observed by Nissl staining. Data are presented as mean ± standard deviation. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. n = 9 mice for each treatment. P-values are provided in the histogram and indicate the significance of differences as compared with sh-NC + oe-NC or sh-JMJD3 + oe-NC.
Discussion
Our results suggest that the demethylase JMJD3 of the H3K27me3 histone may play a protective role in dopaminergic neurons in Parkinson’s disease. JMJD3 augments SNAI2 expression by inhibiting the enrichment of H3K27me3 in the promoter region of SNAI2, activates the YAP/HIF1α signaling axis, and finally decelerates PD progression. Bioinformatics analysis of the current study revealed that JMJD3 may regulate SNAI2 expression activation by affecting the histone methylation in the SNAI2 promoter region. The in vivo and in vitro experiments confirmed that JMJD3 could decrease H3K27me3 enrichment in the promoter region of SNAI2 and resulted in up-regulated SNAI2 expression. Indeed, JMJD3 functions to remove the methyl groups from H3K27me3 specifically, and JMJD3 can reduce H3K27me3 at both enhancers and promoters of genes, thus promoting gene expression (25,26). Consistently, a previous study has shown that JMJD3 can activate SNAI2 expression by reducing H3K27me3 at the promoter of SNAI2 in clear cell renal cell carcinoma cells (27).
Another key observation of the current study indicated that JMJD3 silencing downregulated SNAI2 expression, inhibiting the viability of 6-OHDA-exposed MN9D cells and promoting cell apoptosis. JMJD3 is poorly expressed in the midbrain of aged mice with PD-like behaviors, while suppression of JMJD3 in N9 microglia leads to inhibited M2 polarization and exaggerated M1 microglial inflammatory responses, thus inducing extensive neuron death in vitro (9). In addition, SNAI2 has been reported to be crucial for generating and migrating neural crest cells (28), but the specific role and underlying mechanism still need to be fully elucidated. Our work is the first report revealing the downregulation of SNAI2 expression in PD, and this downregulation can cause suppressed viability of 6-OHDA-exposed MN9D cells and augmented cell apoptosis. Meanwhile, our study further revealed that this effect was associated with decreased YAP expression. In accordance with our results, previous evidence has shown that up-regulated SNAI2 expression is concurrent with the activation of YAP in metanephric mesenchyme cells (29).
Additionally, inhibition of SNAI2 triggers the downregulation of YAP expression in skeletal stem cells (22). Notably, activated YAP can promote midbrain dopaminergic neuron survival in PD by inducing the expression of miR-130a, which suppresses the synthesis of the cell death-associated protein PTEN (14). Thus, the novel SNAI2/YAP axis might exert neuroprotective effects on PD models of cells and mice and create new treatment opportunities for PD.
Additionally, the current research unveiled that YAP functioned as an upstream regulator of HIF1α, downregulated in the mouse and cell PD models, and could up-regulate HIF1α expression, stimulating viability of 6-OHDA-exposed MN9D cells and inhibiting cell apoptosis. In accordance with this finding, YAP has been reported to bind to HIF1α in the nucleus of hepatocellular carcinoma cells and sustain HIF1α protein stability (23). Published data have emphasized the correlation of HIF1α with several processes linked to PD, such as risk factors, gene mutations and molecular pathways, including oxidative stress, mitochondrial dysfunction and protein degradation impairment (30). Previous results revealed that HIF1α can prevent prion protein (PrP)-induced neurotoxicity by activating the expression of β-catenin (31). In addition, HIF1α stabilization is required for reprograming glucose metabolism and sustaining increased mouse cortical neuron proliferation (32).
Meanwhile, HIF1α exhibits low expression in PD, and its augmented expression exerts protective effects on a cellular model of PD (33). Hence, activating the JMJD3/SNAI2/YAP/HIF1α signaling axis may be a favorable treatment approach for curing PD. It is worth discussing whether any drug candidates that directly act on HIF1α affect the progression of PD. Previous evidence has indicated that arbutin, a natural glycoside, can improve the performance of PD mouse models by inhibiting the function of the A2AR and enhancing the effects of cyclic adenosine monophosphate (34). In addition, the natural source of mucuna seed powder formulation is another candidate drug for the treatment of PD due to its rapid onset of action, significant improvement in motor function and few motor disorders and adverse events, as well as its advantages over conventional levodopa preparations in the management of PD (35). Chlorogenic acid has also demonstrated cognitive and neuroprotective effects (36). However, none of them exert anti-PD effects through HIF1α. At the same time, the half-life of the HIF1α protein in the body is extremely short. The current development strategy is mainly to induce the stabilization of HIF1α, but the existing compounds usually have multi-target coverage and poor selectivity (30). Therefore, we believe the focus should be on the upstream regulatory proteins of HIF1α. It will be more efficient to intervene in JMJD3 and increase its expression as a therapeutic target.
Overall, this study identified a novel regulatory signaling axis, JMJD3/SNAI2/YAP/HIF1α, which promotes the viability of neurons and delays neuron apoptosis, ultimately alleviating PD (Fig. 10). The existing drugs for PD treatment, such as dopamine (DA) precursors, DA agonists or monoamine oxidase B (MAO-B) inhibitors, mainly lead to temporary relief of motor symptoms of PD by replenishing the lost DA. Still, the disease progression continues inexorably (37). Therefore, there is an urgent need to find effective therapeutic strategies against PD. In addition, HIF1α can affect DA production, iron metabolism, mitochondrial function, ROS production and autophagy, and it has received extensive attention as a potential candidate target (30). However, there is no drug available currently to interfere with HIF1α directly. The results of this study confirmed the feasibility of indirectly interfering with HIF1α through the JMJD3/SNAI2 axis to regulate abnormal pathways in the treatment of PD, thus providing a new mechanistic basis and molecular targets for the diagnosis and treatment of PD. Despite the contributions, the current study has several limitations. First, it should be noted whether the mechanistic understanding of the JMJD3/SNAI2/YAP/HIF1α signaling axis applies to human beings requires further verification. In addition, experimental models, such as rAAV-α-synuclein and fibril α-synuclein models, mimicking long-term neurodegeneration are needed since PD is a chronic disease. Our in vitro studies only focused on acute cell death and dysfunction in 6-OHDA-exposed MN9D cells.
Schematic representation summarizing the role of JMJD3 in PD. In normal nerve cells, JMJD3 up-regulates the expression of SNAI2 by inhibiting the enrichment of H3K27me3 in the promoter region of SNAI2, thereby activating the YAP/HIF1α signaling axis and alleviating PD. During PD, JMJD3 is down-regulated, leading to increased methylation modification of H3K27me3 in the promoter region of SNAI2, reduced expression of SNAI2 and disruption of the YAP/HIF1α signaling axis. By this mechanism, the progression of PD is promoted.
Materials and Methods
Establishment of a mouse model of MPTP-induced PD
The Animal Ethics Committee of The China Medical University approved animal experiments. ICR mice (10–12 weeks old; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were housed in a specific pathogen-free (SPF) animal room at 24–26°C, with 45–55% humidity. The food and drinking water were used after high-temperature disinfection. To induce PD models, mice received intraperitoneal injections of MPTP at a dose of 20 mg/kg/d for 14 consecutive days (38). The control mice were injected intraperitoneally with an equal volume of normal saline.
RNA extraction and sequencing
Hippocampal tissues from the normal mice and MPTP-lesioned mice were collected, and three samples were taken from each group. First, total RNA was isolated using TRIzol reagent (Invitrogen Inc., Carlsbad, CA). RNA concentration was then determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA; OD260/280) and Qubit® RNA Assay Kit. The total RNA samples exhibiting an RNA Integrity Index of ≥7.0, 28S:18S ratio ≥ 1.5 were used for subsequent experiments.
Sequencing libraries were generated and sequenced by CapitalBio Technology (Beijing, China). A total of 5 μg RNA was used for each sample. Briefly, ribosomal RNA (rRNA) was removed from the total RNA using Ribo-Zero ™ Magnetic Kit (Epicentre Technologies, Madison, Wisconsin). The sequencing libraries were constructed by NEB Next Ultra Directional RNA Library Prep Kit for Illumina. The RNA was then prepared into fragments of approximately 300 base pairs (bp) in length in 5 × NEB Next first-strand synthesis reaction buffer. Next, the first-strand cDNA was synthesized using reverse transcriptase and random primers, and the second-strand cDNA was synthesized in the second-strand synthesis reaction buffer of 10 × dUTP Mix. The end repair of cDNA fragments included the addition of a ployA tail and the connection of sequencing adaptors. After connecting the Illumina sequencing adaptors, the second strand of the cDNA was digested using USER Enzyme (NEB) to construct a strand-specific library. Next, library DNA was amplified, purified and enriched by PCR. Then, the library was identified using Agilent 2100 and quantified using the KAPA library quantification kit (KAPA Biosystems). Finally, paired-end sequencing was performed on a NextSeqCN500 (Illumina) sequencer.
Quality control and reference genome alignment of sequencing data
The quality of paired-end reads of the raw sequencing data was checked using FastQC software (Version 0.11.8). Next, cutadapt software 1.18 was applied for preprocessing of raw data. Next, the Illumina sequencing adaptors and poly (A) tail sequences were removed. Next, the reads with an N content of over 5% were removed using a perl script. Next, the reads with a 70% base mass above 20 were extracted using FASTX Toolkit software (Version 0.0.13). Next, two-end sequences were repaired using BBMap software. Finally, the filtered high-quality reads fragment was aligned with the reference genome using HISAT2 software (Version 0.7.12).
Bioinformatics analysis
Differential analysis was conducted to screen the differentially expressed genes based on mRNA read counts using R ‘edgeR’ package with |log2FC| > 1, P-value < 0.05 as the threshold. KEGG enrichment analysis of the differentially expressed mRNAs was performed using the R ‘ClusterProfiler’ package. The significant enrichment pathways were considered with P < 0.05. STRING database was used for protein–protein interaction (PPI) analysis between genes, and Cytoscape software was applied to visualize the PPI network.
In vivo experimental protocols
Different lentiviral vectors were used in the study. GV287 and GV248 were purchased from Genechem, Shanghai, China, as overexpression and lentiviral interference vectors, respectively. GV287 has a full length of 10.4 kb and contains an ampicillin resistance gene, green fluorescent protein (GFP) marker and FLAG-tagged protein. GV248 has a full length of 11.5 kb and contains an ampicillin resistance gene, GFP marker and FLAG-tagged protein, as well as a random scramble sequence as a control. In addition, lentiviral vectors for JMJD3 knockdown (sh-NC) and HIF-1α overexpression (oe-NC) were also purchased from Genechem, Shanghai, China. In vivo, a transfection experiment was performed on day 12 after birth by injecting 1 μl of lentivirus into the mouse subdural space through the cranial opening at a speed of 0.5 μl/min (39).
A total of 9 mice were included in each group, including the Normal group, PD group, sh-NC + oe-NC group (PD mice treated with both overexpression and interference random scramble lentiviral vectors), sh-JMJD3 + oe-NC group (PD mice treated with overexpression control lentiviral vector and knockdown JMJD3 lentiviral vector), and sh-JMJD3 + oe-HIF1α group (PD mice treated with JMJD3 knockdown and HIF-1α overexpression lentiviral vectors). NC stands for Negative Control, oe stands for overexpression, and sh-RNA stands for short hairpin RNA.
Nissl staining
Mice were euthanized by cervical dislocation under anesthesia, and the brain tissue was harvested for optimum cutting temperature embedding and sectioned. The subsequent detailed steps referred to the previously described method (40).
Western blot
Total protein was extracted from brain tissues or MN9D cells, separated and transferred onto membranes. The membrane was blocked with 5% skim milk powder at room temperature for 1 h and probed overnight at 4°C with primary rabbit antibodies (Supplementary Material, Table S1) and then with secondary HRP-labeled goat anti-rabbit IgG H&L (HRP) (ab97051, 1:2000, Abcam, Cambridge, UK) at room temperature for 1 h. Then chemiluminescence reagent (LAS4000, GE Healthcare, Milwaukee, Wisconsin) was used to visualize the protein bands. Image Pro Plus 6.0 software was applied for band intensity quantification (41).
Immunohistochemistry
Paraffin sections of mouse brain tissues were subjected to antigen retrieval and then blocked with 5% goat serum (Solarbio, Beijing, China). Subsequently, sections were immunostained with primary antibodies (Supplementary Material, Table S1) at 4°C overnight (42). The next day, the sections were incubated with secondary goat anti-rabbit (1:500, ab150077, Abcam) at 37°C for 1 h and developed with DAB (ZSGB-BIO, China). The true color multi-functional cell image analysis management system (Image-Pro Plus, Media Cybernetics, Bethesda, MD) was used for analysis. The percentage of positive cells was counted under an optical microscope and averaged.
ChIP-qPCR assay
EZ ChIP™ kit (Millipore, Germany) was used for this assay. MN9D cells were fixed with 1% formaldehyde and incubated with glycine for 10 min to produce DNA-protein crosslinks. Then, cells were lysed with 100 μl SDS lysis buffer with proteinase inhibitor cocktail II and sonicated to produce 200–1000 bp chromatin fragments. Next, lysates were immunoprecipitated with rabbit polyclonal antibody against JMJD3 (1:200, ab38113, Abcam), rabbit monoclonal antibody against H3K27me3 (1:200, ab192985, Abcam), and NC rabbit antibody against IgG (1:100, ab172730, Abcam). The purified DNA products were subjected to PCR detection to confirm whether the DNA precipitated by the antibody containing the DNA sequence and relative content of the target gene (43). Primers for RT-qPCR are listed in Supplementary Material, Table S2.
In vitro experimental protocols
Parkinson’s disease is characterized by the loss of dopaminergic neurons in the substantia nigra of the human brain, increased oxidative stress and loss of glutathione in the midbrain region. Therefore, we used the mouse midbrain cell line MN9D (44). The mouse MN9D cell line (Cat. No. CRL-1721) was obtained from ATCC, and the supplier confirmed the cell type and the absence of pathogen contamination. The JMJD3 inhibitor GSKJ4 (product number: SML0701) was purchased from Sigma-Aldrich in the USA and cultured in DMEM medium (SLM-243-B, Sigma-Aldrich, USA) containing 10% FBS (Gibco, USA) at 37°C and 5% CO2 in a cell culture incubator.
One day before transfection, MN9D mouse cells were seeded in six-well cell culture plates and incubated in endothelial cell-specific complete growth medium (2 ml) at 37°C incubator with 5% CO2 until the cell density reached 80%. The cells were then infected according to the lentivirus infection protocol provided by GeneChem. A suitable amount of lentivirus with a virus titer (multiplicity of infection, MOI) of 10 was added to the cell culture plate (45) and incubated at 37°C and 5% CO2 overnight. After 48 h of infection, the culture media were replaced with fresh media. After 36 h of infection, cells were treated with ampicillin to select cells with ampicillin-resistant genes (46).
The in vitro cell experiment was divided into the following groups: oe-NC, sh-NC or-JMJD3 (JMJD3 overexpression lentivirus), sh-JMJD3 (JMJD3 interference lentivirus), sh-NC + oe-NC (lentivirus of blank overexpression and random sequence control), sh-JMJD3 + oe-NC (lentivirus of blank control and lentivirus of JMJD3 interference), sh-JMJD3 + oe-SNAI2 (lentivirus of SNAI2 overexpression and lentivirus of JMJD3 interference), sh-JMJD3 + oe-HIF1α (lentivirus of HIF1α overexpression and lentivirus of JMJD3 interference), sh-SNAI2 + oe-NC (lentivirus of blank overexpression control and lentivirus of SNAI2 interference), sh-SNAI2 + oe-YAP (lentivirus of YAP overexpression and lentivirus of SNAI2 interference), sh-YAP + oe-NC (lentivirus of blank overexpression control and lentivirus of YAP interference) and sh-YAP + oe-HIF1α (lentivirus of HIF1α overexpression and lentivirus of YAP interference).
JMJD3 overexpression (oe), JMJD3 interference lentivirus (short hairpin RNA, sh-RNA), SNAI2 overexpression lentivirus, SNAI2 interference lentivirus, YAP overexpression lentivirus, YAP interference lentivirus, HIF-1α overexpression lentivirus and interference control, and blank control lentivirus were all purchased from GeneChem (Shanghai, China).
CCK-8 assay
MN9D cells were seeded in 96-well plates at 1 × 105 cells/ml (100 μl per well) in a 37°C incubator containing 5% CO2. At 12, 24 and 48 h, cells in each well were incubated with 10 μl CCK-8 solution (CA1210, Solarbio) at 37°C for 4 h. The OD value of each well was detected with a microplate reader (elx800, Bio-Tek, Vermont) at 450 nm (47).
Flow cytometry
The suspended cells were directly collected into a 10-ml centrifuge tube at 1–5 × 106/ml and centrifuged at 500–1000 r/min for 5 min. Centrifugation was conducted at 500–1000 r/min for 5 min. Next, cells were resuspended in 100 μl of labeled solution and incubated in the dark for 10–15 min at room temperature. Following centrifugation at 500–1000 r/min for 5 min, cells were incubated with fluorescent (SA-FLOUS) solution at 4°C for 20 min in the dark. Cells were excited at 488 nm and examined at 515 and 560 nm for FITC and PI fluorescence.
Statistical analysis
Measurement data are presented as mean ± standard deviation. All statistical analyses in this study were performed using SPSS 21.0 software (IBM Corp. Armonk, NY). The significance of differences in data obeying normal distribution and homogeneity of variance between the two groups was analyzed using an unpaired t-test. One-way ANOVA with Tukey’s test was applied for multi-group data comparison. Bonferroni-corrected two-way ANOVA was applied to compare data among multiple groups at varied time points. A P-value < 0.05 was considered significant.
Acknowledgement
Not applicable.
Conflict of Interest statement. The authors declare no conflict of interest.
Funding
Not applicable.
Authors’ Contribution
L.D. designed the study. L.D. and L.B.G. collated the data, carried out data analyses and produced the initial draft of the manuscript. L.B.G. contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.
Data Availability
The article’s data will be shared on reasonable request to the corresponding author.
