Salsolinol as an RNA m6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

Dong W., Liu S., Li S., Wang Z.

Abstract Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease. The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease, which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice. However, ethical issues and tumor formation were limitations of its clinical application. Induced pluripotent stem cells can be acquired without sacrificing human embryos, which eliminates the huge ethical barriers of human stem cell therapy. Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons, without the need for intermediate proliferation states, thus avoiding issues of immune rejection and tumor formation. Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease. However, there are also ethical concerns and the risk of tumor formation that need to be addressed. This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease, focusing on the use of induced pluripotent stem cells in cell replacement therapy, including preclinical animal models and progress in clinical research. The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease, as well as the controversy surrounding in vivo reprogramming. These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.

Park J.M., Rahmati M., Lee S.C., Shin J.I., Kim Y.W.

Abstract Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, and although restoring striatal dopamine levels may improve symptoms, no treatment can cure or reverse the disease itself. Stem cell therapy has a regenerative effect and is being actively studied as a candidate for the treatment of Parkinson's disease. Mesenchymal stem cells are considered a promising option due to fewer ethical concerns, a lower risk of immune rejection, and a lower risk of teratogenicity. We performed a meta-analysis to evaluate the therapeutic effects of mesenchymal stem cells and their derivatives on motor function, memory, and preservation of dopaminergic neurons in a Parkinson's disease animal model. We searched bibliographic databases (PubMed/MEDLINE, Embase, CENTRAL, Scopus, and Web of Science) to identify articles and included only peer-reviewed in vivo interventional animal studies published in any language through June 28, 2023. The study utilized the random-effect model to estimate the 95% confidence intervals (CI) of the standard mean differences (SMD) between the treatment and control groups. We use the systematic review center for laboratory animal experimentation's risk of bias tool and the collaborative approach to meta-analysis and review of animal studies checklist for study quality assessment. A total of 33 studies with data from 840 Parkinson's disease model animals were included in the meta-analysis. Treatment with mesenchymal stem cells significantly improved motor function as assessed by the amphetamine-induced rotational test. Among the stem cell types, the bone marrow MSCs with neurotrophic factor group showed largest effect size (SMD [95% CI] = −6.21 [−9.50 to −2.93], P = 0.0001, I 2 = 0.0 %). The stem cell treatment group had significantly more tyrosine hydroxylase positive dopaminergic neurons in the striatum ([95% CI] = 1.04 [0.59 to 1.49], P = 0.0001, I 2 = 65.1 %) and substantia nigra (SMD [95% CI] = 1.38 [0.89 to 1.87], P = 0.0001, I 2 = 75.3 %), indicating a protective effect on dopaminergic neurons. Subgroup analysis of the amphetamine-induced rotation test showed a significant reduction only in the intracranial-striatum route (SMD [95% CI] = −2.59 [−3.25 to −1.94], P = 0.0001, I 2 = 74.4 %). The memory test showed significant improvement only in the intravenous route (SMD [95% CI] = 4.80 [1.84 to 7.76], P = 0.027, I 2 = 79.6 %). Mesenchymal stem cells have been shown to positively impact motor function and memory function and protect dopaminergic neurons in preclinical models of Parkinson's disease. Further research is required to determine the optimal stem cell types, modifications, transplanted cell numbers, and delivery methods for these protocols.

Zhang D., Zhang J., Wang Y., Wang G., Tang P., Liu Y., Zhang Y., Ouyang L.

Wang Y., Wu S., Li Q., Lang W., Li W., Jiang X., Wan Z., Sun H., Wang H.

Endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroiso-quinoline (Salsolinol, SAL) is a dopamine metabolite that is toxic to dopaminergic neurons in vitro and in vivo, and is involved in the pathogenesis of Parkinson’s disease (PD). However, the molecular mechanism by which SAL induces neurotoxicity in PD remains challenging for future investigations. This study found that SAL induced neurotoxicity in SH-SY5Y cells and mice. RNA sequencing (RNAseq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to detect differentially expressed genes in SAL-treated SH-SY5Y cells. We found that NLR family pyrin domain-containing 3 (NLRP3)-dependent pyroptosis was enriched by SAL, which was validated by in vitro and in vivo SAL models. Further, NLRP3 inflammasome-related genes (ASC, NLRP3, active caspase 1, IL-1β, and IL-18) were increased at the mRNA and protein level. Acteoside mitigates SAL-induced neurotoxicity by inhibiting NLRP3 inflammasome-related pyroptosis in in vitro and in vivo PD models. In summary, the present study suggests for the first time that NLRP3-dependent pyroptosis plays a role in the pathogenesis of SAL-induced PD, and acteoside mitigates SAL-induced pyroptosis-dependent neurotoxicity in in vitro and in vivo PD models. The present results demonstrated a new mechanism whereby SAL mediates neurotoxicity by activating NLRP3-dependent pyroptosis, further highlighting SAL-induced pyroptosis-dependent neurotoxicity as a potential therapeutic target in PD.

Yu Z., Huang L., Xia Y., Cheng S., Yang C., Chen C., Zou Z., Wang X., Tian X., Jiang X., Zhou L.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders of aging that impairs predominately dopaminergic neurons. N6-methyladenosine (m6A) is the most prevalent form of internal RNA modification in eukaryotes and it plays an essential role in normal brain development and neurodegenerative diseases. The m6A status is dynamically modulated by diverse types of genes called "writers", "erasers" and "readers". However, whether these m6A regulators are perturbed in PD remains poorly understood. To clarify this point, we established a PD mouse model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The motor as well as learning and memory ability of mice were evaluated through and rotarod and Y maze spontaneous alternation tests. Morphological characteristics of tyrosine hydroxylase (TH)-positive cells were visualized using immunohistochemistry, while expressions of alpha-synuclein (α-syn) and TH were determined by using western blot. Furthermore, the expressions of the m6A regulators in the substantia nigra and striatum were evaluated by using qRT-PCR and western blot. As a result, the MPTP-induced PD mice suffered from learning and memory as well as motor defects. Additionally, there were significant TH+ neuron losses in the substantia nigra and striatum of MPTP-injected mice. In the PD mice, proteins including ALKBH5, IGF2BP2 were up-regulated in the substantia nigra, while YTHDF1 and FMR1 was down-regulated. For the striatum, FMR1 and CBLL1 were up-regulated, while IGF2BP3, METTL3 and RBM15 were down-regulated. The expression of genes at the mRNA level were partially in accordance with the protein changes. These findings indicate the m6A regulators may participate in PD pathogenesis.

Yin S., Liu W., Ji C., Zhu Y., Shan Y., Zhou Z., Chen W., Zhang L., Sun Z., Zhou W., Qian H.

As nanoscale membranous vesicles, human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) have attracted extensive attention in the field of tissue regeneration. Under the premise that the mechanisms of hucMSC-sEVs on the treatment of diabetic kidney disease (DKD) have not been revealed clearly, we constructed DKD rat model with success. After tail vein injection, hucMSC-sEVs effectively reduced blood glucose, maintained body weight and improved renal function in DKD rats. Notably, we found that hucMSC-sEVs suppressed YAP expression in renal cortical regions. Further in vitro experiments, we confirmed that the expression of YAP in the nucleus of renal podocytes was increased, and the level of autophagy was inhibited in the high-glucose environment, which could be reversed by intervention with hucMSC-sEVs. We screened out the key protein 14-3-3ζ, which could not only promote YAP cytoplasmic retention instead of entering the nucleus, but also enhance the level of autophagy in the cytoplasm. Ultimately, excessive YAP protein was removed by autophagy, a classic way of protein degradation. In conclusion, our study provides new strategies for the prevention of DKD and proposes the possibility of hucMSC-sEVs becoming a new treatment for DKD in the future.

Su Q., Chen N., Tang J., Wang J., Chou W., Zheng F., Shao W., Yu G., Cai P., Guo Z., He M., Li H., Wu S.

Paraquat (PQ) is a ubiquitously applied herbicide. Long-term PQ exposure with low dose has been reported to induce abnormal expression of long non-coding RNAs (lncRNAs) in brain nerve cells, which could further lead to Parkinson's disease (PD). N6-methyladenosine (m6A) modification has recently been identified as having an important role in regulating the function of lncRNAs. However, how m6A modification regulates lncRNAs following PQ exposure remains largely unknown. Herein, this study reported m6A modification of lncRNAs in mouse neuroblastoma cells (Neuro-2a) following PQ induced reactive oxide species (ROS). M6A sequencing was performed to explore the m6A modificated pattern of lncRNAs in Neuro-2a cells which were treated with 200 μM PQ for 3 h. It was found that PQ hypermethylated total RNA and changed the expression of m6A methyltransferase and demethylase proteins, which leading to the alteration of m6A modification of lncRNAs. Furthermore, the functional analysis further revealed that N-acetyl-L-cysteine (NAC)，a ROS scavengers, partly reversed PQ-induced distinct m6A modificated pattern of lncRNAs. In addition, tow specific m6A modified lncRNAs were identified: cell division cycle 5-like (lncRNA CDC5L) and signal transducer and activator of transcription 3 (lncRNA STAT3), which could influence downstream autophagy related biological function. In summary, this work could potentially contribute to the new insight of lncRNAs m6A modification mechanism in the field of environmental toxicology.

Shen D., Wang B., Gao Y., Zhao L., Bi Y., Zhang J., Wang N., Kang H., Pang J., Liu Y., Pang L., Chen Z., Zheng Y., Liu H.

N 6-Methyladenosine (m 6 A) is the most abundant internal modification in eukaryotic mRNA, playing critical role in various bioprocesses. Like other epigenetic modifications, m 6 A modification can be catalyzed by the methyltransferase complex and erased dynamically to maintain cells homeostasis. Up to now, only two m 6 A demethylases have been reported, fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5), involving in a wide range of mRNA biological progress, including mRNA shearing, export, metabolism and stability. Furthermore, they participate in many significantly biological signaling pathway, and contribute to the progress and development of cancer along with other diseases. In this review, we focus on the studies about structure, inhibitors development and biological function of FTO and ALKBH5. This review systematically describes the chemical and biological functions of RNA demethylases FTO and ALKBH5, from their crystal structure to inhibitors development and screening, demethylation mechanism and substrates to biological functions and roles in diseases.

Qi Z., Wang S., Li J., Wen Y., Cui R., Zhang K., Liu Y., Yang X., Zhang L., Xu B., Liu W., Xu Z., Deng Y.

One of the major environmental factors that induce PD is Manganese (Mn). Cellular and molecular mechanism of parkinsonism caused by Mn has not been explored clearly. The results of in vivo and in vitro experiments showed that Mn exposure caused abnormal projection of dopaminergic neurons and decreased mRNA expression and protein levels of FTO. This is due to Mn-induced the upregulation of Foxo3a. Using the cell model of overexpression of FTO, we found that FTO could antagonize Mn-induced the down-regulation of axon guidance molecule ephrin-B2 through RNA-seq, MeRIP-qPCR, and RT-qPCR experiments. Through RIP-seq and actinomycin D experiments, it was found that FTO can up-regulate the mRNA m6A level of ephrin-B2, which can be recognized by YTHDF2 and degraded. Finally, it is proved that Mn induces dopaminergic neurons projection injury and motor dysfunction through Foxo3a/FTO/m6A/ephrin-B2/YTHDF2 signal pathway.

Ke W., Huang Z., Peng C., Ke Y.

Reperfusion therapy after acute myocardial infarction can induce myocardial ischemia-reperfusion injury (IRI). Novel evidence has illustrated that N6-methyladenosine (m6A) modification modulates the myocardial IRI progression. Here, our study focuses on the role of m6A methyltransferase fat mass and obesity-associated protein (FTO) in myocardial ischemia/reoxygenation injury and explores potential regulatory mechanisms. Results discovered that FTO down-expressed in myocardial IRI mice and hypoxia/reoxygenation (H/R)-induced cardiomyocytes. Functionally, FTO overexpression attenuated the H/R-induced apoptosis and inflammation of cardiomyocytes. Mechanistically, methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) assay and RIP assay revealed that Yap1 mRNA acted as the target of FTO in cardiomyocytes. Moreover, FTO uninstalled the methylation of Yap1 mRNA, and enforced the stability of Yap1 mRNA. Taken together, our study reveals the role of FTO in H/R-induced myocardial cell injury via m6A-dependent manner, which may provide a new approach to improve myocardial IRI.

Pei T., Luo B., Huang W., Liu D., Li Y., Xiao L., Huang X., Ouyang Y., Zhu H.

We first reported that the Hippo-YAP signaling pathway plays a critical role in the pathogenesis of endometriosis (EMS). Autophagy is also related to the invasion ability of endometrial cells and is involved in the pathogenesis of EMS through multi-levels. However, the precise regulatory mechanism of YAP on autophagy in the eutopic endometrial stromal cells (ESCs) is still unclear. Primary eutopic ESCs of EMS patients (n = 12) and control patients without EMS (n = 9) were isolated and cultured to investigate the expressions of YAP and mTOR, the role of YAP in autophagy, and the effect of the YAP-autophagy signal on the decidualization of the eutopic ESCs. Endometriosis-related sequencing data (GSE51981) in the GEO database were used to find the genes significantly correlated with YAP. We found 155 genes with significant differences in the interaction with YAP in EMS from the dataset, and the autophagy pathway was significantly enriched. Following on from our previous studies of YAP knockdown, overexpression of YAP resulted in an increased expression of mTOR and decreased ratio of LC3-II/LC3-I and autophagy markers, in the eutopic ESCs; transmission electron microscope observation also showed fewer autophagosomes compared with the control cells. Furthermore, ESCs of the Rapamycin-treated group showed significant decidual-like changes with significantly increased decidual prolactin level at 72 h after in vitro decidualization. These results demonstrate that the increased YAP inhibited the level of autophagy by upregulating the mTOR signal in the eutopic ESCs of endometriosis. The YAP-autophagy signal plays an important role in the pathogenesis of endometriosis-associated infertility.

Zhang N., Ding C., Zuo Y., Peng Y., Zuo L.

N6-methyladenosine (m6A) is a dynamic reversible methylation modification of the adenosine N6 position and is the most common chemical epigenetic modification among mRNA post-transcriptional modifications, including methylation, demethylation, and recognition. Post-transcriptional modification involves multiple protein molecules, including METTL3, METTL14, WTAP, KIAA1429, ALKBH5, YTHDF1/2/3, and YTHDC1/2. m6A-related proteins are expressed in almost all cells. However, the abnormal expression of m6A-related proteins may occur in the nervous system, thereby affecting neuritogenesis, brain volume, learning and memory, memory formation and consolidation, etc., and is implicated in the development of diseases, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, depression, epilepsy, and brain tumors. This review focuses on the functions of m6A in the development of central nervous system diseases, thus contributing to a deeper understanding of disease pathogenesis and providing potential clinical therapeutic targets for neurological diseases.

An Y., Duan H.

Metabolic reprogramming is one of the main characteristics of malignant tumors, which is due to the flexible changes of cell metabolism that can meet the needs of cell growth and maintain the homeostasis of tissue environments. Cancer cells can obtain metabolic adaptation through a variety of endogenous and exogenous signaling pathways, which can not only promote the growth of malignant cancer cells, but also start the transformation process of cells to adapt to tumor microenvironment. Studies show that m6A RNA methylation is widely involved in the metabolic recombination of tumor cells. In eukaryotes, m6A methylation is the most abundant modification in mRNA, which is involved in almost all the RNA cycle stages, including regulation the transcription, maturation, translation, degradation and stability of mRNA. M6A RNA methylation can be involved in the regulation of physiological and pathological processes, including cancer. In this review, we discuss the role of m6A RNA methylation modification plays in tumor metabolism-related molecules and pathways, aiming to show the importance of targeting m6A in regulating tumor metabolism.

Li D., Huang C., Zhang G., Zhou L.

N6-methyladenosine (m6A) is the most common internal reversible modification of mRNA, which occurs on the N6 nitrogen of adenosine. Fat mass and obesity-associated (FTO) is a demethylase that erases m6A modification and has recently been linked to cancer. Herein, we explored the role of FTO in oral squamous cell carcinoma (OSCC). High FTO mRNA and protein levels were observed in OSCC cell lines and tissues as compared to normal controls. OSCC patients with high FTO displayed larger tumor size, higher TNM stage, poorer differentiation, and shorter survival time than those with low FTO. Stable knockdown of FTO inhibited OSCC cell viability, colony formation, and tumor growth. Further, FTO depletion increased YAP1 m6A modification at mRNA 3'-untranslated region, accelerating the degradation of YAP1 mRNA, a well-documented oncogene promoting OSCC progression. Importantly, nucleocytoplasmic shuttling of FTO is critical for YAP1 mRNA demethylation and decay following YTHDF2 reading and recognition. Our results highlight the role of FTO in regulating YAP1 mRNA stability, and targeting of FTO/YAP1 axis may be a promising intervention for OSCC patients.

Chen N., Tang J., Su Q., Chou W., Zheng F., Guo Z., Yu G., Shao W., Li H., Wu S.

Paraquat (PQ), a widely used herbicide and well-known oxidative stress inducer, has been linked to numerous neurodegenerative diseases, but the underlying mechanism(s) remains unknown. Circular RNAs (circRNAs) have recently been reported to be associated with oxidative stress in Parkinson's disease. Herein, we performed methylated RNA immunoprecipitation and RNA sequencing assays for mouse neuroblastoma (Neuro-2a) cells and successfully established a positive link between the alteration of circRNAs driven by m6A modification and PQ-induced oxidative stress. We observed oxidative stress and antioxidative stress present distinct m6A modification pattern of circRNAs as well as biological effect. Gene ontology and pathway analysis predicted that differentially m6A-methylated and expressed circRNAs are highly clustered in pathways associated with function and development of nervous system, including axon cargo transport, nervous system development, long-term potentiation, and neurotrophic signaling pathways. Moreover, we demonstrated that the alteration of m6A-methylated circRNAs upon PQ exposure could be partially reversed by N-acetylcysteine pretreatment. The mechanistic analysis further demonstrated that N-acetylcysteine pretreatment attenuated the decreased expression of target genes (UBC and PPP2CA) induced by PQ. These findings revealed distinct patterns of differentially m6A-modified circRNAs, indicating that m6A could participate in a specific regulatory network of circRNAs to modulate the expression of downstream genes in response to PQ-induced oxidative stress. In conclusion, our work established a link between m6A modification of circRNAs and PQ-induced oxidative stress, and further studies are required to explore the underlying molecular mechanisms associated with PQ-induced neurotoxicity.

Liu W., Zou X., Zheng Y., Zhang Y., Cui G., Liu S., Sun C., Peng C.

BackgroundChronic heart failure (CHF) is one of the leading causes of high mortality worldwide. It is characterized by pathological hypertrophy and poses a major threat to human health. Aconiti Lateralis Radix Praeparata is widely used in ancient China to treat CHF. However, the pathology is obscured, necessitating further exploration.MethodsProspective targets were predicted by network analysis. A transverse aortic constriction (TAC) mice model was subsequently constructed to determine the effects of aqueous extract of Aconiti Lateralis Radix Praeparata (AEA) on CHF. The echocardiography was performed to investigate cardiac function. Histopathological analysis of cardiac tissue was conducted to assess myocardial fibrosis. Nontargeted metabolomics was performed to analyze serum metabolites. The phosphorylation level of PI3K and AKT, and downstream targets such as Bnip3, p62, Atg5, and LC3II were measured by Western blotting. In vitro, norepinephrine (NE) was used to stimulate cardiac hypertrophy. Parameters such as reactive oxygen species levels, mitochondrial membrane potential, ATP concentration, and CK/MB content were detected in H9c2 cells.ResultsAEA significantly alleviated CHF. Network analysis indicated the participation of AKT in CHF, and was modulated by Aconiti Lateralis Radix Praeparata. In vivo, AEA administration effectively ameliorated cardiac performance, evidenced by the elevation of ejection fraction. Histopathological analysis displayed a diminishment of collagen fiber. Metabolomics analysis showed that several metabolites such as tetrahydroxycorticosterone, decylubiquinone and taurocholic acid were increased in the TAC mice serum. Additionally, the phosphorylation levels of PI3K and AKT, and expression levels of Drp1, Opa1, Bnip3, p62, Atg5 and LC3II were altered in TAC group. In vitro, NE stimulation increased the cell surface area and deteriorated mitochondrial functions in H9c2 cells. However, AEA administration partially reversed such results, and the mechanism was associated with mitophagy.ConclusionThis study revealed that AEA improved cardiac function via the PI3K/AKT/Bnip3 pathway.

Wang H., Wu S., Jiang X., Li W., Li Q., Sun H., Wang Y.

Luo X., He C., Yang B., Yin S., Li K.

The study was designed to investigate the impact of N6-methyladenosine (m6A) writer Wilms tumor 1-associated protein (WTAP) on the progression of atherosclerosis (AS) and to further elucidate its possible regulatory mechanism. The m6A levels and WTAP expressions were initially assessed through RIP, qRT-PCR, and western blotting. An in vitro model of AS was constructed by ox-LDL treatment in RAW264.7 cells. Next, the impact of WTAP on macrophage pyroptosis and M1 polarization was evaluated. The relationship between WTAP and NLRP3 was then investigated using m6A modification quantification and RIP-qPCR assay. To investigate the effect of WTAP on AS development in vivo, we created an ApoE−/−mouse model of AS by feeding high-fat diet (HFD). Furthermore, the influence of WTAP on macrophage pyroptosis and M1 polarization through NLRP3 was explored by NLRP3 overexpression AAV injection. Here, we found that WTAP was significantly upregulated in peripheral blood mononuclear cells (PBMCs) from AS patients, accompanied by increased total m6A methylation levels. The silencing of WTAP suppressed macrophage pyroptosis and M1 polarization induced by ox-LDL and also ameliorated aortic root lesion damage in AS mice. Mechanistically, m6A modification mediated by WTAP enhanced NLRP3 mRNA stabilization, thereby upregulating NLRP3 expression. Overexpression of NLRP3 was found to enhance macrophage pyroptosis and M1 polarization, contributing to the progression of AS. In conclusion, our findings suggest that WTAP knockdown mitigated AS progression by modulating NLRP3 in an m6A-dependent manner. Our study proposes that targeting WTAP could be a potential preventive and therapeutic strategy for AS patients.

Song L., Liu H., Yang W., Yin H., Wang J., Guo M., Yang Z.