Weizhao Chen,Qiongping Zheng,Qiaoying Huang,Shanshan Ma,Mingtao Li
Lineage reprogramming of resident glial cells to dopaminergic neurons (DAns) is an attractive prospect of the cell-replacement therapy for Parkinson’s disease (PD). However, it is unclear whether repressing polypyrimidine tract binding protein 1 (PTBP1) could efficiently convert astrocyte to DAns in the substantia nigra and striatum. Although reporter-positive DAns were observed in both groups after delivering the adeno-associated virus (AAV) expressing a reporter with shRNA or CRISPR-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage-tracing method. By adopting stringent lineage-tracing strategy, two other studies show that either knockdown or genetic deletion of quiescent astroglial PTBP1 fails to obtain induced DAns under physiological condition. However, the role of reactive astrocytes might be underestimated because upon brain injury, reactive astrocyte can acquire certain stem cell hallmarks that may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage-tracing method to investigate whether reactive astrocytes could be converted to DAns in a 6-hydroxydopamine (6-OHDA) mouse model of PD. However, we found that no astrocyte-originated DAn was generated after effective and persistent knockdown of astroglial PTBP1 either in the substantia nigra or in striatum, while AAV ‘leakage’ to nearby neurons was easily observed. Our results confirm that repressing PTBP1 does not convert astrocytes to DAns, regardless of physiological or PD-related pathological conditions.
Background The RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1), also known as heterogeneous nuclear ribonucleoprotein I (hnRNP I), mediates gene expression through splicing regulation. Its role in virus infection is undefined. Results We show that genetic ablation of PTBP1 renders cell resistant to herpes simplex virus 1 (HSV-1) infection. HSV-1 utilizes 3-O-sulfated heparan sulfate proteoglycans (HSPGs) for attachment and for infection of epithelial cells. We found that knockout of PTBP1 expression resulted in loss of HS3ST3A1 and HS3ST3B1, heparan sulfate glucosaminyl 3-O-sulfotransferase genes for 3-O-sulfation of the heparan sulfate (HS) chains of HSPGs. Each of the HS3ST3A1/HS3ST3B1 genes is composed of 2 exons separated by an extraordinarily long intron whose removal requires PTBP1-associated looping. We found that PTBP1 interacted with the intronic region of HS3ST3A1/HS3ST3B1 pre-mRNAs and modulated their processing to mRNA. The essential role of PTBP1 in functional HS3ST3A1 expression and in HSV-1 infection was demonstrated by ectopic re-expression in the knockout (ko) cells. In addition, we showed that targeting PTBP1 expression by microRNA mimics reduced disease symptoms in a mouse herpetic stromal keratitis (HSK) model. Conclusions The results demonstrate that PTBP1 mediates HSV-1 infection of epithelial cells through splicing regulation of HS3ST3A1/HS3ST3B1. These studies provide a new area for novel therapeutic strategies through splicing regulation. Graphical abstract By Sun et al. RNA-binding protein PTBP1 mediates HSV-1 attachment and infection through regulation of heparan sulfate 3-O-sulfotransferase gene expression.
Hou Xiaoxiao,Chen Ying,Xu Qixun,Zhong Jiaxin,Long Yudan,Liu Kai
Myocardial ischemia-reperfusion (I/R) injury is a complex condition characterized by oxidative stress, inflammation, and mitochondrial dysfunction. Ferroptosis, an iron-dependent form of regulated cell death, plays a critical role in cardiomyocyte damage during I/R. Inhibiting ferroptosis has been shown to reduce myocardial injury and improve cardiac function, making it a promising therapeutic target for enhancing clinical outcomes. An in vivo I/R model was established, and infarct size was assessed using triphenyltetrazolium chloride (TTC) staining. Histological changes were analyzed using hematoxylin and eosin (H&E) staining, Masson, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Cellular experiments included analyses of reactive oxygen species (ROS), lipid peroxidation, and iron content using specific fluorescent probes, as well as enzymatic markers measured with commercial assay kits. Cell viability and senescence were evaluated using the Cell Counting Kit-8 (CCK-8) assay and senescence-associated β-galactosidase (SA–β-gal) staining, respectively. Mitochondrial ultrastructure was examined using transmission electron microscopy (TEM), while molecular mechanisms, including DNA methylation, were investigated using methylation-specific polymerase chain reaction (MSP). Interactions among pleckstrin homology-like domain family A member 3 (PHLDA3), DNA (cytosine-5)-methyltransferase 3 A (DNMT3a), and polypyrimidine tract binding protein 1 (PTBP1) were evaluated using chromatin immunoprecipitation (ChIP) and RNA pull-down assays. Silencing of PHLDA3 demonstrated protective effects against I/R injury by attenuating cardiomyocyte ferroptosis and cardiac microvascular endothelial cells (CMECs) senescence under hypoxia/reoxygenation (H/R) conditions in vitro and I/R injury in vivo. Mechanistically, downregulated DNMT3a was responsible for PHLDA3 hypomethylation. Furthermore, PTBP1 was identified as an upstream RNA-binding protein that destabilized DNMT3a mRNA during H/R injury, indirectly enhancing PHLDA3 expression. Our findings suggest that PTBP1-mediated DNMT3a downregulation contributes to PHLDA3 hypomethylation, which may promote cardiomyocyte ferroptosis and subsequent microvascular endothelial cell senescence during I/R injury.
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