The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC–ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NH4HCO3) as a mobile phase additive for UHPLC–MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7–24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 105 cells. The developed UHPLC–MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.
As is the case with neurodegenerative diseases , abnormal accumulation of aggregated proteins in neurons and glial are also known to implicate in the pathogenesis of ischemic stroke . However, the potential role of protein aggregates in brain ischemia remains largely unknown. Fused in Sarcoma (FUS) protein has a vital role in RNA metabolism and regulating cellular homeostasis . FUS pathology has been demonstrated in the formation of toxic aggregates and critically affecting cell viability in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but whether this also applies to neurological injury following cerebral ischemia is unclear. Herein, we demonstrated a critical role of aggregated FUS in astrocyte activation caused by cerebral ischemia and a possible underlying molecular mechanism. Cerebral ischemic injury significantly induced the formation of cytoplasmic FUS aggregates in reactive astrocytes and injured neurons, thereby aggravating neurofunctional damages and worsening stroke outcomes. Further analysis revealed that extranuclear aggregation of FUS in astrocytes was involved in the induction of excessive autophagy, which contributes to autophagic cell injury or death. In conclusion, our results reveal the important contribution of FUS aggregates in promoting astrocyte activation in stroke pathology independent of its transcriptional regulation activity. We thus propose that aggregation of FUS is an important pathological process in ischemic stroke and targeting FUS aggregates might be of unique therapeutic value in the development of future treatment strategies for ischemic stroke.
Cerebral ischemia/reperfusion (I/R) injury is a devastating neurological disorder with limited treatment options. Emerging evidence suggests that the N6-methyladenosine (m6A) modification and its regulatory factors play pivotal roles in the pathophysiology of I/R. This study aimed to elucidate the function of METTL3-mediated m6A modification of the long non-coding RNA (lncRNA) AU020206 in ferroptosis during cerebral I/R injury and to identify potential molecular targets for neuroprotection. A murine model of middle cerebral artery occlusion/reperfusion (MCAO/R) and N2a cells subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) were established to assess m6A levels and ferroptosis-related changes. Effects of METTL3 overexpression and lncRNA-AU020206 silencing on neuronal apoptosis, inflammation, and ferroptosis were investigated in vitro and in vivo. The interaction between lncRNA-AU020206 and YTHDC2 and the resulting regulation of SLC7A11 mRNA stability and GPX4 expression were evaluated using molecular and biochemical assays. Both MCAO/R mice and OGD/R-treated N2a cells exhibited decreased m6A levels and upregulation of lncRNA-AU020206 accompanied by enhanced ferroptosis. METTL3 overexpression increased the m6A modification of AU020206, promoting its degradation and attenuating neuronal injury, whereas silencing AU020206 or overexpressing YTHDC2 decreased SLC7A11 mRNA stability and enhanced ferroptosis. Restoring the expression of SLC7A11/GPX4 can enhance cell viability, alleviate neuronal apoptosis, and reduce Fe2+overload. Disruption of the METTL3–AU020206–YTHDC2 axis abolished these neuroprotective effects. METTL3-mediated m6A modification of lncRNA-AU020206 restrained ferroptosis and neuronal injury in cerebral I/R by maintaining the stability of the SLC7A11/GPX4 axis via interactions with YTHDC2. Targeting this epitranscriptomic signalling pathway may represent a promising therapeutic strategy for the treatment of ischemic stroke and related neurological disorders.
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