Cellular and Molecular Life Sciences最新文献

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression.

Doxorubicin (DOX) is an effective chemotherapeutic drug, but its use can lead to cardiomyopathy, which is the leading cause of mortality among cancer patients. Macrophages play a role in DOX-induced cardiomyopathy (DCM), but the mechanisms undlerlying this relationship remain unclear. This study aimed to investigate how IKKα regulates macrophage activation and contributes to DCM in a mouse model. Specifically, the role of macrophage IKKα was evaluated in macrophage-specific IKKα knockout mice that received DOX injections. The findings revealed increased expression of IKKα in heart tissues after DOX administration. In mice lacking macrophage IKKα, myocardial injury, ventricular remodeling, inflammation, and proinflammatory macrophage activation worsened in response to DOX administration. Bone marrow transplant studies confirmed that IKKα deficiency exacerbated cardiac dysfunction. Macrophage IKKα knockout also led to mitochondrial damage and metabolic dysfunction in macrophages, thereby resulting in increased cardiomyocyte injury and oxidative stress. Single-cell sequencing analysis revealed that IKKα directly binds to STAT3, leading to the activation of STAT3 phosphorylation at S727. Interestingly, the inhibition of STAT3-S727 phosphorylation suppressed both DCM and cardiomyocyte injury. In conclusion, the IKKα-STAT3-S727 signaling pathway was found to play a crucial role in DOX-induced cardiomyopathy. Targeting this pathway could be a promising therapeutic strategy for treating DOX-related heart failure.

Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111–132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111–132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure–activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120–132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120–132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111–132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111–132) may offer perspectives to restrict autophagy-dependent viruses.

Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

Diabetic kidney disease (DKD) is the predominant type of end-stage renal disease. Increasing evidence suggests thatglomerular mesangial cell (MC) inflammation is pivotal for cell proliferation and DKD progression. However, the exactmechanism of MC inflammation remains largely unknown. This study aims to elucidate the role of inflammatoryfactor high-mobility group box 1 (Hmgb1) in DKD. Inflammatory factors related to DKD progression are screened viaRNA sequencing (RNA-seq). In vivo and in vitro experiments, including db/db diabetic mice model, CCK-8 assay, EdUassay, flow cytometric analysis, Co-IP, FISH, qRT-PCR, western blot, single cell nuclear RNA sequencing (snRNA-seq),are performed to investigate the effects of Hmgb1 on the inflammatory behavior of MCs in DKD. Here, wedemonstrate that Hmgb1 is significantly upregulated in renal tissues of DKD mice and mesangial cells cultured withhigh glucose, and Hmgb1 cytopasmic accumulation promotes MC inflammation and proliferation. Mechanistically,Hmgb1 cytopasmic accumulation is two-way regulated by MC-specific cyto-lncRNA E130307A14Rik interaction andlactate-mediated acetylated and lactylated Hmgb1 nucleocytoplasmic translocation, and accelerates NFκB signalingpathway activation via directly binding to IκBα. Together, this work reveals the promoting role of Hmgb1 on MCinflammation and proliferation in DKD and helps expound the regulation of Hmgb1 cytopasmic accumulation in twoways. In particular, Hmgb1 may be a promising therapeutic target for DKD.

Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3^−/−; 2. C7^−/−; and 3. Cd59ab^−/−) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3^−/−) and MAC formation (in C3^−/−. C7^−/−, and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab^−/−) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3^−/−, C7^−/− mice, and Cd59ab^−/− mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19.

Proliferation of renal tubular epithelial cells (TEC) is essential for restoring tubular integrity and thereby to support renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. Activation of transcriptional factor c-Myc promotes TEC proliferation following KI/R; however, the mechanism regarding c-Myc activation in TEC is incompletely known. Heat shock protein A12A (HSPA12A) is an atypic member of HSP70 family. In this study, we found that KI/R decreased HSPA12A expression in mouse kidneys and TEC, while ablation of HSPA12A in mice impaired TEC proliferation and renal functional recovery following KI/R. Gain-of-functional studies demonstrated that HSPA12A promoted TEC proliferation upon hypoxia/reoxygenation (H/R) through directly interacting with c-Myc and enhancing its nuclear localization to upregulate expression of its target genes related to TEC proliferation. Notably, c-Myc was lactylated in TEC after H/R, and this lactylation was enhanced by HSPA12A overexpression. Importantly, inhibition of c-Myc lactylation attenuated the HSPA12A-induced increases of c-Myc nuclear localization, proliferation-related gene expression, and TEC proliferation. Further experiments revealed that HSPA12A promoted c-Myc lactylation via increasing the glycolysis-derived lactate generation in a Hif1α-dependent manner. The results unraveled a role of HSPA12A in promoting TEC proliferation and facilitating renal recovery following KI/R, and this role of HSPA12A was achieved through increasing lactylation-mediated c-Myc activation. Therefore, targeting HSPA12A in TEC might be a viable strategy to promote renal functional recovery from KI/R injury in patients.

Graphical abstract

HSPA12A facilitated renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. This protective effect of HSPA12A was mediated through directly interacting with c-Myc as well as upregulating the Hif1α-mediated lactate generation, thereby increasing c-Myc lactylation and nuclear localization to drive expression of genes related to cell proliferation, and ultimately promoting TEC proliferation.

The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-β. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca²⁺ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.

Insulin resistance (IR) was found to be a critical element in the pathogenesis of Parkinson’s disease (PD), facilitating abnormal α-synuclein (α-Syn) aggregation in neurons and thus promoting PD development. However, how IR contributes to abnormal α-Syn aggregation remains ill-defined. Here, we analyzed six PD postmortem brain transcriptome datasets to reveal module genes implicated in IR-mediated α-Syn aggregation. In addition, we induced IR in cultured dopaminergic (DA) neurons overexpressing α-Syn to identify IR-modulated differentially expressed genes (DEGs). Integrated analysis of data from PD patients and cultured neurons revealed 226 genes involved in α-Syn aggregation under IR conditions, of which 53 exhibited differential expression between PD patients and controls. Subsequently, we conducted an integrated analysis of the 53 IR-modulated genes employing transcriptome data from PD patients with different Braak stages and DA neuron subclasses with varying α-Syn aggregation scores. Protein tyrosine phosphatase receptor type O (PTPRO) was identified to be closely associated with PD progression and α-Syn aggregation. Experimental validation in a cultured PD cell model confirmed that both mRNA and protein of PTPRO were reduced under IR conditions, and the downregulation of PTPRO significantly facilitated α-Syn aggregation and cell death. Collectively, our findings identified PTPRO as a key regulator in IR-mediated α-Syn aggregation and uncovered its prospective utility as a therapeutic target in PD patients with IR.