Essential roles of exosome and circRNA_101093 on ferroptosis desensitization in lung adenocarcinoma

Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are required for protection against ferroptosis.The purpose of the present studywas to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.Methods:Established human lung fibroblasts MRC-5,WI38,and human LUAD H1650,PC9,H1975,H358,A549,and H1299 cell lines,tumor and matched normal adjacent tissues of LUAD,and plasma from healthy individuals and LUAD patients were used in this study.Immunohistochemistry and immunoblotting were used to analyze protein expression,and quantitative reverse transcription-PCR was used to analyze mRNA expression.Cell viability,cell death,and the lipid reactive oxygen species generationwere measured to evaluate the responses to ferroptosis.Exosomes were observed using transmission electron microscope.The localization of arachidonic acid(AA)was detected using click chemistry labeling followed by confocal microscopy.Interactions between RNAs and proteins were detected using RNA pull-down,RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods.Proteomic analysis was used to investigate RNA-regulated proteins,and metabolomic analysis was performed to analyze metabolites.Cellderived xenograft,patient-derived xenograft,cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.Results:Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis.A potential explanation is that exosomal circRNA_101093(cir93)maintained an elevation in intracellular cir93 in LUAD to modulate AA,a poly-unsaturated fatty acid critical for ferroptosisassociated increased peroxidation in the plasma membrane.Mechanistically,cir93 interacted with and increased fatty acid-binding protein 3(FABP3),which transported AA and facilitated its reaction with taurine.Thus,global AA was reduced,whereas N-arachidonoyl taurine(NAT,the product of AA and taurine)was induced.Notably,the role of NAT in suppressing AA incorporation into the plasma membrane was also revealed.In pre-clinical in vivo models,reducing exosome improved ferroptosis-based treatment.Conclusion:Exosome and cir93 are essential for desensitizing LUAD cells to ferroptosis,and blocking exosome may be helpful for future LUAD treatment.

Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming vari- ous neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in per- forming both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses （WGCNA）, we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neu- ronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.

纳米医学用于CRISPR-Cas9基因编辑技术治疗疾病

In recent years,the clustered regularly interspaced short palindromic repeats-associated protein 9(CRISPR-Cas9)technology that won the 2020 Nobel Prize in Chemistry has served as one of the most prominent and powerful tools in gene editing for biomedical research and the treatment of various diseases[1].Nevertheless,it remains a substantial challenge to effectively and safely deliver CRISPR-Cas9 molecules into target cells[2].

Chimeric cerebral organoids reveal the essentials of neuronal and astrocytic APOE4 for Alzheimer's tau pathology

The apolipoprotein E4(APOE4)genotype is one of the strongest genetic risk factors for Alzheimer’s disease(AD),and is generally believed to cause widespread pathological alterations in various types of brain cells.Here,we developed a novel engineering method of creating the chimeric human cerebral organoids(chCOs)to assess the differential roles of APOE4 in neurons and astrocytes.First,the astrogenic factors NFIB and SOX9 were introduced into induced pluripotent stem cells(iPSCs)to accelerate the induction of astrocytes.Then the above induced iPSCs were mixed and cocultured with noninfected iPSCs under the standard culturing condition of cerebral organoids.As anticipated,the functional astrocytes were detected as early as 45 days,and it helped more neurons matured in chCOs in comparation of the control human cerebral organoids(hCOs).More interestingly,this method enabled us to generate chCOs containing neurons and astrocytes with different genotypes,namely APOE3 or APOE4.Then,it was found in chCOs that astrocytic APOE4 already significantly promoted lipid droplet formation and cholesterol accumulation in neurons while both astrocytic and neuronal APOE4 contributed to the maximum effect.Most notably,we observed that the cooccurrence of astrocytic and neuronal APOE4 were required to elevate neuronal phosphorylated tau levels in chCOs while Aβlevels were increased in chCOs with neuronal APOE4.Altogether,our results not only revealed the essence of both neuronal and astrocytic APOE4 for tau pathology,but also suggested chCOs as a valuable pathological model for AD research and drug discovery.