Activation of spinal cord neural stem cells(NSCs)and subsequent neurogenesis holds a promising alternative for spinal cord injury(SCI)repair.Our previous study demonstrated that complement C3a,derived from reactive as...Activation of spinal cord neural stem cells(NSCs)and subsequent neurogenesis holds a promising alternative for spinal cord injury(SCI)repair.Our previous study demonstrated that complement C3a,derived from reactive astrocytes,inhibits NSC proliferation by suppressing protein aggregate clearance through the deubiquitinating enzyme ubiquitin carboxy-terminal hydrolase L1(UCHL1)-proteasome system post-SCI.However,the potential molecular mechanism by which C3a modulates NSC activation via this pathway remains unclear.Here,we revealed that C3a/C3a receptor(C3aR)signaling activated NF-κB p65,which in turn inhibited Nrf2 activity and UCHL1 expression,resulting in diminished proteasome activity and the accumulation of protein aggregates,and ultimately impaired NSC activation.Both knockdown of NF-κB p65 and Nrf2 upregulation restored UCHL1 expression and proteasome activity in vitro,promoting NSC activation by enhancing protein aggregate clearance.Mechanistically,we found that NF-κB p65 regulated Nrf2 activity through a dual mechanism:(1)promoting Keap1-dependent ubiquitination and proteasome degradation of Nrf2;(2)inhibiting protein kinase C-mediated Nrf2 phosphorylation and nuclear translocation.Using the dual-luciferase reporter assay and chromatin immunoprecipitation(ChIP)analysis,we further identified UCHL1 as a direct transcriptional target of Nrf2.Importantly,in vivo experiments using SCI mice confirmed that either C3aR blockade,NF-κB p65 knockdown,or Nrf2 overexpression could rescue SCI-induced UCHL1 downregulation.Together,this study uncovers the C3a-NF-κB p65-Nrf2-UCHL1-proteasome axis as a critical regulator of NSC activation after SCI.This may provide novel molecular targets and intervention strategies for SCI repair.展开更多
Spinal cord injury(SCI)is one of the most devastating traumas,and the aberrant proliferation of astrocytes usually causes neurological deficits.However,the mechanism underlying astrocyte over-proliferation after SCI i...Spinal cord injury(SCI)is one of the most devastating traumas,and the aberrant proliferation of astrocytes usually causes neurological deficits.However,the mechanism underlying astrocyte over-proliferation after SCI is unclear.Grin2c(glutamate ionotropic receptor type 2c)plays an essential role in cell proliferation.Our bioinformatic analysis indicated that Grin2c and Ca^(2+)transport functions were inhibited in astrocytes after SCI.Suppression of Grin2c stimulated astrocyte proliferation by inhibiting the Ca^(2+)/calmodulin-dependent protein kinase 2b(CaMK2b)pathway in vitro.By screening different inflammatory factors,interleukin 1α(IL1α)was further found to inhibit Grin2c/Ca^(2+)/CaMK2b and enhance astrocyte proliferation in an oxidative damage model.Blockade of IL1αusing neutralizing antibody resulted in increased Grin2c expression and the inhibition of astrocyte proliferation post-SCI.Overall,this study suggests that IL1αpromotes astrocyte proliferation by suppressing the Grin2c/Ca^(2+)/CaMK2b pathway after SCI,revealing a novel pathological mechanism of astrocyte proliferation,and may provide potential targets for SCI repair.展开更多
Spinal cord injury(SCI)triggers a complex cascade of cellular and molecular responses,yet the complex cellular communication remains incompletely understood.This study explored how intercellular communication contribu...Spinal cord injury(SCI)triggers a complex cascade of cellular and molecular responses,yet the complex cellular communication remains incompletely understood.This study explored how intercellular communication contributes to the activation of microglia and astrocytes after SCI.Here,we integrated four datasets using single-cell RNA sequencing(scRNA-seq)or single-nucleus RNA sequencing(snRNA-seq)and constructed a comprehensive cellular atlas of the injured spinal cord.Transcriptomic changes in microglia and astrocytes were analyzed.We identified CD44 as a key receptor in SPP1-mediated microglial activation,which represented a subpopulation involved in inflammatory response in microglia.We defined a gliogenesis subpopulation of astrocytes that emerged at 3 dpi,which became the predominant cell type in the injured spinal cord.These astrocytes highly expressed the Nucleolin(Ncl)gene and interacted via the Pleiotrophin(Ptn)signaling pathway,which is associated with astrocyte proliferation.To validate these findings,we utilized a crush injury model.Flow cytometry of isolated microglia and astrocytes confirmed the upregulation of CD44 in microglia and NCL in astrocytes in response to SCI.In vivo results confirmed that the CD44 positive microglia accumulated and PLA results further confirmed the combination of SPP1 with CD44.In parallel,the upregulated expression of NCL in astrocytes facilitated their proliferation,underscoring the role of the NCL receptor in gliogenesis after SCI.In vitro validation demonstrated that exogenous SPP1 upregulates CD44 expression by promoting the phosphorylation of p65 and activating the NF-κB pathways in BV2 microglia,and that high expression of IL-6 indicates the activation of inflammation.PTN may enhance NCL expression and thus facilitates astrocyte proliferation.Collectively,our study identified key receptors that regulated inflammation responses and gliogenesis.Targeting the CD44 and NCL receptors may provide promising therapeutic strategies to modulate inflammation and promote tissue repair after SCI.展开更多
基金supported by the National Natural Science Foundation of China(82071362 and 82270669)Key Project of the Regional Joint Fund of Guangdong Province(2023B1515120077)+3 种基金Basic Research Program of Shenzhen Science and Technology Innovation Commission(JCYJ20210324123001003 and JCYJ20220530144801003)Shenzhen Key Laboratory of Bone Tissue Repair and Translational Research(ZDSYS20230626091402006)the Innovation and Entrepreneurship Training Program for College Students,Sun Yat-sen University(20242150)the Leading Innovation and Entrepreneurship Team Program of Zhejiang Province,China(2023R01005).
文摘Activation of spinal cord neural stem cells(NSCs)and subsequent neurogenesis holds a promising alternative for spinal cord injury(SCI)repair.Our previous study demonstrated that complement C3a,derived from reactive astrocytes,inhibits NSC proliferation by suppressing protein aggregate clearance through the deubiquitinating enzyme ubiquitin carboxy-terminal hydrolase L1(UCHL1)-proteasome system post-SCI.However,the potential molecular mechanism by which C3a modulates NSC activation via this pathway remains unclear.Here,we revealed that C3a/C3a receptor(C3aR)signaling activated NF-κB p65,which in turn inhibited Nrf2 activity and UCHL1 expression,resulting in diminished proteasome activity and the accumulation of protein aggregates,and ultimately impaired NSC activation.Both knockdown of NF-κB p65 and Nrf2 upregulation restored UCHL1 expression and proteasome activity in vitro,promoting NSC activation by enhancing protein aggregate clearance.Mechanistically,we found that NF-κB p65 regulated Nrf2 activity through a dual mechanism:(1)promoting Keap1-dependent ubiquitination and proteasome degradation of Nrf2;(2)inhibiting protein kinase C-mediated Nrf2 phosphorylation and nuclear translocation.Using the dual-luciferase reporter assay and chromatin immunoprecipitation(ChIP)analysis,we further identified UCHL1 as a direct transcriptional target of Nrf2.Importantly,in vivo experiments using SCI mice confirmed that either C3aR blockade,NF-κB p65 knockdown,or Nrf2 overexpression could rescue SCI-induced UCHL1 downregulation.Together,this study uncovers the C3a-NF-κB p65-Nrf2-UCHL1-proteasome axis as a critical regulator of NSC activation after SCI.This may provide novel molecular targets and intervention strategies for SCI repair.
基金supported by the National Natural Science Foundation of China(82071362,82002899)the Basic Research Project of Shenzhen Science and Technology Innovation Commission(JCYJ202205303001577,JCYJ20190809165201646)Basic Research Projects of Shenzhen Science and Technology Program(JCYJ20180307150610733).
文摘Spinal cord injury(SCI)is one of the most devastating traumas,and the aberrant proliferation of astrocytes usually causes neurological deficits.However,the mechanism underlying astrocyte over-proliferation after SCI is unclear.Grin2c(glutamate ionotropic receptor type 2c)plays an essential role in cell proliferation.Our bioinformatic analysis indicated that Grin2c and Ca^(2+)transport functions were inhibited in astrocytes after SCI.Suppression of Grin2c stimulated astrocyte proliferation by inhibiting the Ca^(2+)/calmodulin-dependent protein kinase 2b(CaMK2b)pathway in vitro.By screening different inflammatory factors,interleukin 1α(IL1α)was further found to inhibit Grin2c/Ca^(2+)/CaMK2b and enhance astrocyte proliferation in an oxidative damage model.Blockade of IL1αusing neutralizing antibody resulted in increased Grin2c expression and the inhibition of astrocyte proliferation post-SCI.Overall,this study suggests that IL1αpromotes astrocyte proliferation by suppressing the Grin2c/Ca^(2+)/CaMK2b pathway after SCI,revealing a novel pathological mechanism of astrocyte proliferation,and may provide potential targets for SCI repair.
基金Basic and Applied Basic Research Fund of Guangdong Province(2023B1515120077)Basic Research Project of Shenzhen Science and Technology Innovation Commission(JCYJ20210324123001003,JCYJ20220530144801003,JCYJ20240813150406009)+2 种基金Natural Science Foundation of Guangdong Province[2024A1515012364,2025A1515010926]National Natural Science Foundation of China(Grant No.82272534,82071362)Shenzhen Key Laboratory of Bone Tissue Repair and Translational Research(NO.ZDSYS20230626091402006).
文摘Spinal cord injury(SCI)triggers a complex cascade of cellular and molecular responses,yet the complex cellular communication remains incompletely understood.This study explored how intercellular communication contributes to the activation of microglia and astrocytes after SCI.Here,we integrated four datasets using single-cell RNA sequencing(scRNA-seq)or single-nucleus RNA sequencing(snRNA-seq)and constructed a comprehensive cellular atlas of the injured spinal cord.Transcriptomic changes in microglia and astrocytes were analyzed.We identified CD44 as a key receptor in SPP1-mediated microglial activation,which represented a subpopulation involved in inflammatory response in microglia.We defined a gliogenesis subpopulation of astrocytes that emerged at 3 dpi,which became the predominant cell type in the injured spinal cord.These astrocytes highly expressed the Nucleolin(Ncl)gene and interacted via the Pleiotrophin(Ptn)signaling pathway,which is associated with astrocyte proliferation.To validate these findings,we utilized a crush injury model.Flow cytometry of isolated microglia and astrocytes confirmed the upregulation of CD44 in microglia and NCL in astrocytes in response to SCI.In vivo results confirmed that the CD44 positive microglia accumulated and PLA results further confirmed the combination of SPP1 with CD44.In parallel,the upregulated expression of NCL in astrocytes facilitated their proliferation,underscoring the role of the NCL receptor in gliogenesis after SCI.In vitro validation demonstrated that exogenous SPP1 upregulates CD44 expression by promoting the phosphorylation of p65 and activating the NF-κB pathways in BV2 microglia,and that high expression of IL-6 indicates the activation of inflammation.PTN may enhance NCL expression and thus facilitates astrocyte proliferation.Collectively,our study identified key receptors that regulated inflammation responses and gliogenesis.Targeting the CD44 and NCL receptors may provide promising therapeutic strategies to modulate inflammation and promote tissue repair after SCI.
