title: Critical role of extracellularly secreted neuronal pentraxin 1 in ischemic neuronal death.
abstract: BACKGROUND
  Developing brain is highly susceptible to hypoxic-ischemic injury leading to severe neurological disabilities in surviving infants and children. Previously we reported induction of neuronal pentraxin 1 (NP1) in hypoxic-ischemic injury in neonatal brain and NP1 co-localization with the excitatory AMPA receptors GluR1 at the synaptic sites. However, how NP1 contributes to hypoxic-ischemic neuronal injury is not completely understood.
  
  
  RESULTS
  Here we report that extracellular secretion of NP1 is required for ischemic neuronal death. Primary cortical neurons at days in vitro (DIV) 12 were subjected to oxygen glucose deprivation (OGD), an in vitro model of ischemic stroke, for different time periods (2-8 h). Oxygen glucose deprivation showed characteristic morphological changes of dying cells, OGD time-dependent induction of NP1 (2-4-fold) and increased neuronal death. In contrast, the NP1-KO cortical neurons were healthy and showed no sign of dying cells under similar conditions. NP1gene silencing by NP1-specific small interfering RNA (NP1-siRNA) protected cortical neurons from OGD-induced death. Conditioned media (CM) collected from OGD exposed WT cortical cultures caused neurotoxicity when added to a subset of DIV 12 normoxia control WT cortical cultures. In contrast, CM from OGD-exposed NP1-KO cultures did not induce cell toxicity in control WT cultures, suggesting a role for extracellular NP1 in neuronal death. However, NP1-KO neurons, which showed normal neuronal morphology and protection against OGD, sustained enhanced death following incubation with CM from WT OGD-exposed cultures. Western blot analysis of OGD exposed WT CM showed temporal increase of NP1 protein levels in the CM. Most strikingly, in contrast to NP1-KO CM, incubation of normal cortical cultures with CM from OGD exposed NP2-KO cultures showed neurotoxicity similar to that observed with CM from OGD exposed WT neuronal cultures. Western immunoblotting further confirmed the increased presence of NP1 protein in OGD-exposed NP2-KO CM. Live immunofluorescence analysis show intense cell surface clustering of NP1 with AMPA GluR1 receptors.
  
  
  CONCLUSIONS
  Collectively, our results demonstrate that extracellular release of NP1 promote hypoxic-ischemic neuronal death possibly via surface clustering with GluR1 at synaptic sites and that NP1, not its family member NP2, is involved in the neuronal death mechanisms.
authors.affiliation: Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD, 21205, USA. thatipamula@kennedykrieger.org.
authors.forename: Shabarish
authors.initials: S
authors.lastname: Thatipamula
authors.affiliation: Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD, 21205, USA. hossain@kennedykrieger.org.|Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. hossain@kennedykrieger.org.|Department of Neurology, The Kennedy Krieger Institute, 707 North Broadway, Room 400-N, MD, 21205, Baltimore, USA. hossain@kennedykrieger.org.
authors.forename: Mir Ahamed
authors.initials: MA
authors.lastname: Hossain
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000473:pathology / Q000503:physiopathology*; D002097:C-Reactive Protein / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000473:pathology / Q000503:physiopathology*; D017077:Culture Media, Conditioned / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D005947:Glucose / Q000172:deficiency; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000473:pathology / Q000502:physiology*; D018091:Receptors, AMPA / Q000378:metabolism; D013569:Synapses / Q000502:physiology; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D017077:Culture Media, Conditioned; D005947:Glucose; D009419:Nerve Tissue Proteins; D018091:Receptors, AMPA; D000099153:Pentraxins