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. 2004 Apr 28;24(17):4187-96.
doi: 10.1523/JNEUROSCI.0347-04.2004.

Neuronal pentraxin 1: a novel mediator of hypoxic-ischemic injury in neonatal brain

Mir Ahamed Hossain  1 , Juliet C Russell, Richard O'Brien, John Laterra
Affiliations

Neuronal pentraxin 1: a novel mediator of hypoxic-ischemic injury in neonatal brain

Mir Ahamed Hossain et al. J Neurosci. .

Abstract

Neonatal hypoxic-ischemic brain injury is a major cause of neurological disability and mortality. Its therapy will likely require a greater understanding of the discrete neurotoxic molecular mechanism(s) triggered by hypoxia-ischemia (HI). Here, we investigated the role of neuronal pentraxin 1 (NP1), a member of a newly recognized subfamily of "long pentraxins," in the HI injury cascade. Neonatal brains developed marked infarcts in the ipsilateral cerebral hemisphere at 24 hr and showed significant loss of ipsilateral striatal, cortical, and hippocampal volumes at 7 d after HI compared with the contralateral hemisphere and sham controls. Immunofluorescence analyses revealed elevated neuronal expression of NP1 in the ipsilateral cerebral cortex from 6 hr to 7 d and in the hippocampal CA1 and CA3 regions from 24 hr to 7 d after HI. These same brain areas developed infarcts and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells within 24-48 hr of HI. In primary cortical neurons, NP1 protein was induced >2.5-fold (p < 0.001) after their exposure to hypoxia that caused approximately 30-40% neuronal death. Transfecting cortical neurons with antisense oligodeoxyribonucleotides directed against NP1 mRNA (NP1AS) significantly inhibited (p < 0.01) hypoxia-induced NP1 protein induction and neuronal death (p < 0.001), demonstrating a specific requirement of NP1 in hypoxic neuronal injury. NP1 protein colocalized and coimmunoprecipitated with the fast excitatory AMPA glutamate receptor subunit (GluR1) in primary cortical neurons, and hypoxia induced a time-dependent increase in NP1-GluR1 interactions. NPIAS also protected against AMPA-induced neuronal death (p < 0.05), implicating a role for NP1 in the excitotoxic cascade. Our results show that NP1 induction mediates hypoxic-ischemic injury probably by interacting with and modulating GluR1 and potentially other excitatory glutamate receptors.

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Figures

Figure 1.
Figure 1.
Cerebral hypoxia-ischemia (HI)-induced injury in neonatal rat brain. The right CCA of neonatal rats was ligated. Rats were then subjected to 2 hr of hypoxia as described in Materials and Methods. A, Pups were killed at 24 and 48 hr after HI for TTC staining. Marked infarction (white areas) in the right cerebral cortex and hippocampal CA1 and CA3 areas was found compared with sham controls. B, Brain injury was quantified 7 d after HI by measuring cortical, striatal, and hippocampal volumes from Nissl-stained coronal sections (25 μm) using MCID Elite software according to our published method (Hossain et al., 1998). Morphometric quantification of representative coronal sections stained with 0.5% cresyl violet revealed significant loss of ipsilateral striatal (Str), cortical (Ctx), and hippocampal (Hippo) volumes compared with the contralateral side and that of sham controls. Representative coronal brain sections are shown. Data represent mean ± SEM (n = 10; *p < 0.05; **p < 0.01).
Figure 2.
Figure 2.
Temporal patterns of neuronal expression of NP1 after neonatal hypoxic-ischemic brain injury. Neonatal rats were killed at 6, 12, 24, and 48 hr after HI. Representative coronal sections (20 μm) from sham controls and HI rat brain were subjected to immunofluorescence using mouse anti-rat NP1 monoclonal primary and FITC-conjugated (green fluorescence) secondary antibodies. In A, NP1-specific immunoreactivity appeared at 6 hr after HI in the ipsilateral cortex, and more intense NP1 immunofluorescence was detected from 12 to 48 hr after HI compared with the contralateral hemisphere and sham controls (viewed at 40× magnification). In C, NP1-specific immunofluorescence was observed in the hippocampal pyramidal layer of CA1 and CA3 (shown by arrows) but not in the DG at 24 and 48 hr after HI. Representative brain sections are shown at 5× magnification. In B and D, Fluorometric TUNEL histochemistry of adjacent brain sections obtained from sham controls and at corresponding time periods after HI. Sections were counterstained with DAPI (dark blue) after TUNEL staining (green). Enhanced fluorescence of TUNEL-positive cells with darkly stained round chromatin (apoptotic cells; yellow arrows) and diffusely stained chromatin (necrotic cells; red arrows) was observed in the ipsilateral cerebral cortex at 12, 24, and 48 hr (B) and in the hippocampal CA1 (shown) (D) at 24 and 48 hr after HI. No TUNEL-positive cells were found in the sham controls and in the contralateral hemisphere during the indicated time after HI (viewed at 40×). Scale bars: A, B, D, 20 μm; C, 200 μm.
Figure 3.
Figure 3.
NP1 induction in the ipsilateral cerebral cortex and hippocampus persists for a longer time period after neonatal HI. Representative coronal brain sections (20 μm) from sham controls and 7 d post-HI rats were analyzed for NP1-specific immunofluorescence as described in Materials and Methods. A, A high level of NPI immunoreactivity was observed in the ipsilateral hippocampus (pyramidal layer of CA3 and CA1 but not in DG) relative to the contralateral hemisphere and sham controls. NP1-specific immunofluorescence (green) is shown by arrows at low (5×; left panels) and high (100×; right panels) magnifications. B, A similar increase in NP1 immunoreactivity was observed in the frontal and parietal cortex of brain sections from animals subjected to HI. Scale bars: A, 200 μm; B, 20 μm.
Figure 4.
Figure 4.
In vitro hypoxia causes substantial neuronal death and induces NP1 protein expression in primary cortical cultures. Primary cortical neurons cultured for 6-8 d in vitro were placed in neurobasal medium without B27 supplement for 2 hr before exposure to hypoxia. A, Morphological evidence of injury and death in cortical neurons exposed to hypoxia for 12-14 hr. Scale bar, 20 μm. Quantification of cell death by LDH release (B), by TUNEL-positive staining (C), and by MTT reduction (D) revealed ∼30-40% cell death in response to hypoxia. Data are expressed as percentage of control normoxic cells (mean ± SEM; n = 8; *p < 0.001). E, Total cellular proteins (20 μg per lane) were analyzed by SDS-PAGE and immunoblotted for NP1 expression in cortical cultures exposed to 0, 3, and 6 hr of hypoxia and normoxia controls. Chemiluminescent detection shows an intense NP1-immunoreactive protein band at ∼47kDa. Digitized images of NP1 band were quantified by densitometry and normalized to actin. Data represents percentage of normoxic controls (mean ± SEM; n = 5-6; *p < 0.001). Blots shown are from representative experiments. N, Normoxia; H, hypoxia.
Figure 5.
Figure 5.
Inhibition of hypoxia-induced NP1 protein expression by NP1 antisense (NP1AS) ODNs. Cortical neuronal cultures at 6 DIV were transfected with phosphorothioated antisense (NP1AS) or sense (NP1S) ODNs (0.5 μg), respectively, as described in Materials and Methods. Approximately 30 hr later cells were exposed to hypoxia. Control transfected cells were incubated under normoxic condition. A, Immunoblotting for NP1 expression in cortical neurons transfected with either NP1 antisense or sense ODNs. NP1AS completely inhibited hypoxia-induced NP1 protein expression. The graph summarizes densitometric quantification of NP1 protein, normalized to actin, and is expressed as percentage of control normoxic cells (mean ± SEM; n = 4; *p < 0.05 vs normoxia; +p < 0.01 vs hypoxia). Representative immunoblots for NP1 and actin are displayed. B, Phase-contrast photomicrographs of primary cortical neurons exposed to hypoxia. Cortical neurons transfected with NP1AS maintained normal morphology with intact processes after hypoxia relative to normoxic cells. NPIS failed to preserve characteristics of normal cellular morphology after hypoxia. Scale bar, 20 μm.
Figure 6.
Figure 6.
NP1 antisense (NP1AS) oligodeoxyribonucleotides prevent hypoxia-induced neuronal death in primary cortical cultures. Degree of cell death was assessed by LDH release (A), TUNEL-staining (B), and mitochondrial MTT reduction (C) in cortical neurons transfected with NP1AS (antisense) or NP1S (sense) oligonucleotides. NP1AS significantly inhibited hypoxia-induced neuronal death in cortical cultures but not the NP1S. Data represent mean ± SEM (n = 8; *p < 0.001 vs normoxia control; +p < 0.001 vs hypoxia group).
Figure 7.
Figure 7.
Colocalization and coimmunoprecipitation of NP1 protein with GluR1 AMPA receptor subunits. Primary cortical cultures were established at low density (30-40 × 103 cells/cm2) on coverslips as described in Materials and Methods. A, Live immunostaining of 7 d cultured cortical neurons with antibodies to NP1 (1:200) and GluR1 (1:150). Neurons were permeabilized and stained with secondary antibodies (FITC-conjugated goat anti-mouse for NP1; green fluorescence) and Texas Red-conjugated goat anti-rabbit for GluR1; red fluorescence). Digitized individual and overlapped fluorescence images show colocalization of NP1 and GluR1 subunits (top panels). Cultures transfected with NP1AS showed a chromatic decrease in NP1-specific immunofluorescence and a near total absence of NP1-GluR1 colocalization (bottom panels) relative to sense-treated neurons. B, Double immunofluorescence staining of NP1 and GluR1 in rat brain sections from control and HI animals visualized under confocal microscopy. Merged images (100×) show NP1 and GluR1 colocalization. C, SDS-PAGE and immunoblotting of GluR1 immunoprecipitates from total cellular extracts revealed a time-dependent increase in NP1 co-precipitation in response to hypoxia (0-6 hr) compared with normoxic neurons. IgG immunoprecipitates showed no evidence of NP1- and GluR1-specific bands. The ratio of NP1 over actin intensity was expressed as percentage of control normoxia. Data represent mean ± SEM (n = 4; *p < 0.01; **p < 0.001 vs control normoxia). Representative immunoblots for NP1 and GluR1 are displayed.
Figure 8.
Figure 8.
NP1 antisense ODNs inhibit AMPA-induced neuronal death. A, Primary cortical neurons cultured for 6-8 d in vitro were exposed to various concentrations of AMPA (10-300 μm) for 24 hr. Measurement of neuronal viability by MTT assay revealed an AMPA concentration-dependent increase in neuronal death in primary cortical cultures. B, Protection of cortical neurons against AMPA-induced neuronal death by CNQX, a specific AMPA receptor antagonist, and by NP1AS. Neuronal cultures were transfected with either NP1AS or NP1S and ∼30 hr later cells were exposed to 100 μm AMPA or PBS as control in the presence or absence of CNQX. Pretreatment with CNQX completely attenuated AMPA-induced death, and cortical cultures transfected with NP1AS were partially protected against AMPA-induced death as determined by MTT assay. Data represent mean ± SEM (n = 8; *p < 0.001 vs normoxia controls; ++p < 0.001, +p = 0.039 vs AMPA).
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