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. 2018 Dec 5;15(1):336.
doi: 10.1186/s12974-018-1367-2.

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure

Ekaterina P Demina  1 , Wyston C Pierre  1 , Annie L A Nguyen  1 , Irene Londono  1 , Bela Reiz  2 , Chunxia Zou  2 , Radhika Chakraberty  2 , Christopher W Cairo  2 , Alexey V Pshezhetsky  3   4   5 , Gregory A Lodygensky  6   7   8   9
Affiliations

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure

Ekaterina P Demina et al. J Neuroinflammation. .

Abstract

Background: The extension of sepsis encompassing the preterm newborn's brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins.

Methods: Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids.

Results: At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II-III) of somatosensory cortex had a reduced surface content of polysialic acid.

Conclusions: Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

Keywords: Lysosomal dysfunction; Neonatal neuroinflammation; Neonatal rat model; Neuronal neuraminidase 1; Sialic acid.

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Conflict of interest statement

Authors’ information

Not applicable.

Ethics approval

Approval for the animal experimentation was granted by the Animal Care and Use Committee of the Montreal Heart Institute and by the Animal Care and Use Committee of the CHU Sainte-Justine.

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All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. All authors gave their consent for this publication.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
LAMP-1 protein level was increased 21 days after LPS exposure. LAMP-1 protein levels were measured by Western blot in the brain homogenates of rat pups injected with LPS (n = 8) and Sham (n = 8) 1 day (P4) and 21 days (P24) after injection, *p < 0.05 (a, b). LAMP-1 distribution in upper layers of somatosensory cortex (I–II) in neurons (NeuN-positive cells) (c) and microglia (ILB4-positive cells) (d) of LPS and Sham animals at P4 and P24 was analyzed by immunofluorescent confocal microscopy. Cortex I–II, cortical layers according to [64]
Fig. 2
Fig. 2
Neuraminidase activity is increased in brains of rat pups exposed to LPS. β-galactosidase (a), β-hexosaminidase (b), and neuraminidase (c) activities were measured in the brain homogenates of rat pups injected with LPS (n = 8) and Sham (n = 8) 1 day (P4) and 21 days (P24) after injection, and in the brain homogenates of 8-month-old MPSIIIC (Hgsnat KO, n = 4) and C57BL16 WT (n = 4) mice. *p < 0.05, ** p < 0.01, ***p < 0.001
Fig. 3
Fig. 3
Pan-neuraminidase activity against the X-Neu5Ac substrate is increased in the cortex of LPS-injected rats and shows a pattern different from that of activated microglial cells. Panels (a) and (b) show representative coronal brain slices of Sham (n = 3) and LPS-injected (n = 3) rat pups 1 day (P4) after injection and panels (c) and (d), 21 days (P24) after injection. The bar graphs (e) and (f) show a relative size of X-Neu5Ac-stained areas (% of total areas) measured in seven different regions for P4 brains: corpus callosum, hippocampus (left and right), and the upper (layers II–III) and lower (layers V–VI) somatosensory cortices (left and right), and in nine different regions for P24 brains: corpus callosum, hippocampus (left and right), the upper (layers II–III) and lower (layers V–VI) somatosensory cortices (left and right), and RSGb (left and right). g Cortical distribution of activated microglia marked with ILB4 at P4 (scale bar = 200 μm) *p < 0.05, *** p < 0.001
Fig. 4
Fig. 4
Neu1 mRNA is increased in brains of rat pups exposed to LPS. Expression of Neu1, Neu3, and Neu4 mRNA was measured in brain tissues of animals injected with LPS (n = 5) or saline (Sham) (n = 7) at (a) 24 h (P4) and (b) 21 days (P24) after injection. Total RNA was extracted from tissues of LPS-injected and Sham rats at P4 and P24 to analyze the expression of neuraminidases by RT-qPCR. The values were normalized for the level of control Gapdh mRNA. **p < 0.01
Fig. 5
Fig. 5
a-d Representative HPLC chromatograms of glycan chains of rat brain GSLs labelled with anthranilic acid. Panels show representative chromatograms of P4 Sham (n=2) (a), P24 Sham (n=2) (b), P4 LPS (n=2) (c) and P24 LPS (n=2) (d) samples, with fluorescence intensity (arbitrary) shown on the y-axis. e, f Levels of gangliosides measured by quantification of HPLC chromatograms. (e) Comparison of relative compositions of LacCer, GM3, GM1, GD3, GD1a, GD1b, GT1a, GT1b and GQ1 gangliosides in P4 LPS and Sham brains. (f) Relative compositions of LacCer, GM3, GM1, GD3, GD1a, GD1b, GT1a and GT1b gangliosides in P24 LPS and Sham brains. Values represent means ± S.D. of duplicate (P4) or triplicate (P24) measurements
Fig. 6
Fig. 6
Profile of N-glycans from brain tissues of LPS-exposed and saline-treated (Sham) animals 21 days after injection. Fluorescence chromatograms of N-glycans cleaved from proteins in homogenates of brains from Sham and LPS-injected rat pups were analyzed by LC-MS after fluorescent labeling. Samples were fractionated on an anion exchange column, where glycans with higher content of sialic acid elute at later retention times. Shaded regions indicate the retention times of glycans containing 0 (< 14.5 min), 1 (14.5–20 min), and 2 (> 20 min) sialic acid residues. The traces show fluorescence intensity (arbitrary units). Individual peaks were assigned to specific glycan structures using the MS data and quantified by integrating the areas under the chromatograms (table in the inset). Figure shows representative profiles of duplicate experiments
Fig. 7
Fig. 7
21 days after LPS injection, rat brain tissue glycoproteins show reduced staining for MAL-II lectin and increased staining for PNA lectin consistent with their desialylation. a, b Representative glycoprotein patterns of brain tissue homogenates of Sham (lanes 2–3) and LPS-injected (lanes 4–5) rat pups at P24 after injection. Following SDS-PAGE, blots were stained with biotinylated peanut agglutinin (PNA) lectin, specific for Gal-GalNAc residues and biotinylated Maackia amurensis II (MAL II) lectin, specific for α2–3-linked sialic acids. The bands showing apparent difference in the intensity between LPS and Sham samples are boxed. c, d Intensity of lectin-positive bands normalized to the level of total protein. For statistical analysis, five animals from each group were used; *p < 0.05, **p < 0.01
Fig. 8
Fig. 8
21 days after LPS exposure, upper cortical neurons show reduced PSA immunostaining. a Neurons with decreased PSA immunostaining are detected in the upper somatosensory cortex (layers II–III) of LPS-injected rat pups at P24 (n = 3) after injections as compared to Sham animals (n = 3). PSA, green; neuronal marker NeuN, red. Bar graphs (b) show average PSA immunostaining intensity in cortical neurons counted for nine adjacent 0.0042-mm2 sections of upper and lower layers of the somatosensory cortex. Bar represents 20 μm. ****p < 0.0001. n.s., non-significant, Cx, cortex; I–VI, cortical layers according to [64]
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