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. 2019 Apr 1;129(4):1779-1784.
doi: 10.1172/JCI124485. Epub 2019 Mar 18.

Circulating Heparan Sulfate Fragments Mediate Septic Cognitive Dysfunction

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Free PMC article

Circulating Heparan Sulfate Fragments Mediate Septic Cognitive Dysfunction

Joseph A Hippensteel et al. J Clin Invest. .
Free PMC article

Abstract

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.

Keywords: Extracellular matrix; Glycobiology; Neuroscience; growth factors.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Cognitive impairment in mouse survivors of endotoxemia or sepsis is BDNF and TrkB responsive.
(A) Memory impairment occurred in mice 7 days after i.p. administration of LPS (10 μg/g BW), as demonstrated by loss of freezing behavior (indicating a fearful memory of a previous paw shock) after contextual fear conditioning. (B) Living hippocampal slices isolated from mice 7 days after i.p. LPS administration (as compared with saline) showed impaired LTP, the BDNF-dependent neuronal process responsible for spatial memory. (C) A similar loss of LTP was also seen 7 days after CLP, a model of polymicrobial peritonitis–induced sepsis. Loss of LTP occurred 7 days after LPS, despite (D) maintenance of hippocampal BDNF content and (E) preserved responsiveness to excess (100 ng/ml) exogenous BDNF. (F) Maintenance of the BDNF-responsive molecular machinery of learning after sepsis was further demonstrated by reversal of memory deficits in post-LPS mice treated daily with 7,8-DHF (5 μg/g i.p.), a direct agonist of the BDNF receptor TrkB. *P < 0.05 and **P < 0.01, by t test. For LTP measurements, the left panels represent the mean ± SEM of groups; the right panels represent the average change from baseline over the final 10 minutes of measurement (each data point represents a unique biological replicate). TBS, theta burst stimulation.
Figure 2
Figure 2. Sepsis-associated circulating heparan sulfate fragments penetrate the hippocampus and impede LTP.
Liquid chromatography–tandem mass spectrometry MRM (LC-MS/MS MRM) analyses demonstrated shedding of heparan sulfate (HS) into the plasma of (A) mice 24 hours after i.p. LPS administration (10 μg/g BW vs. saline control) and (B) human patients with sepsis (enrolled in the MESSI cohort and followed longitudinally; control samples represent normal blood donors). (C) Accordingly, an increase in heparan sulfate content was detected in the hippocampus of LPS-injected mice that persisted for 7 days after injection. (D) Fluorescein-labeled, highly sulfated heparan sulfate (heparin) octasaccharides (250 μg) administered i.v. to mice 24 hours after i.p. LPS (10 μg/g) or saline treatment penetrated the hippocampal blood-brain barrier, as observed by confocal microscopy of freshly isolated hippocampal slices. Scale bars: 100 μm. (E) Highly sulfated heparan sulfate (heparin) octasaccharides (degree of polymerization 8 [dp8]) induced loss of LTP when perfused (2.5 μg/ml) over hippocampal slices isolated from healthy (non-septic mice). LTP was rescued by simultaneous perfusion with the TrkB agonist 7,8-DHF (250 nM). *P < 0.05, **P < 0.01, and ***P < 0.001, by ANOVA with Tukey’s correction for multiple comparisons (A, C, and E) or Kruskal-Wallis with Dunn’s test for multiple comparisons (B and D).
Figure 3
Figure 3. Importance of heparan disaccharide sulfation in BDNF binding and septic cognitive impairment.
(A) Heparan sulfate disaccharides may be sulfated at the N (NS) and 6-O (6S) positions of glucosamine and the 2-O (2S) position of iduronic acid. (B) Glycoarray analyses showed that heparan sulfate oligosaccharides, which bind to BDNF (n = 12), were enriched in NS and 2S compared with low-affinity (n = 40) oligosaccharides. 3S sulfation (found only in heparin) was not associated with binding. (C and D) Circulating heparan sulfate fragments detected by mass spectrometry in plasma collected (C) 24 hours after i.p. LPS administration (10 μg/g BW) in mice or (D) at the time of ICU admission in patients (n = 20) with sepsis were enriched in N sulfation (including NS, NS2S, NS6S, and TriS disaccharides) and 2-O sulfation (NS2S, 2S, 2S6S, and TriS). Control patients were normal blood donors (n = 9). (E) Mouse hippocampi after sepsis were enriched in N- and 2-O sulfation on day 7 (n = 3 for all time points and groups). (F) Patients who showed moderate to severe cognitive impairment (MoCA score <21 or cognitive inability to perform the test, n = 6) at the time of hospital discharge (or on day 14 after ICU discharge) were those who previously had (at the time of ICU admission, day = 0) circulating heparan sulfate enriched in NS2S disaccharides or a combination of NS, NS2S, and NS6S disaccharides. n = 14 patients with normal to mild impairment. Horizontal lines in the boxes represent the median, borders represent the 25th and 75th percentiles, and whiskers represent the upper and lower adjacent values (1.5 × interquartile range, Tukey’s method). Outside values were included in the analyses. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by t test for single comparisons (BD), ANOVA with Tukey’s correction for multiple comparisons (E), or Wilcoxon rank-sum test (F).

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