Narcolepsy patients have antibodies that stain distinct cell populations in rat brain and influence sleep patterns - PubMed (original) (raw)

. 2014 Sep 2;111(35):E3735-44.

doi: 10.1073/pnas.1412189111. Epub 2014 Aug 18.

Csaba Adori 2, Szilvia Vas 3, Ylva Kai-Larsen 4, Tomi Sarkanen 5, Andreas Cederlund 6, Birgitta Agerberth 7, Ilkka Julkunen 8, Beata Horvath 9, Diana Kostyalik 9, Lajos Kalmár 10, Gyorgy Bagdy 3, Anne Huutoniemi 11, Markku Partinen 12, Tomas Hökfelt 2

Affiliations

Narcolepsy patients have antibodies that stain distinct cell populations in rat brain and influence sleep patterns

Peter Bergman et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Narcolepsy is a chronic sleep disorder, likely with an autoimmune component. During 2009 and 2010, a link between A(H1N1)pdm09 Pandemrix vaccination and onset of narcolepsy was suggested in Scandinavia. In this study, we searched for autoantibodies related to narcolepsy using a neuroanatomical array: rat brain sections were processed for immunohistochemistry/double labeling using patient sera/cerebrospinal fluid as primary antibodies. Sera from 89 narcoleptic patients, 52 patients with other sleep-related disorders (OSRDs), and 137 healthy controls were examined. Three distinct patterns of immunoreactivity were of particular interest: pattern A, hypothalamic melanin-concentrating hormone and proopiomelanocortin but not hypocretin/orexin neurons; pattern B, GABAergic cortical interneurons; and pattern C, mainly globus pallidus neurons. Altogether, 24 of 89 (27%) narcoleptics exhibited pattern A or B or C. None of the patterns were exclusive for narcolepsy but were also detected in the OSRD group at significantly lower numbers. Also, some healthy controls exhibited these patterns. The antigen of pattern A autoantibodies was identified as the common C-terminal epitope of neuropeptide glutamic acid-isoleucine/α-melanocyte-stimulating hormone (NEI/αMSH) peptides. Passive transfer experiments on rat showed significant effects of pattern A human IgGs on rapid eye movement and slow-wave sleep time parameters in the inactive phase and EEG θ-power in the active phase. We suggest that NEI/αMSH autoantibodies may interfere with the fine regulation of sleep, contributing to the complex pathogenesis of narcolepsy and OSRDs. Also, patterns B and C are potentially interesting, because recent data suggest a relevance of those brain regions/neuron populations in the regulation of sleep/arousal.

Keywords: H1N1 vaccination; POMC neurons; autoantigen; neurotransmitter.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Demonstration of the NEI/αMSH staining pattern. (A) Low-power overview from a glycosidase predigested section of a colchicine-treated rat brain incubated with group A serum: a distinct neuron population is present in the ZI-LH region and Arc. (B) A dense fiber network but no cell bodies are seen in rats not treated with colchicine. (Scale bars: A, 500 µm; B, 200 µm.)

Fig. 2.

Fig. 2.

Characterization of the NEI/αMSH staining pattern in the ZI-LH region (group A). (A and A*) Fluorescent cells are identified as the MCH+ population. (C and C*) These cells are never immunoreactive for Hcrt/Orx. (B) High-power magnification reveals a dot-like subcellular pattern (100× objective, 1.5 digital zoom; merge of six _Z_-stack 0.5-µm-thick optical-layer micrographs). (B*) Serum staining was distinctly colocalized with syntaxin-6, a Golgi apparatus marker. Green channel, serum staining; red channel, MCH/Hcrt/syntaxin-6. (Scale bars: A and C, 200 µm; B and B*, 10 µm; A* and C*, 30 µm.)

Fig. 3.

Fig. 3.

Characterization of the NEI/αMSH staining pattern in the Arc (group A) and the cortical interneuron staining pattern (group B). (A–C) Serum+ cells are identical to the POMC-immunoreactive Arc neurons. (D) Only a few serum+ cortical fibers are double stained for MCH (insular cortex). The dense serum+ fiber plexus in the (E) paraventricular thalamic nucleus and (F) ventrolateral/lateral periaqueductal gray strongly colocalizes β-endorphin. (G–I) All pattern B serum+ interneurons express GAD67-EGFP; (J–L) most but not all pattern B interneurons (parietal cortex) are positive for (J) calbindin but never (L) calretinin, and (K) a few are somatostatin+. (M–O) In one group B case (insular cortex), however, nearly all serum+ interneurons are (O) calretinin+ and (N) occasionally, vasoactive intestinal polypeptide+ (VIP+) but never (M) calbindin+. Green channel: (A, C, D–F, and J–O) serum, (H and I) GAD67-EGFP; red channel: (G and I) serum, (B, C, E, and F) β-endorphin, (D) MCH, (J and M) calbindin, (K) somatostatin, (L and O) calretinin, or (N) VIP. (Scale bars: 100 μm.)

Fig. 4.

Fig. 4.

Characterization of patterns B and C neurons. Cortical bi- and multipolar interneurons are seen in (A) the hippocampus and (B) parietal cortex after incubation with group B serum, and (C) in the parietal cortex after incubation with group B CSF. Multipolar neurons with dendritic processes and axons are stained in the (D) globus pallidus and (E) piriform cortex after incubation with group C serum. (Scale bars: A–C and E, 100 µm; D, 200 µm.)

Fig. 5.

Fig. 5.

Characterization of the immunogenic peptides. Pattern A IgGs recognize a common epitope in the C terminus of αMSH and NEI. (A) Pattern A serum (1:5,000) recognizes αMSH- and NEI-matured peptides (0.6 nmol) and less strongly, NEI-MCH peptide (0.6 nmol; preproMCH [131–165]) but not the matured MCH peptide in the dot blot test. (B) Preincubation of pattern A serum (1:5,000) with 10−5 or 10−6 M αMSH or NEI peptides distinctly attenuates or completely abolishes the binding of serum to NEI or αMSH peptide (0.6 nmol). Preincubation of the same pattern A serum with 10−6–10−3M PV-NH2 or PI-NH2 dipeptides concentration-dependently decreases the binding of serum to NEI or αMSH peptide (0.6 nmol). (C) Summary of adsorption experiments: +, staining intensity does not change; ↓, staining intensity is decreased; ↓↓, staining intensity is highly decreased; 0, staining is completely abolished. (D) Schematic presentation of proMCH and the enzymatic processes leading to formation of matured NEI and MCH peptide. Prohormone-convertase (PC) first cleaves on the C-terminal side of the KR and RR sequences. Carboxypeptidase-C (CpE) removes the RR dibasic extensions. Then, peptidylglycine-α-amidating-monooxygenase (PAM) generates an amide group of G and the C terminus of I in the NEI peptide. (Amidated NEI peptide is the active form in the brain.) Note that the C terminus of αMSH peptide contains an amidated PV C-terminal motif (double underlined). *Acetylation site on the αMSH peptide. Modified from ref. . (E and F) Note the high structural similarities between (E) PV-NH2 and (F) PI-NH2 dipeptides (the difference is only a methylene group; circled). (G–N) Representative micrographs of adsorption experiments showing (G–J) the perifornical region (MCH neurons) and (K–N) Arc (POMC neurons). Note that NEI and αMSH peptides at 10−5 M completely abolish the immunostaining, whereas NEI-MCH peptide at 10−4 M concentration distinctly decreases the staining. (Scale bars: G–J, 200 µm; K–N, 100 µm.)

Fig. 6.

Fig. 6.

(A–I) Physiological effects of pattern A IgG on the sleep architecture. Graphical representation of different vigilance stages and sleep fragmentation [first 6 h, inactive (light) phase of D2] after icv. injection of IgG preparations (65 mg/mL) from (A, D, and G) an HC case (HC-IgG), (B, E, and H) a narcoleptic case with no staining pattern (NP-IgG), and (C, F, and I) a narcoleptic case with an staining pattern (NEI/αMSH-IgG) compared with the BL recordings. Sleep fragmentation is increased in the cases of both narcoleptic IgG preparations (B and C vs. A). The SWS2/SWS1 ratio and the time spent in REM are selectively decreased only in the case of the NEI/αMSH-IgG (F vs. D and E and I vs. G and H; SWS2/SWS1 and time spent in REM, respectively). Statistical analysis: two-way repeated measure ANOVA matched by pairs (repeated factor: hours 1–6); each group was compared with its own BL. *P < 0.05, Bonferroni multiple comparisons posthoc test for every 1 h. (J–L) Graphical representation of the number of REM episodes during the first 6 h of the inactive phase (D2 and D15). The number of REM episodes significantly decreased only in the NEI/αMSH-IgG group at both time points (L vs. J and K). Statistical analysis: one-way repeated measure ANOVA. *P < 0.05, Tukey posthoc test. (M–O) Graphical representation of the numbers of NREM → wake and NREM → REM transitions (first 6 h, inactive phase, and D2) after the injection (N and O vs. M). The number of NREM → wake transitions increased with both narcoleptic sera, whereas the number of NREM → REM decreased selectively with the NEI/αMSH pattern IgG (O vs. M and N). Statistical analysis: one-way repeated measure ANOVA. *P < 0.05, Tukey posthoc test. (P) Summary of the statistically significant alterations in sleep architecture compared with BL recordings. *Significant results of two-way repeated measure ANOVA or one-way repeated measure ANOVA. In the HC and NP groups, no significant effects were observed at D15 (not shown). n.s., not significant. (Q–S) Representative micrographs of the LH (colchicine-treated rat) stained with (Q) serum, (R) CSF, or (S) IgG preparation from the same group A patient all showing the same NEI/αMSH. (Scale bar: Q–S, 200 µm.)

Fig. 7.

Fig. 7.

Effect of icv.-injected IgG from (A and B) an HC, from (C and D) an NC without the NEI/αMSH pattern, and (E and F) an NC with the NEI/αMSH pattern on the distribution and density of EEG power on a 2D-state space heat map in passive and active phases. Plotting the spectral ratios of EEG power data (ratio 1 on the x axis and ratio 2 on the y axis) separated three distinct clusters of EEG power points: right, REM sleep; upper left, NREM sleep; lower left, wake. Each plot represents EEG power data (including all animals per groups) of 6-h recordings on color-coded density maps (digits on the scale show the number of overlapping epochs on a given area). Centroids of different sleep stages are indicated by circles (black, BL; white, day 2). Noteworthy, REM point of NEI/αMSH group shifted toward wake in the active (dark) phase on day 2 vs. BL. The method is modified from ref. .

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