IGFBP3 colocalizes with and regulates hypocretin (orexin)

Abstract

Background: The sleep disorder narcolepsy is caused by a vast reduction in neurons producing the hypocretin (orexin) neuropeptides. Based on the tight association with HLA, narcolepsy is believed to result from an autoimmune attack, but the cause of hypocretin cell loss is still unknown. We performed gene expression profiling in the hypothalamus to identify novel genes dysregulated in narcolepsy, as these may be the target of autoimmune attack or modulate hypocretin gene expression.

Methodology/principal findings: We used microarrays to compare the transcriptome in the posterior hypothalamus of (1) narcoleptic versus control postmortem human brains and (2) transgenic mice lacking hypocretin neurons versus wild type mice. Hypocretin was the most downregulated gene in human narcolepsy brains. Among many additional candidates, only one, insulin-like growth factor binding protein 3 (IGFBP3), was downregulated in both human and mouse models and co-expressed in hypocretin neurons. Functional analysis indicated decreased hypocretin messenger RNA and peptide content, and increased sleep in transgenic mice overexpressing human IGFBP3, an effect possibly mediated through decreased hypocretin promotor activity in the presence of excessive IGFBP3. Although we found no IGFBP3 autoantibodies nor a genetic association with IGFBP3 polymorphisms in human narcolepsy, we found that an IGFBP3 polymorphism known to increase serum IGFBP3 levels was associated with lower CSF hypocretin-1 in normal individuals.

Conclusions/significance: Comparison of the transcriptome in narcolepsy and narcolepsy model mouse brains revealed a novel dysregulated gene which colocalized in hypocretin cells. Functional analysis indicated that the identified IGFBP3 is a new regulator of hypocretin cell physiology that may be involved not only in the pathophysiology of narcolepsy, but also in the regulation of sleep in normal individuals, most notably during adolescence. Further studies are required to address the hypothesis that excessive IGFBP3 expression may initiate hypocretin cell death and cause narcolepsy.

Figure 1. IGFBP3 signals in wild type, ataxin-3 hemizygous, and hypocretin KO mice.

The upper panel shows IGFBP3 ISH staining in wild type (A: WT), ataxin-3 hemizygous (B:HZ) and HCRT knockout (C: KO) mice. HCRT staining in neurons (arrowheads) is markedly reduced or absent in the ataxin-3 mouse. The lower panel shows IGFPB3 ISH signal (D: purple; digoxigenin staining with BCIP/NBT), HCRT fluorescence (E: red; Alexa Fluor) immunostaining, and a composite picture (F), indicating that many hypocretin neurons (asterisks) are positive for IGFBP3 in a WT mouse. Scale bar 20 µm.

Figure 2. Colocalization of IGFBP3 in HCRT cells in control and narcolepsy human brain.

Upper panel: Distribution of hypocretin cells and fibers in the perifornical area of human hypothalamus. (A) In control brains, HCRT cells and fibers were densely stained by an anti-HCRT monoclonal antibody (red fluorescence: VectorRed), while in narcolepsy brains, staining was markedly reduced (B) Lower panel: HCRT immunoreactivity (C: red fluorescence) and IGFBP3 immunoreactivity (D: green fluorescene; Q-dot525) and a composite picture (E) arrows indicate HCRT cells colocalized with IGFBP3). Note non-neuronal autofluorescent elements. Scale bar: 500 µm in A and B, 100 µm in C, D and E.

Figure 3. IGFBP3 inhibits HCRT production in vivo and in vitro.

(A, B) Hypocretin-1 peptide content is significantly decreased in both hypothalamus and brainstem of IGFBP3 overexpressing transgenic mice (hIGFBP3 TG). In mutant IGFBP3 overexpressing mice (hmutIGFBP3 TG), the hypocretin-1 peptide shows slight but significant decrease only in brainstem, not in hypothalamus (A, B). Hypocretin mRNA is also significantly decreased in IGFBP3 (hIGFBP3 TG) mice but not in hmutIGFBP3 TG mice. (C) MCH mRNA level is not affected in hIGFBP3 TG or hmutIGFBP3 TG TG mice.(D) IGFBP3 expression reduces preprohypocretin promotor activity in the SH-SY5Y neuroblastoma cell line, but not in non-neural cell lines (HeLa, HEK, SF126, Becker). (F). In human subjects, rs2854744, −202 C, a promotor polymorphism allele known to be associated with reduced IGFBP3 production, is dose dependently associated with increased CSF hypocretin-1 levels. A, B: *: p<0.05; **, p<0.01; ***:p<0.001; C: *: p<0.001 versus without 3.2 kb promotor activity, #:p<0.001 versus promotor activity without IGFBP3; F: *: p<0.05 using ANOVA with genotype as a grouping factor. Sample numbers were indicated in parentheses.

Figure 4. Decreased wakfeluness in human IGFBP3 transgenic mice.

(A) Wakefulness is decreased in the second half of the dark/active period in human IGFBP3 transgenic mouse (hIGFBP3 TG) versus wild type littermates (WT). Reduced sleep rebound is also observed at the same Zeitgeber times (ZT) after sleep deprivation. (B) Cumulative NREM plots at baseline and during recovery after 6 hours of sleep deprivation. Note increased sleep in IGFBP3 transgenic mice in the second half of the active period, ZT17-24. **: p<0.01 versus WT; ***: p<0.001 versus WT. Temperature and locomotion were also affected, see Fig. S2.