(A) RT-PCR experiments performed on total RNAs from a transgenic mouse hypothalamus (Tg+), showing the presence of both the construct and hypocretin (Hcrt) transcripts. Four different primer pairs were used, each generating a PCR fragment of the size predicted by its cDNA sequence: 5′ Hcrt/Flag (467 bp), Pabpc1/IRES (333 bp), GFP (611 bp), and Hcrt (189 bp). (B) Quantitative RT-PCR experiments showing similar relative RNA expression levels for Flag-tagged Pabpc1, GFP, and Hcrt in transgenic mice hypothalamus. Only the Hcrt transcript was detected in nontransgenic (Tg-) mouse hypothalamus as expected. The normalized relative values (+ 1 SD) obtained correspond to the mean values from 4 biological replicates per condition. (C) Western blot analysis demonstrating the presence of the Flag protein in the hypothalamic (hypoth.) extract (obtained from 3 transgenic mice) and its absence in the cortical fraction (from the same 3 mice). The loading control is a nonspecific band generated by the mouse monoclonal anti-Flag M2 antibody. Photomicrographs illustrating the distribution of (D and F) Hcrt- and (E and G) Flag-expressing cells on a coronal section from the tuberal hypothalamus. (F–H) Higher magnification views of the dashed regions in D and E confirm that Hcrt and Flag are coexpressed in the same neurons. Arrows indicate both single Hcrt or Flag or double Hcrt-Flag staining. Almost all Hcrt neurons were found to express the Flag-containing construct. (H) Merged image of F and G. cp, cerebral peduncle; fx, fornix; mtt, mammilo-thalamic tract; 3V, third ventricle. Scale bar: 100 μm (D and E); 20 μm (F–H).

(A) Each symbol corresponds to the serum of a single subject. Mean ± 1 SD of each group is shown next to the individual values. The dotted horizontal line indicates the mean Trib2-specific antibody titer in healthy control subjects plus 2 SD. All values are relative to the optical density of a healthy control subject (which is equal to 1). P values correspond to independent t-tests between indicated groups. OIND, other inflammatory neurological diseases. (B) Mean ± 1 SD of Trib2-specific antibody titers at different intervals from the disease onset. The solid line and dotted lines indicate mean titer ± 1 SD in normal control subjects. Numbers indicate the number of narcolepsy patients at each interval. Note the sharp decrease in titers within the first 2–3 years, reaching normal values. From 5 up to 30 years after disease onset, the titers of narcolepsy patients remain stable but significantly (1 SD, P < 5 × 10^–5) higher than those of healthy control subjects (n = 42).

(A) Coronal section through the mouse hypothalamus, stained with anti-hypocretin antibody. (B) The same section stained with the patient’s serum. (C) Merged hypocretin-serum staining showing that the vast majority of hypocretin-positive neurons are costained by the patient’s serum. (D–F) Higher magnification views of the dashed regions in A–C confirm the double labeling of hypocretin neurons. Overall, 87% of hypocretin neurons are costained by the serum. (G) Another mouse hypothalamic section stained with anti-hypocretin antibody. (H) Immunoreactivity with the same patient’s serum as in B but after preabsorption of Trib2-specific antibodies with excess Trib2 peptide. (I) Merged image showing that the vast majority of hypocretin neurons are only stained with hypocretin antibody. Only 8% of hypocretin neurons are double labeled. (J–L) Higher magnification views of the dashed regions in G–I confirm that serum depletion of Trib2-specific antibodies results in the absence of double staining. Scale bar: 20 μm.