Autoimmune demyelination alters hypothalamic transcriptome and endocrine function

Abstract

The hypothalamus is a brain structure that is deputed to maintain organism homeostasis by regulating autonomic function and hormonal production as part of the neuroendocrine system. Dysfunction in hypothalamic activity results in behavioral alterations, depression, metabolic syndromes, fatigue, and infertility. Remarkably, many of these symptoms are associated with multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS) characterized by focal demyelination, immune cell infiltration into the brain parenchyma, and neurodegeneration. Furthermore, altered hormonal levels have been documented in MS patients, suggesting the putative involvement of hypothalamic deficits in MS clinical manifestations. Yet, a systematic analysis of hypothalamic function in response to neuroinflammatory stress is still lacking. To fill this gap, here we performed a longitudinal profiling of the hypothalamic transcriptome upon experimental autoimmune encephalomyelitis (EAE)-a murine disease model recapitulating key MS phenotypes at both histopathological and molecular levels. We show that changes in gene expression connected with an anti-inflammatory response start already at pre-onset and persist along EAE progression. Altered levels of hypothalamic neuropeptides were also detected, which possibly underlie homeostatic responses to stress and aberrant feeding behaviors. Last, a thorough investigation of the principal endocrine glands highlighted defects in the main steroidogenic pathways upon disease. Collectively, our findings corroborate the central role of hypothalamic dysfunction in CNS autoimmunity.

Keywords: Endocrine system; Experimental autoimmune encephalomyelitis; Hypothalamus; Multiple sclerosis.

Fig. 1

Transcriptomic profiling of the hypothalamus along EAE progression. A–C Heatmaps of normalized counts (Z-scores) for the significant differentially expressed genes (DEGs) in the hypothalamus between EAE and control mice at 10, 20, and 40 days post-immunization (dpi). Hierarchical clustering can differentiate the two experimental conditions in each cross-sectional comparison. In the heatmaps, each row represents a DEG and each column a sample. D–F Histograms showing the top 5 most enriched gene ontology (GO) terms among the significant DEGs at the different time points. G, H Most significant protein interaction MCODE networks associated with the DEGs at 10 and 40 dpi. I Validation of RNA-seq results by qRT-PCR on selected DEGs from each time point. All genes except Fabp7, Zfpm1 and Hmgcs1 show significant differences between EAE and control conditions. The data are expressed as log2 transformed fold change (log2FC) and plotted as means ± SEM (N = 3 per group from one EAE immunization). Differences between experimental groups were assessed by two-tailed Student’s t-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 2

Interferon beta signaling in the hypothalamus upon EAE. A Heatmap of the levels of 45 cytokines in the hypothalamus of EAE and control mice at 20 dpi. Each cell represents the average concentration value expressed in pg/mL. B RT-PCR analysis of Ifnb1 and Ifna2 expression in the hypothalamus upon EAE induction. While Ifna2 was detected at all time points, no Infnb1 transcript was identified in the EAE hypothalamus. Gapdh was used as an internal control. Liver (LV), spleen (SP), adipose (AD) and lung (LG) tissues were used as additional controls. (C) Confocal analysis of co-localization patterns between IFNAR1 (red) and cell-specific markers (green) for neurons (NeuN), astrocytes (GFAP), microglia (CD11b), and oligodendrocytes (OLIG2). Nuclei were counterstained with DAPI (blue). IFNAR1 is expressed in all main cytotypes of the hypothalamus. Differences between experimental groups (N = 5 per group from one EAE immunization) were assessed by Mann–Whitney U-test. Magnification = 80 ×, scale bar = 25 μm. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 3

Histopathologic analysis of the adrenal glands upon EAE. A, B Bar plots showing the distributions of weight and adreno-somatic index values for adrenal glands isolated from EAE and control mice at 20 dpi. C Representative H&E sections at low and high magnification of adrenal glands from EAE and control mice at 20 dpi. Cortex (C) and medulla (M) are indicated in the low magnification images, while the zona glomerulosa (ZG), zona fasciculata (ZF) and X-zone (XZ) layers are identified in the high magnification images. Magnification = 10 ×, scale bar = 200 μm (top) and magnification = 40 ×, scale bar = 50 μm (bottom). D Quantification of the size of whole adrenal glands and sub-anatomical regions between EAE and control mice. E Quantification of the thickness of the different layers in adrenal glands between EAE and control mice. Differences between experimental groups (N = 5–6 per group from two EAE immunizations) were assessed by Mann–Whitney U-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 4

Histopathologic analysis of the ovaries upon EAE. A, B Bar plots showing the distributions of weight and gonado-somatic index values for ovaries isolated from EAE and control mice at 20 dpi. C Representative H&E sections at low and high magnification of ovaries from EAE and control mice at 20 dpi. Magnification = 10 ×, scale bar = 200 μm (top) and magnification = 40 ×, scale bar = 50 μm (bottom). D, E Quantification of viable follicles and corpora lutea between EAE and control mice. F Quantification of viable follicles within each maturation stage between EAE and control mice. Differences between experimental groups (N = 5–6 per group from two EAE immunizations) were assessed by Mann–Whitney U-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 5

Histopathologic analysis of the thyroid upon EAE. A, B Bar plots showing the distributions of weight and thyroid-somatic index values for thyroid glands isolated from EAE and control mice at 20 dpi. C Representative H&E sections at low and high magnification of thyroid glands from EAE and control mice at 20 dpi. Magnification = 10 ×, scale bar = 200 μm (top) and magnification = 40 ×, scale bar = 50 μm (bottom). D, E Quantification of mean and individual follicle size between EAE and control mice. Differences between experimental groups (N = 5–6 per group from two EAE immunizations) were assessed by Mann–Whitney U-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 6

Hormonal biosynthetic pathway activity upon EAE. A The expression levels of key enzymes for hormonal biosynthesis were analyzed by qRT-PCR in the adrenal glands from EAE and control mice at 20 dpi. The levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1 (Cyp11a1) and cytochrome P450 11B1 (Cyp11b1) are increased in EAE mice compared to controls. No differences were found in the levels of steroid 5 alpha-reductase 2 (Srd5a2), cytochrome P450 17A1 (Cyp17a1), cytochrome P450 11B2 (Cyp11b2), and cytochrome P450 19A1 (Cyp19a1). B Expression analysis of the same enzymes in the ovaries. The levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 17A1 (Cyp17a1) and cytochrome P450 19A1 (Cyp19a1) are increased in EAE mice. No differences were found in the expression levels of 17-beta dehydrogenase 1 (Hsd17b1), cytochrome P450 11A1 (Cyp11a1), and steroid 5 alpha-reductase 2 (Srd5a2). C Expression analysis for enzymes involved in thyroid hormonal biosynthesis. No differences were detected in the levels of hyroglobulin (Tg), thyroid peroxidase (Tpo), forkhead box protein E1 (Foxe1), and NK2 homeobox 1 (Nkx2-1). The data are expressed as log2 transformed fold change (log2FC) and plotted as means ± SEM. Differences between experimental groups (N = 5 per group from one EAE immunization) were assessed by two-tailed Student’s t-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005

Fig. 7

Quantification of serum hormones upon EAE. A–C Bar plots showing the concentration of the pituitary releasing-hormones adrenocorticotropic hormone (ACTH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) in the serum of EAE mice and controls at 20 dpi. D, E Plots showing the serum concentration of corticosterone and estradiol at the same time points. Data are plotted as means ± SEM and differences between experimental groups (N = 5 per group from one EAE immunization) were assessed by Mann–Whitney U-test. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.005