Fig 2: Hypothalamic microgliosis in AD mice occurs in a nucleus-specific manner but is unaffected by sex or HF diet. Labeling of tissue sections including the hypothalamus was performed for Iba1. Quantification was performed for percent area positive for each label in the arcuate nucleus (a), ventromedial nucleus (b), dorsomedial nucleus (c), paraventricular nucleus (d), and lateral hypothalamus area (e). Representative images for labeling with Iba1 are also shown (f). N = 3–5/group. ^ = AD effect p < 0.05; ^^ = AD effect p < 0.01; ^^^ = AD effect p < 0.001

Fig 3: Numbers of neurons but not microglia are increased in the TRN in adult Casp1-/- mice. This figure is supported by Extended Data Figure 4-1. A, Enlarged image of the GFP+PI+ cluster shown in Figure 3. Mice were injected with PI as shown in Figure 3A, and fetal brain slices were stained with anti-Pax6 antibody. Images of Cx3cr1-GFP (top left), PI (top right), anti-Pax6 (bottom left), and merged signals are shown. Scale bar: 50 µm. B, Representative images of anti-NeuN staining signals from male Casp1-/- (N = 3, right, from 2 litters) and sex/age-matched WT (N = 3, left, from 2 litters) adult brains are shown. TRN regions are outlined in yellow. Scale bar: 200 µm. C, Average anti-NeuN signals from Casp1-/- (red triangles) and WT (black circles) mice are shown as cell number/mm2. Dots indicate individual animals and are the average cell numbers from a total of six serial sections stained as shown in B; p = 0.0209, df = 4, t = 3.694. Error bars indicate SEM; *p < 0.05. D, Representative images of anti-Iba1 antibody staining in the TRNs (regions outlined in yellow) of male WT (N = 3, left, from 2 litters) and Casp1-/- (N = 3, right, 2 litters) mice are shown. Scale bar: 200 µm. E, Average number of Iba1+ cells in Casp1-/- (red triangles) and WT (black circles) mice are shown as mean cell number/mm2. Dots indicate individual animals and are the average of cell numbers from a total of six serial sections stained as shown in D; p = 0.7533, df = 4, t = 0.336. Error bars indicate SEM; n.s., not significant.

Fig 4: A 2-week therapeutic treatment with BLZ945 after a 5-week cuprizone intoxication period reduced microglia numbers but enhanced astrocytes. a Representative pictures from immunohistological stainings detecting the microglia marker Iba1 and glial fibrillary acidic protein (GFAP) astrocytes in the cortex for the different treatment groups at week 7 (see Fig. 2a for the experimental setup and groups). b, c, d Corresponding quantitative analysis of the immunohistochemistry for Iba1-positive microglia numbers and GFAP-positive astrocyte stained area in the cortex, striatum and corpus callosum/external capsule. Values were normalized to those of control, vehicle-treated mice. Group sizes: For all treatments n = 7. Data are shown as means±SEM. Scale bars: 100 µm. Statistics: Turkey’s multiple comparison test one-way ANOVA (**: p < 0.01, ***: p < 0.001, ****: p < 0.0001, n.s.: not significant), cpz: cuprizone, cc: corpus callosum, ec: external capsule

Fig 5: Sex differences in the effects of AD and diet on peripheral inflammation and hypothalamic expression of inflammation-related genes. a–d Plasma concentrations of cytokines. Blood was collected following a 5 h fasting period just prior to euthanasia and assayed for 23 cytokines, with significant group differences seen for a IL-10, b IL-12 (p40), c MIP-1a, and d MIP-1ß. N = 3–4/group for all plasma markers. e–g Gene expression levels in visceral fat related to inflammation, including e TNF-a, f IL-1ß, g IL-6. h–l Gene expression levels in homogenate of the whole hypothalamus related to inflammation. Hypothalamus was collected following a 5-h fasting period just prior to euthanasia and assayed for h TNF-a, i IL-1ß, j IL-6, k Iba1, l GFAP. N = 5–8/group for all gene expression analyses in the hypothalamus. * = diet effect p < 0.05; *** = diet effect p < 0.001; **** = diet effect p < 0.0001; $ = sex effect p < 0.05; $$ = sex effect p < 0.01; $$$$ = sex effect p < 0.0001; ^ = AD effect p < 0.05; ^^ = AD effect p < 0.01; ^^^ = AD effect p < 0.001; ^^^^ = AD effect p < 0.0001