Fig 2: Astrocytic expression of leptin and LepR in the cerebral cortex of young (10–12 week) and old- (48–52 week) 5XFAD (Tg) mice. Thioflavin-S-stained amyloid plaques (arrows) are present in the cerebral cortex of old-Tg and to a lesser extent in young-Tg mice. LepR and leptin was detected in neurons and astrocytes in the cerebral cortex of young- (a and e, respectively) and old-Tg (i and m, respectively) mice. Panels (b,f,j,n) depict astrocytes labelled with GFAP. Panels (c,g,k,o) depict nuclei staining with DAPI. Colocalization of LepR with GFAP (l) and leptin with GFAP (p) labelled astrocytes of old-Tg mice was observed. Co-expression of LepR (d) and leptin (h) with GFAP was also observed to a lesser extent in young-Tg mice. Scale bar = 20 µm.

Fig 3: Colocalization of leptin and LepR with glial fibrillary acidic protein (GFAP) in astrocytes of young (10–12 week) and old (48–52 week) 5XFAD (Tg) mice. Thioflavin-S was used to stain for amyloid plaques. LepR and leptin were detected in astrocytes of both young- (a and e, respectively) and old-Tg mice (l and m, respectively). Astrocytes in old-Tg mice had high expression of GFAP (j,n) compared to young mice (b,f). A high degree of colocalization of GFAP with LepR and leptin in astrocytes was observed in old (k and o, respectively) compared to young-Tg (c and g, respectively) mice. The line expression profile of GFAP and LepR (d) in the astrocytes of young-Tg mice presented similar peaks and troughs along the same relative distance (yellow line). The expression profile of GFAP and leptin (h) shows low levels of leptin (yellow line) in astrocytes of young-Tg mice. Expression profile of GFAP and LepR (l) in the astrocytes of old-Tg mice had similar intensities to young-Tg mice. The intensity profile of GFAP and leptin (p) showed increased expression of leptin in the astrocytes of old-Tg mice. Scale bar = 10µ m.

Fig 4: Leptin and LepR with Thioflavin-S and glial fibrillary acidic protein (GFAP) staining on the hippocampi of young (10–12 week) and old (48–52 week) 5XFAD (Tg) mice. Amyloid plaques stained by thioflavin-S (ThS) are indicated by white arrows. Plaques were more prevalent in the hippocampi of old-Tg (i,j,o,p) compared to young-Tg mice (a,b,g,h). Leptin and LepR was detected in the hippocampus of young-Tg (a and b, respectively) and to a lesser extent in old-Tg mice (i and j, respectively). Both young- (c,d) and old-Tg (k,l) mice also had astrogliosis identified by GFAP staining. Furthermore, astrocytic expression of leptin (o) and LepR (p) with GFAP was observed in the hippocampi of old-Tg mice compared to young-Tg mice (g and h, respectively). Young- (e,f) and old-Tg (m,n) sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 200 µm.

Fig 5: (A) Fluorescence micrographs show the expression of LepR (red) in FRCs from Leprcre;tdTomato mice LNs. Scale bar, 10µm. (B) The expression of LepR in FRCs was confirmed by Western blot. (C) Immunoblot analysis of pSTAT3 (Y705) and STAT3 in FRCs exposed to 100 ng/ml leptin for 15 min and 30 min. GAPDH was used as a loading control. (D) Quantitative RT-PCR analysis was performed on FRCs RNA to examine the expression of iNOS, IDO, and PD-L1 in untreated FRCs and FRCs exposed to 100 ng/ml of leptin for 24 hrs. The data are representative of two independent experiments with five mice/group. All the data are presented as mean ± SEM, Student’s t test, **p < 0.01, n.s., not significant. (E) Cell growth curve. Proliferation rate of FRCs from WT and db/db mice, as measured by cell count. The values are expressed as three independent measurements. All the data are presented as mean ± SEM, Student’s t test, **p < 0.01. (F) Immunoblotting analysis of the cell survival markers cyclinD1, survivin, and Bcl-2 in FRCs exposed to 100 ng/ml leptin for 0.5 hr, 2 hr, 6 hr, and 12 hr. GAPDH was used as a loading control. (G) Quantitative RT-PCR analysis was performed to examine inflammatory response-related gene expression in WT and db/db FRCs. The data are representative of three independent experiments. All the data are presented as mean ± SEM, Student’s t test, **p < 0.01, ***p < 0.001.