Fig 2: TX14(A) acts via GPR37L1 and GPR37 to protect primary astrocytes against toxicity induced by H2O2, staurosporine or rotenone. (a) Pre-exposure to stressors (5 hr) drastically reduce relative wound density recorded at 48 hr in PDM. TX14(A) (100 nM) rescued astrocytes bringing wound density close to normal (compare to Figure 3). GPR37L1/GPR37 knock-down prevented the protective effect of TX14(A), while the control vector had no effect (n = 6, triplicates, ***p < .001 vs. indicated group). (b) LDH release was used as a measure of cytotoxicity, 24 hr after exposure of astrocytes to oxidative stress. In PDM, manipulation of GPR37L1/GPR37 had no effect. TX14(A) (100 nM) protected them from damage but only when they were expressing GPR37L1/GPR37, (n = 6, triplicates, **p < .01, ***p < .001 vs. control group, for example, PDM groups). One-way ANOVA with Bonferroni's post hoc analysis [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: Adoptive transfer of OCN-treated macrophages confers protection against LPS stimulation. (A) Experimental design, to test whether adoptive transfer of macrophages pretreated with OCN, could attenuate LPS-induced acute inflammation. Peritoneal macrophages were isolated from WT or GPR37-/- mice and then treated with PBS or OCN (10 nM) for 24 h, followed by a washout of OCN and an adoptive transfer of macrophages (I.P.) into GPR37-/- mice challenged with LPS for 1 h. (B,C) ELISA analysis of serum IL-6 (B) and TNFa (C) levels, in GPR37-/- mice with adoptive transfer, of macrophages derived from WT or GPR37-/- mice. Sample sizes are indicated as dots in each column. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01, as compared to PBS; n.s. indicates not significant.

Fig 4: A schematic diagram illustrating that the bone-derived hormone OCN, via the activation of GPR37 in macrophages, plays a protective function against LPS challenge through the regulation of macrophage inflammatory reactions and phagocytic function.

Fig 5: Co-cultured astrocytes protect cortical neurons against oxidative toxicity partially through GPR37L1/GPR37 signaling in astrocytes. (a) Experimental design: (A—stressed neurons (blue) in absence or presence of astrocytes (green) on a culture insert; B—depletion of PSAP from the media with anti-PSAP antibodies; C—GPR37L1 and GPR37 knock-down in astrocytes co-cultured with neurons; D—GPR37L1 and GPR37 knock-down in neurons in the co-culture system. Neurons were treated with H2O2 (250 µM) for 1 hr, or staurosporine (100 nM) or rotenone (50 µM) for 2 hr. Stressors were then removed and inserts with astrocytes were introduced. LDH assay was carried out 24 hr later. (b) Astrocytes protect cortical neurons against H2O2-induced stress, the effect saturates at ~75 k astrocytes per co-culture (n = 5, triplicates, ***p < .001 vs. control [no astrocytes insert], ****p < .0001 vs. groups with less than 50 k astrocytes, one-way ANOVA with Tukey's post hoc analysis). (c) TX14(A) has a weak protective effect on neurons against oxidative stress (n = 6, triplicates, ***p < .001, ****p < .0001 vs. 50 nM TX14(A) group (effect of 50 nM is not significant, one-way ANOVA with Bonferoni's post hoc analysis). D: PSAP depletion or GPR37L1/GPR37 knock-down selectively in astrocytes significantly attenuates the protective effect of astrocytes on neurons pre-exposed to oxidative stressors (n = 6, triplicates, ***p < .001 vs. indicated group, one-way ANOVA with Turkey's post hoc analysis) [Color figure can be viewed at wileyonlinelibrary.com]