Fig 2: PSAP/GPR37L1/GPR37–mediated signaling is essential for migration of astrocytes in the scratch assay and the effect of PSAP is mimicked by TX14(A). (a) Astrocytes move into a wound in media containing 5% FBS and in PDM (prosaposin‐depleted media) supplemented with 100 nM TX14(A) but the movement is inhibited in absence of PSAP. Representative images at 0, 24, and 48 hr post scratch. See also Supporting Information Movies SS1–SS3. (b) Dynamics of the relative wound density under conditions shown in a (n = 3, triplicates). (c) Relative wound density 48 hr post scratch for astrocytes incubated in FBS‐containing media, PDM, PDM + TX14(A), PDM + TX14(A) + both knock‐down vectors and PDM + TX14(A) + knock‐down control vector n = 6, triplicates, ***p < .001 vs. indicated group, one‐way ANOVA with Turkey's post hoc analysis). (d): DAPI staining revealed no significant differences in the cell density between astrocytes cultured in media (+FBS), PDM and PDM supplemented with TX14(A) (100 nM; n = 15). (e): The addition of TX14(A) to PDM does not affect the numbers of new astrocytes based on BrdU staining (n = 7, triplicates) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: TX14(A) acting on GPR37L1/GPR37 reduces cAMP levels in astrocytes. (a) Layout of the adenoviral vectors for knock‐down of GPR37L1 and GPR37. Each vector allows co‐cistronic expression of three pre‐miRNAs targeting different regions of the target gene. AVV = human adenoviral vectors serotype 5; CMV = human cytomegalovirus promoter; EmGFP = Emerald green fluorescent protein; miR155 = flanking pre‐miRNA sequence derived from miR‐155. (b) Western blot confirms that AVV–CMV–EmGFP–miR155/GPR37L1 and AVV–CMV–EmGFP–miR155/GPR37 (MOI 10) efficiently knock‐down GPR37L1 and GPR37 in astrocytes. AVV–CMV–EmGFP–miR155/negative is a control vector with hairpin sequence relevant to no known vertebrate gene. (c) Concentration–response curves for inhibition of cAMP production by TX14(A) in astrocytes pretreated with 1 μM NKH477. Cells were transduced with AVV–CMV–Glosensor and either AVV–CMV–EmGFP (control), a mixture of AVV–CMV–EmGFP–miR155/GPR37L1 and AVV–CMV–EmGFP–miR155/GPR37 to knock‐down GPR37L1/GPR37, or with AVV–CMV–EmGFP–miR155/negative (n = 4, triplicates). (d) AVV–CMV–Glosensor transduced astrocytes were pretreated with PTX (20 hr, 100 ng/mL). About 100 nM TX14(A)‐induced cAMP reduction in astrocytes was PTX sensitive (n = 12, ***p < .001 vs. indicated group, one‐way ANOVA with Turkey's post hoc analysis). (e): Astrocytes expressing an EPAC‐based cAMP sensor were kept in PSAP‐depleted media overnight and were stimulated with NKH477 (0.5 μM) in the absence or presence of TX14(A) (100 nM). TX14(A) decreased the transient cAMP signal; average of 58 astrocytes from four experiments. (f) Pooled data from (e) shows significantly decreased FRET ratio peaks with TX14(A) (n = 58 = ****p < .0001, paired t test) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: GPR37L1 and GPR37 are non‐responsive to prosaptide TX14(A) in PRESTO‐Tango assay in HEK293 cells. PRESTO‐Tango uses clones of numerous human GPCR, C‐terminally tagged with a special signaling element. These receptors need to be expressed in a specially designed clone of HEK293 cells. Agonist binding triggers receptor internalization and eventually leads to expression of luciferase and luminescence. Two concentrations of plasmid DNA were used to express the tagged receptors. GPR37 exhibits strong constitutive activity, especially when using 0.1 μg/μL. Values obtained with 0.1 μg of GPR37 DNA could not be fitted with the regression algorithm of Prism software, hence no line is shown (n = 6) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 5: Working model of the neuroprotective role of astrocytic GPR37L1/GPR37 based on the evidence presented in this study. Damaged neurons release diffusible “SOS” factor(s) which trigger release of PSAP. PSAP acts on GPR37L1 on astrocytes and activates release of diffusible neuroprotective factor(s) [Color figure can be viewed at wileyonlinelibrary.com]