Inhibition of WNK in mice enhances learning and memory.

A) Model shows WNK downstream signaling. B) Quantification shows pOSR1 (Ser325)/total OSR1 from hippocampi of mice treated with WNK463 (PO; 6 mg/kg) or vehicle; n=4. C) Diagram representing Novel Object Recognition test protocol. D) Quantification of discrimination index in mice administered with WNK463 (PO; 6mg/kg) (n=13) or vehicle (n=19) tested on the Novel Object Recognition protocol. E) Diagram representing Context-Cue Fear Conditioning test protocol. F) Quantification of % Freezing in Contextual Fear Conditioning test in mice administered WNK463 (PO; 6 mg/kg) (n=11) or vehicle: n=9. G) Quantification of % Freezing in Cued Fear Conditioning test in mice administered WNK463 (PO; 6mg/kg) (n=11) or vehicle: n=9. H) Diagram representing locomotor test protocol. I) Quantification of locomotory activity in mice administered WNK463 (PO; 6mg/kg) or vehicle; n=6. J) Model shows the effect of WNK inhibition on learning and memory in mice. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test or one-way ANOVA. ns: non-significant, *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

Inhibition of hippocampal WNK enhances anxiety-related behavior in mice.

A) Diagram representing Open Field test protocol. B) Graph representing cumulative time spent in the center by mice treated with vehicle or WNK463 (PO; 6 mg/kg) in the Open Field test; n=7. C) Graph representing the number of entries (frequency) in the center by mice treated with vehicle or WNK463 (PO; 6 mg/kg) in the Open Field test; n=7. D) Diagram representing Elevated Plus Maze test protocol. E) Graph representing cumulative time spent in the open arms by mice treated with vehicle or WNK463 (PO; 6 mg/kg) in the Elevated Plus Maze test; n=8. F) Graph representing the number of entries (frequency) in the open arms by mice treated with vehicle or WNK463 (PO; 6 mg/kg) in the Elevated Plus Maze; n=8. G) Graph representing cumulative time spent in the center by mice treated with vehicle (n=8) or WNK463 (PO; 6 mg/kg) (n=7) in the Open Field test after mice received electric foot-shocks. H) Graph representing the number of entries (frequency) in the center by mice treated with vehicle (n=8) or WNK463 (PO; 6 mg/kg) (n=7) in the Open Field test after mice received the electric foot-shocks. I) Graph representing cumulative time spent in the open arms by mice treated with vehicle (n=7) or WNK463 (PO; 6 mg/kg) (n=6) in the Elevated Plus Maze test after mice received electric foot-shocks. J) Graph representing the number of entries (frequency) in the open arms by mice treated with vehicle (n=7) or WNK463 (PO; 6 mg/kg) (n=6) in the Elevated Plus Maze after mice received electric foot-shocks. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. ns: non-significant; p>0.05.

Inhibition of WNK augments glucose uptake via GLUT4.

A) Model shows downstream effects of insulin and the impact of WNKs on insulin signaling. B) Graph representing quantification of in vivo radioactive 2-deoxyglucose uptake per mg hippocampal weight in mice treated with vehicle or WNK463 (PO; 6 mg/kg); n=4. C) Graph representing enhanced radioactive 2-deoxyglucose uptake in hippocampal slice culture from C57BL/6J whole brains treated with WNK63 (1 µM); n=4. D) Graph representing enhanced radioactive 2-deoxyglucose uptake in crude synaptosome from C57BL/6J whole brains treated with WNK63 (1 µM); n=3. E) Graph shows in vitro radioactive 2-deoxyglucose uptake in SH-SY5Y cells treated with WNK463 (1 µM), insulin (10 nM); n=7. F) Graph representing enhanced radioactive in vitro 2-deoxyglucose uptake in differentiated SH-SY5Y cells treated with insulin (10 nM) ± WNK463 (1 µM) ± indinavir (10 nM); n=5. G) Representative Western blot shows surface and total GLUT4 protein fraction from mice hippocampal slices treated with insulin (10 nM) and/or WNK463 (1 µM). H) Corresponding quantification of ‘G’ shows enhanced surface GLUT4 (measured as a fraction of total GLUT4) with WNK463 ± insulin treatment; n=4. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

Inhibition of WNK enhances insulin signaling in the hippocampus and cell culture.

A) Model shows insulin signaling and the impact of WNK kinases on insulin signaling. B) Quantification of pAKT/AKT from hippocampi of mice treated with WNK463 (PO; 6 mg/kg) or vehicle; n=4. C) Representative Western blot shows pAKT, AKT and pOSR1 in the presence of WNK463 (1 µM) ± insulin (10 nM) in SH-SY5Y cells. D) Quantification of pAKT/AKT in differentiated SH-SY5Y cells treated with insulin (10 nM) ± WNK463 (1 µM); n=4. E) Representative Western blot shows pAKT and GAPDH in presence of WNK463 (1 µM) ± insulin (10 nM) in mouse cortical primary cells. F) Quantification of pAKT/GAPDH in cortical primary cells treated with insulin (10 nM) ± WNK463 (1 µM); n=2 for DMSO and insulin and n=3 for WNK463±insulin. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

OSR1 interacts with molecular mediators involved in GLUT4 trafficking.

A) Model shows balanced regulation of sortilin and AS160 by the WNK/OSR1/SPAK pathway to regulate GLUT4 trafficking. B) Representative Western Blot shows co-immunoprecipitation of endogenous OSR1 with AS160 from whole brain C57BL/6J mouse lysates; n=3. C) Representative Western blot shows co-immunoprecipitation of AS160 with OSR1 upon treatment with WNK63 (1 µM) and/or insulin (10 nM) in differentiated SH-SY5Y cells. D) Corresponding quantification of ‘C’ shows decreased association of AS160 and OSR1 in cells treated with insulin (10 nM) + WNK463 (1 µM) compared to insulin alone; n=6. E) Representative Western blot shows pAS160, AS160 and GAPDH in differentiated SH-SY5Y cells treated with insulin (10 nM) ± WNK463 (1 µM). F) Corresponding quantification of ‘E’ shows increased pAS160 in cells treated with insulin (10 nM) or WNK463 (1 µM) compared to DMSO; n=4. G) R-x-F-x-V-containing blocking peptide (BP: WNK1 1253-1265; NH +-SAGRRFIVSPVPE-COO−; 100 µM) decreases interaction of overexpressed OSR1/SPAK CCT bait protein fragment (aa 50-545) with myc-AS160 protein fragment (aa 193-446) in vitro; n=3. I) Corresponding quantification of ‘G’. I) Bright-field (left) or confocal images shows co-localization of endogenous OSR1 with sortilin in differentiated SH-SY5Y cells. OSR1 (red), sortilin (green), merged (yellow), scale bar=10 µm; n=3. J) Representative endogenous co-immunoprecipitation of OSR1 with sortilin in differentiated SH-SY5Y cells; n=5. K) Yeast two-hybrid assay shows binding of the conserved C-terminus (CCT) of OSR1 with the C-terminus (C-t) of sortilin (3); binding of full-length and CCT of OSR1 to the C-terminus of WNK1 as positive controls (5,7). N-t: N-terminus. L) Representative Western blot shows co-immunoprecipitation of OSR1 and Flag-sortilin in HEK cells is diminished upon co-incubation with the blocking peptide (BP) SAGRRFIVSPVPE; n=3. M) Corresponding graphical representation for ‘L’; n=8. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

A) Representative Western blot shows change in body weights of C57BL/6J mice treated with vehicle or WNK463 (PO; 6mg/kg) for 1 week; n=10. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test. ns: non-significant; p>0.05.

A) Quantification of total exploration time during Novel Object Recognition test protocol in mice administered with WNK463 (PO; 6 mg/kg); n=13 or vehicle; n=19. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test. ns: non-significant; p>0.05.

A) Quantification of total distance moved (cm) in the Open Field test in mice administered with WNK463 (PO; 6 mg/kg) or vehicle; n=7. B) Quantification of total distance moved (cm) in the Open Field test in mice administered with WNK463 (PO; 6 mg/kg) (n=7) or vehicle (n=8) in mice pre-exposed to electric foot-shocks. C) Quantification of total distance moved (cm) in the Elevated Plus Maze test in mice administered with WNK463 (PO; 6 mg/kg) or vehicle; n=8. D) Quantification of total distance moved (cm) in the Elevated Plus Maze test in mice administered with WNK463 (PO; 6 mg/kg) or vehicle in mice pre-exposed to electric foot-shocks; n=7. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test. *p<0.05, **p<0.005 and *** p<0.0005. ns: non-significant; p>0.05.

A) Representative Western blot shows surface and total GLUT4 protein fraction in differentiated SH-SY5Y cells treated with insulin (10 nM) and/or WNK463 (1 µM). B) Corresponding quantification of ‘A’ shows enhanced surface GLUT4 (measured as a fraction of total GLUT4) with WNK463 ± insulin treatment; n=3. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t-test. *p<0.05, **p<0.005 and *** p<0.0005.

A) Graph representing the binding affinities of WT-sortilin and sortilin mutant peptides with OSR1/SPAK CCT determined by fluorescence anisotropy. Unlabeled peptides displace labeled peptide (NH3+-NLVGRF-[DAP-FAM]-VSPVPE-COO−) [diaminopropionic acid (DAP)]. Labeled peptide held constant at 25 nM, OSR1 CCT held constant at 3.0 μM; n=3.