Fig 2: The beneficial effect of blocking the nNOS–CAPON interaction might be due to rescue of the ERK–CREB–BDNF pathway. (a) pression of spinophilin and BDNF and decreased phosphorylation of ERK and CREB in the hippocampus, and these changes were recovered by TAT‐CAPONi (n = 6, * p < .05 compared to the 4‐month‐old C57BL/6J mouse group, # p < .05 compared to the 4‐month‐old APP/PS1 mice group). (b) Beneficial effects could be observed in vitro and were abolished with an ERK1/2 inhibitor but not with a PKA inhibitor or a TrkB inhibitor (n = 6, * p < .05 compared to the control group, # p < .05 compared to the oligo‐Aβ group, & p < .05 compared to the oligo‐Aβ+TAT‐CAPONi group)

Fig 3: EGF-ERK1/2 activation in Müller glia depends on retinal cell death. Dual-phosphorylation (P) of Tyr204/Y187 of p44/42 mitogen-activated protein kinases p44/42 (MAPK3, MAPK1), also called P-ERK1/2 (extracellular signal-regulated kinases 1 and 2), correlates with experimentally induced cell death in retinal explant culture (related to Figs 2–5): (a) P-ERK1/2 expression in Müller glia depends on retinal explant protocol (cC versus dC protocol, see Fig. 2) and experimental retinal damage (stab wound, SW, see Fig. 3, Table S1). Images and quantitative analysis of retinal explant sections immunostained for the MG marker SOX2 and P-ERK1/2 expression showed lower amounts of P-ERK1/2+ MG in retinal explants with reduced baseline cell death (continuously cooled protocol, cC) compared to higher baseline cell death (discontinuously cooled protocol, dC). Stab wound injury (SW) applied to retinal explants with low baseline cell death (cC protocol) increased P-ERK1/2 expression in MG in the lesion area compared to unharmed neighboring control areas ( > 600 µm distant to lesion area; dC vs cC, N = 4; SW, N = 3). (b) P-ERK1/2 expression in Müller glia was differentially reduced upon application of cell death pathway inhibitors, which efficiently prevented retinal cell death and MG proliferation (Figs 4–5, N ≥ 3, Tables S1 and S4). (c) Western blot analysis of whole retinal explant lysates revealed decreased ERK1/2 activation in experimental paradigms with lower retinal cell death, comparing cC /dC methods and upon cell death inhibition by Nec1+ ZVAD (one data point was removed as an outlier: 2.5-fold different than the mean, P < 0.05 Grubb’s test; complete data shown in Fig. S6c). P-ERK1/2 levels in SW lesioned retinas are lower than in control retinas. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. DEV, days ex vivo. Data are presented as mean ± SD (Western blot) and ± SEM all others. *P < 0.05; **P < 0.01; ***P < 0.001 with Student’s t-test (unpaired, two-tailed). Scale bars: 50 µm.

Fig 4: EGFR signaling is necessary for Müller glia proliferation and ERK1/2 activity. (a) Potential EGFR activity in Müller glia (MG) in EGF-stimulated retinal explant culture upon indicated treatments and days ex vivo. For analysis, retinal sections were immunostained for the MG marker SOX2 and dual phosphorylation (P) of Tyr204 / Y187 of p44/42 mitogen-activated protein kinases 3 and 1 (MAPK3, MAPK1), also called P-ERK1/2 (extracellular signal-regulated kinases 1 and 2). EdU was given cumulatively throughout explant culture to monitor cell proliferation. EdU+ SOX2+ double-positive nuclei indicate MG proliferation. (a) Images and quantitative analysis showed that retinal explant culture induced a strong upregulation of P-ERK1/2 immunolabeling with radial morphology. Early on P-ERK1/2 overlaps with SOX2+ MG nuclei located in the INL, and later P-ERK1/2+ SOX2+ MG are also found in the ONL (arrow). Dashed white boxes show regions of interest at higher magnification (DEV 0 & 2, N = 7; DEV 0.75, N = 5; DEV 4, N = 3)). (b,c) Application of EGFR inhibitors PD153035 or PD158780 throughout retinal explant culture blocked (b) P-ERK1/2 upregulation, shown by IHC (N = 4) and Western blot analysis (N = 5), and (c) MG proliferation (N = 3) each in comparison to solvent control (CTRL). Bar graph color scheme indicates: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer or cell across all layers (total cells). DEV, days ex vivo. Data are presented as mean ± SD (Western blot) and ± SEM all others; N ≥ 3 (see Table S4). *P < 0.05; **P < 0.01; ***P < 0.001 with Student’s t-test (unpaired, two-tailed). Scale bars: 50 µm. See Fig. S6.

Fig 5: C1q regulates ERK1/2 phosphorylation in IC-stimulated BMDM and THP-1. a BMDM were adhered to 4 μg/mL HSA (control) or C1q for 30 min and then incubated with IC for an additional 0, 10 min, or 30 min. After the indicated time, cells were lysed and levels of phospho-proteins and control proteins were detected by western blot. One representative western blot from 3 to 6 replicates is shown b BMDM were processed as described in a and phosphorylated ERK1/2 (p-ERK) was normalized to either total ERK1/2 (n = 3) or actin (n = 3) loading controls at 0 and 10 min using ImageJ. Bars represent the mean of 6 individual experiments with SEM and points represent individual experiments. *p < 0.05 paired student’s t-test for time zero control versus C1q. c THP-1 cells were processed as described in a and one representative blot out of three is shown. All western blot images were cropped to display relevant bands. Corresponding uncropped full-length blots are included in supplementary Fig. 2