Fig 2: Gamma frequency light flicker suppresses KCC2 internalization and subsequent degradation via regulating both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. (a) Cortex extracted from 6‐month‐old WT and APP/PS1 littermates treated with 7 days of 1 h/day dark or 40 Hz light flicker immunoprecipitated with an anti‐KCC2 antibody (IP: KCC2) and probed with anti‐ubiquitin antibody. (b) Quantification of the ubiquitinated KCC2 (Ub‐KCC2) for each group (n = 3 mice per group). Data are presented as mean ± SEM. *p < 0.05 vs. WT group, #p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (c) Cortex isolated from 6‐month‐old WT and APP/PS1 littermates with or without 7 days of 1 h/day 40 Hz light flicker immunoprecipitated with an anti‐KCC2 antibody (IP: KCC2) and probed with anti‐phospho‐Tyrosine antibody. (d) Quantification of phosphorylated KCC2 on tyrosine (p‐KCC2 (Tyr)) for each group (n = 3 mice per group). Data are presented as mean ± SEM. #p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (e) Representative immunoblots of KCC2 incubated with MG132 at different concentrations. (f) Representative immunoblots of membrane proteins from 6‐month‐old WT or APP/PS1 mice treated with or without 7 days of 1 h/day 40 Hz light flicker and MG132. (g) Relative immunoreactivity of surface‐KCC2 normalized to Na/K‐ATPase (n = 3). Data are presented as mean ± SEM. *p < 0.05 vs. control group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test

Fig 3: KCC2 C-terminal is essential for Zn2+-dependent upregulation of transport(A) Immunoblot analysis of KCC2, ΔNTD-KCC2, or ΔCTD-KCC2 expression in HEK293 cells. Blots were exposed to a KCC2 antibody together with an anti-FLAG antibody, and normalized to actin as loading control. The KCC2 antibody, which is targeted to the C-terminus, works well with both the WT and ΔNTD KCC2. The FLAG antibody recognizes the WT KCC2 and ΔCTD truncated mutant but works poorly with the ΔNTD. White arrows on the blot point to the monomeric forms of each of the constructs at their expected molecular weight. Right panel shows quantification of expression level, and a schematic drawing of the KCC2 constructs used. (p = 0.0008, Welch’s t test).(B) HEK293 cells co-transfected with GPR39 and KCC2 or KCC2 alone were loaded with the pH-sensitive fluorescent indicator BCECF (1 μM). Cells were washed with K+-free Ringer’s solution, baseline, and addition of NH4Cl (15 mM) resulted in increase in fluorescence due to NH3 diffusion. Subsequent NH4+ transport, used as surrogate by K+ transporter, resulted in acidification (Hershfinkel et al., 2009). Shown are representative fluorescent signal traces from cells treated with Zn2+ (200 μM, 2 min) compared to control cells or cells treated with the KCC inhibitor DIOA (100 μM). Right panel shows averaged rates of NH4+ transport, mediated by KCC2, obtained over initial 100 s from maximal signal.(∗∗∗p< 0.0001, t test).(C) BCECF fluorescent signal traces from cells co-transfected with GPR39 and ΔNTD-KCC2 or ΔCTD-KCC2, treated with or without Zn2+. Averaged rates of NH4+ transport, representing KCC2 activity, are shown in right panel. (∗∗p = 0.0032, t test). Dotted line indicates the rate of transport in cells expressing GPR39 and WT KCC2 that were treated with Zn2+ as in (B).(D) Biotinylation assay of KCC2 variants in HEK293 cells co-transfected with GPR39 and KCC2 or ΔNTD-KCC2 or ΔCTD-KCC2 (upper panel). Comparison of surface expression between cells treated with Zn2+ (200 μM, 2 min) and control cells, using KCC2 and FLAG antibodies (as in A). Loading controls for surface expression were the membrane protein transferrin receptor and cytoplasmic tubulin. Total protein levels (lower panel) determined in cell lysates were used for normalization, tubulin was used as loading control. The arrows mark the protein isoform, quantified in the right panel, at the expected molecular weight. Quantification of normalized membrane expression of KCC2 variants in Zn2+-treated cells versus control (∗∗p = 0.0064 KCC2 with and without Zn2+, ∗∗p = 0.0012 ΔNTD-KCC2 with and without Zn2+, Mann-Whitney test). All bar graphs values are averages ± SD, with all data points presented.

Fig 4: ZnR/GPR39-dependent upregulation of KCC2 activity is not mediated through its phosphorylation(A) Scheme of residues that are putative phosphorylation sites on KCC2.(B) BCECF fluorescent signal traces from HEK293 cells co-transfected with GPR39 and WT KCC2 or KCC2 mutant S940A, treated with Zn2+ (200 μM, 2 min) and compared to controls. Cells were treated with NH4Cl and rates of acidification following NH4+ transport were determined. Bar graph shows averaged rates of NH4+ transport, calculated over initial 100 s from maximal signal.(∗∗∗p<0.0001 Mann-Whitney test).(C) BCECF fluorescent signal traces from HEK293 cells co-transfected with WT KCC2 and GPR39, pretreated with the PKC inhibitor staurosporine and then with Zn2+, compared to controls. Right panel shows averaged rates of acidification following NH4+ transport in control cells or cells treated with the PKC inhibitors staurosporine (1 nM) or GO6983 (10 μM). (∗∗∗p<0.0001 KCC2 with and without Zn2+, ∗∗p = 0.0022 staurosporine with and without Zn2+, ∗∗∗p = 0.0005 GO6983 with and without Zn2+, Mann-Whitney test).(D and E) Averaged rates of acidification following NH4+ transport monitored in HEK293 cells co-transfected with GPR39 and the indicated KCC2 mutants. Cells were treated with Zn2+ or without it. (all comparisons are between control and Zn2+-treated mutants, panel D using t test: p = 0.0046 KCC2, p = 0.0212 T1007E, p = 0.0088 T906E; for panel E, t test was used: p<0.0001 KCC2, p = 0.0002 S988A, p = 0.0002 S986/988A, p = 0.0003 S986/988D, p<0.0001 T584A, p = 0.0002 S868A, p = 0.0274 SPVSS; and p = 0.0041 S986A using Mann-Whitney test). All bar graphs values are averages ± SD, with all data points presented.

Fig 5: Model shows the potential mechanism by which 40 Hz light flicker reduces Aβ levels. Phosphorylation of APP induced by PKC activation under the treatment of 40 Hz light flicker led to maintained plasma membrane levels of full‐length APP as well as decreased trafficking to endosomes, which ultimately inhibited BACE1 cleavage pathway. Moreover, on the basis of PKC‐induced serine phosphorylation of KCC2, the tyrosine phosphorylation and degradation of KCC2 were further limited by a direct interaction with full‐length APP anchored within the plasma membrane, which contributed to the upregulation of surface GABAA receptor α1 levels. In addition, the increase of ATP caused by 40 Hz light flicker promoted PLC/DAG signaling cascade, which is likely to be involved in the activation of PKC