Decreased D-serine Level, Increased Stargazin-SR Interaction, and Decreased Stargazin pT321 Level in the Penumbral Tissue after MCAO/R injury
To identify SR catalytic activity after MCAO/R, we established an experimental model of MCAO/R in male SD rats and subsequently performed gas/liquid chromatography analysis of brain homogenate samples from penumbral tissue. Compared with the sham group, the D-serine level was decreased at 3 h after MCAO/R and rebounded at 6 h and 24 h (Fig. 1a and b) (Sham: 1.00 ± 0.15, MCAO/R 1 h: 1.03 ± 0.17, MCAO/R 3 h: 0.79 ± 0.13, MCAO/R 6 h: 0.99 ± 0.12, MCAO/R 24 h: 1.00 ± 0.13; one-way ANOVA, F (4, 25) = 3.078, P < 0.05). Additionally, we used immunofluorescence labeling to determine the effects of MCAO/R on the stargazin-SR interaction. Our results indicated that the ratio of SR colocalized with stargazin was increased in the penumbral tissues of MCAO/R rats compared with that of sham rats (Fig. 1c and 1d) (Sham: 1.00 ± 0.67, MCAO/R 3 h: 1.69 ± 0.34; Student’s t test, t (2.25) = 10, P < 0.05). The results of the co-immunoprecipitation assays consistently showed that the stargazin-SR interaction was significantly higher 3 h after MCAO/R compared with the sham group, providing further evidence for an increase in the stargazin-SR binding in the penumbral tissues of MCAO/R rats (Fig. 1e and 1f) (Sham: 1.00 ± 0.29, MCAO/R 3 h: 1.51 ± 0.38; Student’s t test, t (2.58) = 10, P < 0.05).
It has been established that the binding between stargazin and SR is regulated by phosphorylation of stargazin at Thr-321 [17]. We therefore sought to investigate whether the stargazin pT321 level changed as a result of stroke damage. Western blotting was subsequently performed to examine the protein levels of stargazin and stargazin pT321. Our results showed that MCAO/R 3 h increased the level of stargazin (Fig. 1g and 1i) (Sham: 1.00 ± 0.18, MCAO/R 3 h: 1.70 ± 0.09, Student’s t test, t (8.60) = 10, P < 0.01), but it decreased the level of stargazin pT321 in the penumbrae compared with the sham group (Fig. 1h and 1j) (Sham: 1.00 ± 0.13, MCAO/R 3 h: 0.73 ± 0.09; Student’s t test, t (4.19) = 10, P < 0.01). Thus, the ratio of stargazin pT321 to stargazin decreased by approximately 60% in the MCAO/R group (Fig. 1k) (Sham: 1.04 ± 0.3, MCAO/R 3 h: 0.43 ± 0.04, Student’s t test, t (5.03) = 10, P < 0.01).
Genetic Knockdown of Stargazin Reduces the Stargazin-SR Interaction and Increases the D-serine Level in the Penumbral Tissue of Rats Subjected to MCAO/R
We next sought to determine whether the increases in stargazin-SR binding regulates ischemic insult. To this end, we used lentivirus-mediated expression of specific short hairpin RNAs (shRNAs) against stargazin. The transduction efficiency of stargazin lentivirus was examined by fluorescence assays in vivo and in vitro (Fig. 2a and 2b). Then, we verified the interfering effects of the stargazin lentivirus using western blotting. Our results showed that compared with the negative control group, the level of stargazin was significantly decreased in the LV-ShRNA-STG group (Fig. 2c and 2d) (Sham: 1.00 ± 0.20, MCAO/R 3 h: 1.60 ± 0.14, MCAO/R 3 h + Ctr ShRNA: 1.64 ± 0.14, MCAO/R 3 h + ShRNA-STG: 1.06 ± 0.18, one-way ANOVA, F (3, 20) = 24.82, P < 0.01). Next, we examined whether the stargazin-SR interaction was affected by stargazin knockdown. The results of fluorescent labeling showed a decreased proportion of SR colocalized with stargazin in penumbral-tissue neurons of the stargazin-downregulated rats compared with those of the control rats (Fig. 2e and 2f) (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.20, MCAO/R 3 h + ShRNA-STG: 0.57 ± 0.22, Student’s t test, t (3.57) = 10, P < 0.05). Moreover, the results of the coimmunoprecipitation assay (Fig. 2g and 2h) showed a reduction in the interaction between stargazin and SR in the stargazin-downregulated group compared with that in the negative control group (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.06, MCAO/R 3 h + ShRNA-STG: 0.75 ± 0.13; Student’s t test, t (4.26) = 10, P < 0.01). We next employed gas/liquid chromatography analysis to confirm the role of the stargazin-SR interaction downregulation in SR activity. Our results showed that the level of D-serine level was increased in the stargazing-downregulated group compared with that in the negative control group (Fig. 2i and 2j) (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.08, MCAO/R 3 h + ShRNA-STG: 1.15 ± 0.11; Student’s t test, t (2.73) = 10, P < 0.05). These findings suggest that downregulation of stargazin increased the expression of D-serine, which may be associated with a reduction in the stargazin-SR interaction.
Genetic Knockdown of Stargazin Aggravates Ischemic Neuronal Death and Neurological Function after MCAO/R in Rats
Having determined that SR activity is affected by binding of stargazin to SR, we then explored whether stargazin downregulation affected neurological functions and brain damage in rats subjected to MCAO/R (Fig. 3a). Assessment of cerebral infarction 24 h after MCAO/R using TTC staining showed that total infarction volume in stargazin-knockdown rats was increased compared with the control rats (Fig. 3b and 3c) (MCAO/R 24 h: 0.27 ± 0.07, MCAO/R 24 h + Ctr ShRNA: 0.27 ± 0.06, MCAO/R 24 h + ShRNA-STG: 0.36 ± 0.03; one-way ANOVA, F (2, 15) = 7.592, P < 0.05). Furthermore, mNSS was significantly higher in the stargazing-downregulated group compared with the negative control group (Fig. 3d) (Sham: 0.00 ± 0.00, MCAO/R 24 h: 9.67 ± 2.65, MCAO/R 24 h + Ctr ShRNA: 10.10 ± 2.33, MCAO/R 24 h + ShRNA-STG: 14.25 ± 2.61; one-way ANOVA, F (3, 34) = 80.72, P < 0.0001). To further evaluate the functional-recovery effect of stargazin downregulation in rats subjected to MCAO/R, we used the rotarod test to assess the post-stroke locomotor activity, the adhesive-removal test to evaluate forelimb sensorimotor asymmetries, and the Morris water maze test to examine the spatial learning and memory function. Untreated MCAO/R rats performed significantly worse in all three tests compared with the sham-treated rats (Fig. 3e–3k). Moreover, rats treated with shRNA-stargazin showed a significant increase in the time spent on the accelerating rotarod compared with the MCAO/R +LV-shRNA-control rats (Fig. 3e) (ShRNA-STG [F (3, 196) = 204.00, P < 0.0001], test day [F (4, 196) = 124.20, P < 0.0001], with an interaction observed between these two factors [F (12, 196) = 16.63, P < 0.0001]). The sensorimotor function was aggravated in MCAO/R + LV-shRNA-stargazin rats compared with that of the MCAO/R+LV-shRNA-control rats, as demonstrated by the significantly shortened contact time (Fig. 3f) (ShRNA-STG [F (3, 196) = 101.00, P < 0.0001], test day [F (4, 196) = 91.86, P < 0.0001], with an interaction observed between these two factors [F (12, 196) = 11.12, P < 0.0001]) and removal time (Fig. 3g) (ShRNA-STG (F (3, 196) = 140.10, P < 0.0001), test day (F (4, 196) = 128.90, P < 0.0001), with an interaction observed between these two factors (F (12, 196) = 15.70, P < 0.0001]). The Morris water maze test, performed 22 to 27 days post-MCAO/R (Fig. 3a), showed that stargazin downregulation aggravated spatial learning, as evidenced by the decreased time spent seeking out the underwater hidden platform compared with the time taken by the lentivirus-negative control rats (Fig. 3h and 3i) (ShRNA-STG [F (3, 190) = 29.64, P < 0.0001], test day [F (4, 190) = 21.37, P < 0.0001], with an interaction observed between these two factors (F (12, 190) = 0.21, P = 1.00]). Unlike the other tests used in our study, the memory test indicated no difference in memory function between the MCAO/R +LV-shRNA-stargazin group and the MCAO/R + LV-shRNA-control group. Additionally, the swimming speed was similar among the rats used in our present study (Fig. 3j) (Sham: 27.04 ± 4.35, MCAO/R: 27.59 ± 3.04, MCAO/R + Ctr ShRNA: 27.32 ± 4.06, MCAO/R + ShRNA-STG: 27.11 ± 5.68; one-way ANOVA, F (3, 37) = 0.03, P = 0.99). On day 27 after sham or MCAO/R surgery, the platform was removed and the rats were evaluated using the memory test. Compared with the performance of the rats in the sham group, the rats in the MCAO/R group showed a spatial memory deficit, as demonstrated by a reduced numbers of passes over the previous location of the platform (Fig. 3k) (Sham: 2.42 ± 1.00, MCAO/R: 1.30 ± 0.95, MCAO/R + Ctr ShRNA: 1.18 ± 0.75, MCAO/R + ShRNA-STG: 1.11 ± 0.78; one-way ANOVA, F (3, 38) = 5.52, P < 0.05). These results indicated that downregulation of stargazin aggravated stroke outcomes, which may be associated with increased expression of D-serine regulated by stargazin-SR interaction.
Inhibition of Phosphorylation of Stargazin-Thr-321 Increases Stargazin-SR Interaction and Decreases D-serine Expression in Penumbral Tissue of Rats Subjected to MCAO/R
It has been reported that the interaction between stargazin and PSD-95 is inhibited by phosphorylation of stargazin at Thr-321[19]. It also has been demonstrated that mutation of stargazin-Thr-321 to aspartate or glutamate, mimicking phosphorylation, abolishes binding to SR [17]. Moreover, the stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA) [17]. Thus, we used H89, an inhibitor of PKA, to inhibit the phosphorylation of stargazin-Thr-321 and to determine whether this inhibition affected the stargazin-SR interaction and expression of D-serine. We intraperitoneally injected H89 in MCAO/R rats. The increased level of stargazin in penumbrae was unchanged in the MCAO/R 3 h + H89 group compared with those of the MCAO/R 3 h + DMSO group (Fig. 4a and 4c) (MCAO/R 3 h + DMSO: 1.01 ± 0.32, MCAO/R 3 h + H89: 1.43 ± 0.15; Student’s t test, t (2.93) = 10, P < 0.05); however, the level of phosphorylation of stargazin-Thr-321 in the penumbrae was significantly decreased in the MCAO/R 3 h + H89 group compared with the MCAO/R 3 h + DMSO group (Fig. 4b and 4d) (MCAO/R 3 h + DMSO: 1.00 ± 0.12, MCAO/R 3 h + H89: 0.78 ± 0.12; Student’s t test, t (3.14) = 10, P < 0.05). The ratio of stargazin pT321 to stargazin also decreased in the MCAO/R group (MCAO/R 3 h + DMSO: 1.10 ± 0.42, MCAO/R 3 h + H89: 0.55 ± 0.12; Student’s t test, t (3.07) = 10, P < 0.05). Next, we used fluorescent labeling to investigate whether the stargazin-SR interaction is inhibited by H89. The results showed an increased proportion of SR colocalized with stargazin in penumbral-tissue neurons of the MCAO/R 3 h + H89 rats compared to that of the MCAO/R 3 h + DMSO rats (Fig. 4f and g) (MCAO/R 3 h + DMSO: 0.66 ± 0.40, MCAO/R 3 h + H89: 1.25 ± 0.39, Student’s t test, t (2.63) = 10, P < 0.05). Additionally, a coimmunoprecipitation assay was used to analyze the brain-tissue homogenates from H89-treated and DMSO-treated rats to determine whether the protein levels of phosphorylation of stargazin-Thr-321 affected the interaction between stargazin and SR. As shown in Fig. 4h and 4i, increased interaction between stargazin and SR was observed in the MCAO/R 3 h + H89 group compared with those in the MCAO/R 3 h + DMSO group (MCAO/R 3 h + DMSO: 1.00 ± 0.35, MCAO/R 3 h + H89: 2.10 ± 0.69; Student’s t test, t (3.47) = 10, P < 0.01). Moreover, decreased D-serine was observed in the MCAO/R 3 h + H89 group compared with that in the MCAO/R 3 h + DMSO group (Fig. 4j and 4k) (MCAO/R 3 h + DMSO: 1.00 ± 0.14, MCAO/R 3 h + H89: 0.75 ± 0.06, Student’s t test, t (4.01) = 10, P < 0.01). These findings suggested that inhibition of stargazin-Thr-321 phosphorylation decreased the expression of D-serine, which might be associated with increased stargazin-SR interaction.
Inhibition of Stargazin-Thr-321 Phosphorylation Protects Against Ischemic Neuronal Death and Improves Neurological Function after MCAO/R in Rats
We then investigated whether the inhibition of stargazin-Thr-321 phosphorylation affected neurological functions and brain damage in rats subjected to MCAO/R (Fig. 5a). Assessment of cerebral infarction 24 h after MCAO/R using TTC staining and mNSS score showed that total infarction volume in H89-treated rats was reduced compared with that in the DMSO-treated rats (Fig. 5b and 5c) (MCAO/R 3 h + DMSO: 25.17 ± 3.51, MCAO/R 3 h + H89: 15.59 ± 5.85, Student’s t test, t (3.44) = 10, P < 0.01). Furthermore, mNSS was significantly lower in the MCAO/R + H89 group than in the MCAO/R +DMSO group (Fig. 5d) (MCAO/R 3 h + DMSO: 8.67 ± 2.00, MCAO/R 3 h + H89: 6.11 ± 2.26, Student’s t test, t (2.54) = 16, P < 0.05). To further evaluate the functional recovery effect of the inhibition of stargazin-Thr-321 phosphorylation in rats subjected to MCAO/R, we used the rotarod test to assess the post-stroke locomotor activity, the adhesive-removal test to evaluate forelimb sensorimotor asymmetries, and the Morris water maze test to examine the spatial learning and memory function. Rats treated with H89 showed a significant increase in the time spent on the accelerating rotarod compared with that of DMSO-treated rats (Fig. 5e) (H89 [F (1, 94) = 10.08, P < 0.01], test day [F (4, 94) = 55.49, P < 0.0001], with an interaction observed between these two factors [F (4, 94) = 0.75, P = 0.56]). Moreover, the sensorimotor function was improved in MCAO/R + H89 rats compared with that of the MCAO/R + DMSO rats, as demonstrated by a significantly shortened contact time (Fig. 5f) (H89 [F (1, 94) = 5.36, P < 0.05], test day [F (4, 94) = 24.21, P < 0.0001], with an interaction observed between these two factors [F (4, 94) = 0.50, P = 0.73]) and removal time (Fig. 5g) (H89 [F (1, 94) = 8.59, P < 0.01], test day [F (4, 94) = 40.91, P < 0.0001], with an interaction observed between these two factors [F (4, 94) = 0.96, P = 0.43]). The Morris water maze test, performed 22 to 27 days post-MCAO/R (Fig. 5a), showed that H89-treated rats rescued spatial learning, as evidenced by the decreased time spent seeking out the underwater hidden platform compared with that of the DMSO-treated rats (Fig. 5h and 5i) (H89 [F (1, 90) = 12.53, P < 0.05], test day [F (4, 90) = 19.81, P < 0.0001], with an interaction observed between these two factors [F (4, 90) = 0.29, P = 0.88]). Unlike the other tests used in our study, the memory test indicated no significant difference in memory function between the MCAO/R + H89 group and the MCAO/R + DMSO group (Fig. 5k) (MCAO/R 3 h + DMSO: 1.30 ± 0.82, MCAO/R 3 h + H89: 1.60 ± 0.70; Student’s t test, t (0.88) = 18, P = 0.39). Additionally, the swimming speed was similar among the rats used in our study (Fig. 5j) (MCAO/R 3 h + DMSO: 28.25 ± 4.39, MCAO/R 3 h + H89: 28.04 ± 3.32, Student’s t test, t (0.13) = 18, P = 0.90). These results indicated that the inhibition of stargazin-Thr-321 phosphorylation improved stroke outcomes, which may be associated with decreased expression of D-serine regulated by stargazin-SR interaction.