To study the role of STEP in Aβ-mediated NMDAR endocytosis, we took advantage of STEP KO cultures.11
We examined Aβ-mediated NMDAR endocytosis in STEP KO cultures using two complementary techniques: surface biotinylation and immunofluoresence as described previously.4,9
We used 7PA2-conditioned medium (7PA2-CM) as a source of Aβ. These cell secrete soluble amyloid-beta oligomers into the medium that were concentrated for use in these experiments.12,13
Cortical cultures derived from WT and STEP KO (18 DIV) were treated with 7PA2 (Aβ-enriched) or CHO (control) medium and GluN1 and GluN2B levels were determined by surface biotinylation. The surface GluN1 and GluN2B levels were significantly decreased in WT cultures after 7PA2-CM (Aβ-enriched) treatment compared to control (GluN1: 75.2 ± 1.8%; GluN2B: 64.8 ± 3.7%) (). As previously shown, there was no change in the surface GluN2A levels upon 7PA2-CM treatment9
(data not shown). As expected, STEP KO cultures showed increase in basal levels of GluN1 and GluN2B compared to WT (GluN1: 138.9 ± 3.5%; GluN2B: 130.0 ± 4.4%) and 7PA2 treatment failed to significantly decrease the surface GluN1 and GluN2B receptors (GluN1: 129.9 ± 3.7%; GluN2B: 129.6 ± 10.4%) (). Preincubation with WT TAT-STEP protein during 7PA2-CM treatment rescued GluN1/GLUN2B internalization (GluN1: 89.2 ± 3.3%; GluN2B: 63.7 ± 5.6%) in STEP KO cultures compared to CHO controls ().
Next, we examined the Aβ-mediated NMDAR endocytosis in WT and STEP KO cortical cultures using immunofluorescence to measure the colocalization of GluN1 with the presynaptic marker synapsin I.4
Treatment of neuronal cultures from WT mice with 7PA2-CM significantly decreased surface GluN1 staining (4.78 ± 0.24 puncta per 10 µm, p < 0.001) as compared to untreated WT (8.69 U ± 0.47 puncta per 10 µm) cultures ( and B
). Untreated STEP KO cultures had significantly higher numbers of GluN1 puncta (15.33 ± 0.83 puncta per 10 µm, p < 0.001) compared to WT cultures ( and C
). Aβ-mediated endocytosis was abolished in STEP KO cultures (16.31 U± 0.78 puncta per 10 µm, p > 0.99 as compared to KO control) ( and D
Figure 2 STEP KO cultures show no decrease in GluN1 surface levels after 7PA2-CM (Aβ-enriched) treatment. (A–D) representative images from STEP WT and KO cortical cultures either untreated or treated with 7PA2-CM (Aβ-enriched) showing Glun1 (more ...)
We examined whether we could rescue Aβ-induced GluN1 endocytosis by restoring STEP protein to the KO cultures. The WT cortical cultures treated with WT TAT-STEP were similar to untreated WT cultures either in the presence or absence of 7PA2-CM (). Treatment of KO cortical cultures with WT TAT-STEP reduced GluN1 surface expression (12.89 ± 0.81 puncta per 10 µm, p > 0.20 as compared to KO control, ) and resulted in significant Aβ-induced GluN1 internalization (10.39 ± U0.67 puncta per 10 µm, p < 0.001) as compared to both KO cultures and KO cultures treated with 7PA2-CM (). The surface GluN1 levels in KO cultures treated with WT TAT-STEP followed by 7PA2-CM were not significantly different from WT cultures (p > 0.74), suggesting that restoration of WT STEP protein in KO cultures rescues Aβ-induced GluN1 endocytosis. In contrast, treatment of KO cortical cultures with inactive TAT-STEP C-S fusion protein resulted in a significant increase in surface expression of GluN1 receptors (19.17 ± 1.07 puncta per 10 µm, p < 0.002 as compared to KO control, ) and the inactive STEP (C-S) fusion protein did not rescue Aβ-induced endocytosis of GluN1 subunits (17.36 U± 0.68 puncta per 10 µm, p > 0.99) (). The WT cultures treated with TAT-STEP C-S showed a significant increase in GluN1 surface expression and partially blocked the Aβ mediated GluN1 internalization ().
In summary, these studies indicate that active STEP protein is required for Aβ-induced internalization of NMDARs. It also suggests a new therapeutic strategy by which inhibiting STEP activity could lead to the development of a new family of therapeutic agents in Alzheimer disease.