The role of STEP in Alzheimer's disease

Abstract

Amyloid beta (Aβ), the putative causative agent in Alzheimer's disease, is known to affect glutamate receptor trafficking. Previous studies have shown that Aβ downregulates the surface expression of N-methyl D-aspartate type glutamate receptors (NMDARs) by the activation of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP₆₁). More recent findings confirm that STEP₆₁ plays an important role in Aβ-induced NMDAR endocytosis. STEP levels are elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The increase in STEP₆₁ levels and activity contribute to the removal of GluN1/GluN2B receptor complexes from the neuronal surface membranes. The elevation of STEP₆₁ is due to disruption in the normal degradation of STEP₆₁ by the ubiquitin proteasome system. Here, we briefly discuss additional studies in support of our hypothesis that STEP₆₁ contributes to aspects of the pathophysiology in Alzheimer's disease. Exogenous application of Aβ-enriched conditioned medium (7PA2-CM) to wild-type cortical cultures results in a loss of GluN1/GluN2B subunits from neuronal membranes. Abeta-mediated NMDAR internalization does not occur in STEP knock-out cultures, but is rescued by the addition of active TAT-STEP to the cultures prior to Aβ treatment.

Figure 1

STEP activity regulates GluN1/GluN2B trafficking. (A) Surface biotinylation of wild-type cortical cultures after treatment with WT TAT-STEP OR an inactive variant TAT-STEP C-S and probed for surface and total GluN1 and GluN2B with respective antibodies. Biotinylated receptors were normalized to total receptor level and then to loading control to determine percent change. Quantitation shows a significant decrease in surface GluN1 and GluN2B subunits in WT TAT-STEP treated, compared to TAT-STEP C-S or untreated control (*p < 0.05; one-way ANOVA with post hoc tukey's test; n = 3). (B) Surface biotinylation of wild-type and STEP KO cortical cultures after treatment with CHO (control) or 7PA2-CM (Aβ-enriched) medium in the presence or absence of WT TAT-STEP protein and probed for surface and total GluN1 and GluN2B with respective antibodies. Quantitation show a significant decrease in surface GluN1 and GluN2B subunits in wild-type cultures upon 7PA2-CM or 7PA2-CM + WT TAT-STEP treatment compared to CHO treated, whereas the effect of 7PA2-CM was absent in STEP KO cultures and was rescued in the presence of WT TAT-STEP protein (*p < 0.05, **p < 0.01; one-way ANOVA with post hoc Tukey's test; n = 4).

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 surface staining (green) and the pre-synaptic marker synapsin I (red). (E and F) STEP KO cortical cultures pretreated with WT TAT-STEP alone or WT TAT-STEP followed by 7PA2-CM. (G and H) STEP KO cortical cultures pretreated with WT TAT-STEP C-S (inactive) alone or TAT-STEP C-S followed by 7PA2-CM. (I) Histogram showing the quantification of the surface GluN1 staining that colocalized with the presynaptic marker, synapsin I. STEP KO cultures showed significantly higher levels of surface GluN1 compared to WT controls. Treatment of WT cultures with 7PA2-CM resulted in a significant loss of GluN1; however, this was abolished in the STEP KO cultures. Restoration of STEP WT protein, but not inactive STEP C-S, rescued the 7PA2-CM-mediated endocytosis of GluN1 in the KO cultures (***p < 0.001 as compared to WT control; ^###p < 0.001 or ^##p < 0.01 as compared to KO control, two-way ANOVA, n = 4 independent experiments, scale bar: 5 µm).