Genetic reduction of striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer's disease mouse model

Abstract

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder. Early in the pathophysiology of AD, synaptic function is disrupted by soluble Aβ oligomers, possibly through Aβ-mediated internalization of NMDA receptors. Striatal-enriched phosphatase (STEP) is a tyrosine phosphatase that regulates the internalization of NMDA receptors. Recent work shows that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Here, we use genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model and show that a decrease in STEP levels reverses cognitive and cellular deficits observed in these mice. Our results suggest that STEP inhibitors may prove therapeutic for this devastating disorder.

Fig. 1.

Reduction of STEP levels improves cognitive function in 3xTg-AD mice. (A) In the hidden platform trials of the Morris water maze, 6-mo-old wild-type (WT; n = 11), STEP^−/− (n = 9), and 3xTg-AD/STEP^−/− [n = 17; double mutant (DM)] mice significantly outperformed 3xTg-AD mice (n = 25) in escape latencies [one-way (genotype) repeated measures (session) ANOVA with Fisher's least significant difference post hoc tests; **P < 0.002]. DM mice did not significantly differ from WT or STEP^−/− mice (P > 0.05). (B) In probe trials performed 90 min after the final training trial on days 3, 6, and 9, WT, STEP^−/−, and DM mice spent more time in the target quadrant than 3xTg-AD [one-way (genotype) repeated measures (day) ANOVA with Fisher's LSD post hoc tests; *P < 0.05]. The same effect was seen in a final probe trial 24 h after the last training trial (one-way ANOVA with Fisher's post hoc; *P < 0.05). (C) No significant differences were detected among groups in the visible platform trials [one-way (genotype) repeated measures (day) ANOVA with Fisher's LSD post hoc tests; P > 0.05]. (D) 3xTg-AD mice (n = 31) exhibited an impairment in spontaneous alternation in a Y maze compared with all other groups (one-way ANOVA with Fisher's post hoc; *P < 0.02). DM mice (n = 20) did not differ in spontaneous alternation compared with WT (n = 18) and STEP^−/− (n = 18) mice (P > 0.05). (E) In an object recognition task, 3xTg-AD mice (n = 10) were outperformed by all other groups (one-way ANOVA with Fisher's post hoc tests; P < 0.05) and did not exhibit a preference for the novel object relative to chance (dashed line at 15 s; one-sample t test; P > 0.05). In contrast, WT (n = 10), STEP^−/− (n = 8), and DM (n = 7) mice spent significantly more time than chance with the novel object (one-sample t test; *P < 0.02).

Fig. 2.

STEP knockout restores synaptosomal membrane NR1/NR2B levels in 3xTg-AD mice. NMDAR levels were analyzed in hippocampal synaptosomal fractions (LP1) of WT, 3xTg-AD, STEP^−/−, and DM mice. Representative immunoblots from 6-mo-old mice show pNR2B, NR2B, NR1, and NR2A. Histograms are shown in Lower panel. 3xTg-AD mice showed significantly lower levels of synaptosomal pNR2B Y¹⁴⁷², NR2B, and NR1 compared with WT (pNR2B Y¹⁴⁷², NR2B: *P < 0.05; NR1: **P < 0.01). No differences were found in NR2A levels (P > 0.05; n = 8). STEP^−/− had significantly higher levels of synaptosomal pNR2B Y¹⁴⁷², NR2B, and NR1 compared with WT (*P < 0.05) but not NR2A (P > 0.05; n = 8). In DM mice, pNR2B Y¹⁴⁷², NR2B, and NR1 synaptosomal levels were increased compared with WT (*P < 0.05; n = 8) and 3xTg-AD mice (P < 0.05; n = 8) but were not significantly different from STEP^−/− (P > 0.05; n = 8). Normalization was to GAPDH. STEP₆₁ levels were significantly increased in 3xTg-AD brain membrane fractions compared with WT littermates (*P < 0.05; n = 4).

Fig. 3.

Absence of STEP in 3xTg-AD does not alter Aβ or tau levels. (A) 3xTg-AD and DM had no significant differences in total Aβ_1–42 levels as measured by ELISA (P > 0.05; n = 4). (B) Immunoprecipitation from mouse brain homogenates used 6E10 antibody, and samples were also blotted with 6E10 antibody. CTFs and Aβ are indicated by arrowheads. Representative Aβ-enriched conditioned medium (7PA2-CM) immunoreactivity showing Aβ-monomer, dimer, and trimers (Right). 3xTg-AD and DM brain samples showed no significant difference in Aβ levels (P > 0.05). (C) In hippocampus, 3xTg-AD and DM 6-mo-old mice have similar patterns of intraneuronal and extracellular staining with 6E10. STEP^−/− and WT mice showed no detectable staining with 6E10. (D) tau pathology was evaluated with an antibody that recognizes p-Ser235 and p-Thr231 (AT180) and (E) a human-specific tau antibody (HT7). 3xTg-AD or DM mice had no significant differences in immunoreactivity staining with either tau antibody. STEP^−/− and WT mice showed no detectable staining with these antibodies. (Magnification: 10×; scale bar: 200 μm.) CA, cornu ammonis; Fi, fissure. (F) Quantification of 6E10 staining revealed no significant difference between 3xTg-AD and DM mice (one-way ANOVA with Fisher's LSD post hoc tests; P > 0.05). (G) Quantification of p-tau (AT180) and total tau (HT7) staining also revealed no significant differences between 3xTg-AD and DM mice (one-way ANOVA with Fisher's LSD post hoc tests; P > 0.05). (H) Mouse brain membrane fractions were probed with p-tau antibody and total tau antibody (P > 0.05; n = 4).

Fig. 4.

Hippocampal synaptic plasticity is enhanced in 3xTg-AD mice lacking STEP. (A) Field potentials were recorded in the CA1 region of hippocampal slices from 10-mo-old 3xTg-AD mice with or without STEP. A stable baseline was recorded for at least 20 min before LTP induction by θ-burst stimulation (TBS). Inset shows traces before and after TBS. The slope of the EPSP relative to the pre-TBS level is plotted as a function of time. For 3xTg-AD, seven slices from three animals were used; for DM, six slices from three animals were used. Data are means ± SEM from separate slices. The increase in EPSP between 30 and 60 min post-TBS was greater in DM slices than 3xTg-AD slices (repeated measures ANOVA; P < 0.05). (B and C) Basal synaptic transmission and paired pulse response were indistinguishable between 3xTg-AD and DM mice.