There are currently 4 million individuals suffering from Alzheimer's disease (AD) in the USA, with an annual cost of over $200 billion per year. Although some drugs are currently available to treat AD, none provide more than temporary relief from the relentless progression of cognitive deficits. The discovery of new drugs that improve cognitive function is a critically important area in translational research in AD.
The Lombroso Lab studies the function of a protein tyrosine phosphatase STEP61 that has been implicated in the pathophysiology of AD (Snyder et al., 2005; Kurup et al., 2010; Zhang et al., in press). STEP61 is targeted in part to post-synaptic compartments of cortical and hippocampal neurons. The toxic peptide A-beta induces an increase in STEP61 in human prefrontal cortex of patients with AD as well as in several AD mouse models (Kurup et al., 2010; Chin et al., 2005). The increase in STEP61 results in the internalization of NR1/NR2B and GluR1/GluR2 glutamate receptors, which we propose contributes to the cognitive deficits in AD.
Reducing STEP levels reverses cognitive deficits in an AD mouse model: STEP61 is elevated in the prefrontal cortex of patients with AD (Fig. 1), and the increase is due to an A-beta-mediated inhibition of the proteasome. STEP is normally ubiquitinated and then degraded by the proteasome, and inhibition of this organelle leads to increased levels of STEP61. STEP61 levels are also increased in the cortex and hippocampus of three different mouse models of AD (J20, Tg2576 and 3xTg-AD mice; Chin et al., 2004; Kurup et al., 2010; Zhang et al., in press). Higher levels of active STEP61 led to a decrease in NR2B p-tyr1472 and a decrease in NR1 and NR2B subunits in synaptosomal membrane fractions.
Figure 1. STEP61 levels are increased in Tg2576 mice and human AD brains. A, immunoblot of STEP61 and total Ab levels in WT and Tg2576 mice cortex at 3-, 6-, 9-, and 12-months of age. B , Quantitation of STEP61 protein levels in WT and Tg2576 mice normalized to ERK2 levels. STEP61 levels were increased in 6-, 9-, 12-month old Tg2576 mice compared to age matched WT controls (*p<0.05, two-way ANOVA with post hoc Tukey's test; n=4). C, STEP61 is increased in human AD brain samples. Representative immunoblot of prefrontal cortex from control a nd AD brains probed with anti-human STEP antibody. D, Quantitation of STEP61 control (n=10) and AD (n=18) groups normalized to beta-actin levels. Data are expressed as percentage increase in STEP61 compared to controls. E, Histogram of data (**p<0.01, Student's t-test; n=3).
We reasoned that a genetic reduction of STEP might reverse the loss of these receptors and the cognitive deficits in 3xTg-AD mice. To this end, we crossed STEP KO mice with 3xTg-AD mice to generate progeny null for STEP but with the AD mutations still present (double mutants; DM). These mice no longer show significant loss of NR1/NR2B receptors from synaptosomal-associated membrane compartments, despite elevated A-beta levels (Zhang et al., 2010).
But a more interesting question was how would they perform on cognitive tasks? We tested these mice for cognitive improvements in the Y-maze, object recognition, and Morris water maze. Progeny null for STEP function significantly better in all of these behavioral tests compared to 3xTg-AD mice, and perform comparably to WT mice (Zhang et al., 2010).
Figure 2: Spatial memory assessed in the hidden platform trials of a water maze task. Testing consisted of 9 days of hidden platform trials (4 trials/session/day). The main effect of group was significant (p<0.001), and post hoc tests revealed that Alz mice had memory impairment relative to all groups (p<0.002). There were no differences among the WT, STEP KO, and DM mice (p>0.05).
|Wild-Type Mouse||Alzheimer's Mouse with STEP||Alzheimer's Mouse without STEP|
DM Mice also performed better than 3xTg-AD mice when memory was assessed in probe trials (Fig. 3). Probe trials were performed 90 min after the last hidden platform training trial on days 3, 6, and 9. Across these trials, the main effect of genotype was significant (p<0.05) and post hoc tests revealed that 3xTg-AD mice spent significantly less time in the target quadrant than other groups (p<0.05). There were no differences between the DM, WT, and STEP-/- mice (p>0.05), and the genotype by day interaction was not significant (p>0.05). On a final probe trial, completed 24 hours after the last hidden platform session, the main effect of genotype was again significant (p<0.05), and post hoc tests revealed that 3xTg-AD mice spent significantly less time in the target quadrant than all other groups (p<0.05).
Figure 3: In probe trials performed 90 min after the final training trial on day 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 hrs after the last training trial (one way ANOVA with Fisher's post hoc; *p<0.05).
We also tested the progeny in an object recognition task (Fig. 4). As expected, Alz mice failed to exhibit memory retention at a 24-hour delay, as they did not show a preference for the novel object relative to chance (dashed line at 15 sec; p>0.05). In contrast, DM mice showed a significant preference for the novel object, indicating intact object memory retention (p<0.01). There were no significant differences between DM mice and WT and STEP KO mice, all of which exhibited memory retention at a 24-hour delay (p<0.01). We also tested spontaneous alternation in WT, Alz, STEP KO, and DM mice in the Y-maze and found that Alz mice were impaired compared to all other groups (p<0.05; data not shown), and that the DM mice were not significantly different from WT and STEP KOs. These results indicate that a genetic reduction of STEP in 6-month old 3xTg-AD mice significantly improved cognitive functioning in these three behavioral tasks.
Figure 4: Genetic reduction of STEP improves performance in an OR task in 3xTg-AD mice. 6-month old mice of WT, STEP-KO, 3xTg-AD (Alz) or 3x Tg-AD/STEP-KO (DM) were tested in a novel object recognition (OR) task. At a 24-hour retention delay, Alz mice do not exhibit memory retention (p>0.05, n=9), whereas DM (n=7), WT (n=10) and STEP-KO (n=8) show intact object memory retention (**p<0.01, one sample t-test) (Zhang et al., in press).
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- Kurup P, Zhang YF, Xu J, Venkitaramani DV, Haroutunian V, Nairn A, Lombroso PJ (2010) Abeta-mediated NMDA receptor endocytosis in Alzheimer's disease involves ubiquitination of the tyrosinie phosphatase STEP61. Journal of Neuroscience 30:5948-5957. [PDF]
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- Zhang YF, Kurup P, Xu J, Carty N, Fernandez S, Nygaard HB, Pittenger C, Greengard P, Strittmatter S, Nairn AC and Lombroso PJ (2010) Genetic reduction of STEP reverses cognitive and cellular deficits in a mouse model of Alzheimer's disease. PNAS. [PDF]