Skip to Main Content

Paul Lombroso on STEP and Alzheimer's Disease

Concept and Scientific Content by Paul Lombroso, MD - Yale University

Animation by John Liebler at

Music by Matt Berky -

Alzheimer's disease

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., 2010). STEP61, the only STEP isoform present in cortex and hippocampus, is significantly elevated in post-synaptic compartments of cortical and hippocampal neurons AD as well as in several AD mouse models (Kurup et al., 2010; Chin et al., 2005). We showed that one mechanism that leads to the increase in STEP61 activity is an inhibition of the proteasome by the toxic peptide A-beta. The increase in STEP61 results in the internalization of GluN1/GluN2B (Zhang et al., 2010) and GluA1/GluA2 (Zhang et al., 2011) glutamate receptors, which we propose contributes to the cognitive deficits in AD.

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 and 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).

Reducing STEP levels reverses cognitive deficits in an AD mouse model: STEP 61 is elevated in the prefrontal cortex of patients with AD (Fig. 1 C-E), and the increase is due in part 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 STEP 61. STEP 61 levels are also increased in the cortex and hippocampus of Tg2576 (Fig. 1A, B; Kurup et al., 2010) and two additional mouse models of AD (J20 and 3xTg-AD mice; Chin et al., 2004; Zhang et al., 2010). Higher levels of active STEP 61 led to a decrease in GluN2B p-tyr 1472 and a decrease in GluN1 and GluN2B subunits in synaptosomal membrane fractions.

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).

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 GluN1/GluN2B 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 were indistinguishable from WT in their performance (Fig. 2; Zhang et al., 2010).

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).

Double mutant (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 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).

We also tested the progeny in an object recognition task (Fig. 4). As expected, AD 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, AD, STEP KO, and DM mice in the Y-maze and found that AD 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.

Drug discovery: The above papers found that genetic reduction of STEP expression is sufficient to reverse cognitive and behavioral deficits in mouse AD models. This work validated STEP as a target for drug discovery. The laboratory entered into collaboration with Marcei Clicksman and Greg Cuny at the Laboratory for Drug Discovery in Neurodegeneration (Harvard University) to discover STEP inhibitors. These efforts were led by Jian Xu, PhD and we soon recruited the invaluable assistance of Jonathan Ellman, PhD, Professor of Chemistry and Angus Nairn, PhD, Professor of Psychiatry. We screened over 150,000 small molecules and one compound that emerged was the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6 amine hydrochloride (known as TC-2153), which was a potent STEP inhibitor with an IC-50 of 24.6 nM (Xu et al., 2014). TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increases the tyrosinephosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibits no toxicity in cortical cultures. Validation and specificity experiments performed in WT and STEP KO cortical cells and in vivo in WT and STEP KO mice found a high degree of specificity of the STEP inhibitor compared to its ability to inhibit highly homologous tyrosine phosphatases.

TC-2153 improves cognitive function in 6- and 12-month old triple transgenic AD mice, with no change in beta amyloid and phospho-tau levels (Xu et al., 2014). This is a major contribution, as the search for inhibitors of the family of PTPs has proven quite difficult, and our results demonstrate that STEP inhibition is sufficient to significantly improve cognitive deficits. This work has generated considerable interest from pharma companies, and we are now testing this compound in additional mouse, rat, and monkey models in which STEP 61 levels are elevated (SZ, fragile x syndrome, and MPTP-induced Parkinson's disease).



NOTE: It was recently brought to our attention that certain individuals are trying to sell on the internet a nutritional approach to inhibit STEP activity. This nutritional approach is claimed to have "cured" the cognitive deficits in treated Alzheimer's patients. They cite our recent paper on TC-2153 and the finding that it reverses cognitive deficits in a mouse model of Alzheimer's disease. Anyone who sees this advertisement is strongly advised to question its validity, as the only experiments we have conducted to date have been on mice. There is no validity to claims that TC-2153 works in humans nor that the treatment proposed in the advertisement has any relationship to TC-2153 or to our research.