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.
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.
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).
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).
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).
- Xu J, Chatterjee M, Baguley TD, Brouillette J, Kurup P, Ghosh D, Kanyo J, Zhang Y, Seyb K, Ononenyi C, Foscue E, Anderson GM, Gresack J, Cuny GD, Glicksman MA, Greengard P, Lam TT, Tautz L, Nairn AC, Ellman JA, Lombroso PJ: Inhibitor of the tyrosine phosphatase STEP reverses cognitive deficits in a mouse model of Alzheimer's disease. PLoS Biol. 2014 Aug 5; 2014 Aug 5. PMID: 25093460
- Zhang Y, Kurup P, Xu J, Anderson GM, Greengard P, Nairn AC, Lombroso PJ: Reduced levels of the tyrosine phosphatase STEP block β amyloid-mediated GluA1/GluA2 receptor internalization. J Neurochem. 2011 Nov; 2011 Sep 21. PMID: 21883219
- Zhang Y, Kurup P, Xu J, Carty N, Fernandez SM, Nygaard HB, Pittenger C, Greengard P, Strittmatter SM, Nairn AC, Lombroso PJ: Genetic reduction of striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer's disease mouse model. Proc Natl Acad Sci U S A. 2010 Nov 2; 2010 Oct 18. PMID: 20956308
- Kurup P, Zhang Y, Xu J, Venkitaramani DV, Haroutunian V, Greengard P, Nairn AC, Lombroso PJ: Abeta-mediated NMDA receptor endocytosis in Alzheimer's disease involves ubiquitination of the tyrosine phosphatase STEP61. J Neurosci. 2010 Apr 28. PMID: 20427654
- Chin J, Palop JJ, Puoliväli J, Massaro C, Bien-Ly N, Gerstein H, Scearce-Levie K, Masliah E, Mucke L: Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease. J Neurosci. 2005 Oct 19. PMID: 16237174
- Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P: Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci. 2005 Aug; 2005 Jul 17. PMID: 16025111