Gleevec, the cancer "wonder drug" that has proven effective for gastrointestinal stromal tumors might also be useful for Alzheimer's patients, if only one could deliver it to the brain effectively, according to a paper in this week's early online edition of PNAS.

Principal author Paul Greengard, and colleagues from The Rockefeller University and the Memorial Sloan-Kettering Center, both in New York, report that the kinase inhibitor, whose chemical name is imatinib mesylate, reduces γ-secretase cleavage of Aβ precursor protein (AβPP) without affecting Notch processing. The latter is fortuitous, as potential interference with Notch signaling and other pathways is one of the obstacles impeding the search for γ-secretase-targeted therapeutics (see ARF related news story), ARF news story, and scroll to Shearman in Titisee conference report).

Gleevec inhibits Abl, and several other tyrosine kinases, by blocking the enzymes' ATP binding site. Greengard and colleagues had previously shown that in a cell-free system consisting of mouse N2a neuroblastoma membranes, the γ-secretase cleavage of AβPP is ATP dependent. This prompted joint first authors William Netzer, Fei Dou, and Dongming Cai to try blocking the ATP-dependent step in the production of Aβ. When the authors added Gleevec to either the cell-free system or to N2a cells themselves, production of Aβ decreased by 50 percent. In contrast, the quantity of Notch's intracellular domain produced by the N2a cells remained constant.

The authors found that Gleevec was primarily, if not exclusively, affecting the γ-secretase step of Aβ production because it had no effect on the level of the β-secretase-generated C-terminal fragment (CTF) of AβPP. In addition, when they added Gleevec to N2a cells expressing this CTF, Aβ production was still blocked, though not as robustly as in cells expressing full-length AβPP.

When Netzer and colleagues added the kinase inhibitor to cultured primary neurons, they found a similar effect. Five μM Gleevec inhibited Aβ production by 75 percent, while the similar ATP site blocker PD173955 worked even better, inhibiting by 80 percent at a fivefold lower dose. In vivo, the kinase inhibitors were also effective. When the authors administered as little as 0.2 mg/Kg of either drug into the brains of adult albino guinea pigs, cortical levels of Aβ dropped by over half, while the levels of the β-secretase CTF rose by as much as fourfold. These results, which were statistically significant, suggest that γ-secretase cleavage can be effectively halted in vivo.

How Gleevec works in this context is uncertain. The authors show that it is just as effective at inhibiting γ-secretase in Abl-negative cells, and suggest that other tyrosine protein kinases, such as platelet-derived growth factor receptor (PDGFR) or Src kinase, may be involved. Intriguingly, Tommaso Russo's group has just reported that PDGFR and Src kinase may play a role in Aβ production (see Gianni et al., 2003).

Gleevec has FDA approval, and the authors write that "the safety of Gleevec, demonstrated by its successful application to […] tumors, and its inability to inhibit Notch-1 cleavage by γ-secretase, make this class of compounds attractive as potentially safe, Aβ-lowering drugs." If, that is, ways can be found to get them across the blood-brain barrier.—Tom Fagan

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  1. The findings described in the paper of Netzer et Al. demonstrating that STI571/Gleevec inhibits Ab production in vivo are of significant interest at least for two reasons: the drug does not affect Notch processing and it is currently used in humans for the treatment of CML. However, another reason why this observation is of interest is that it probably contributes to our understanding of the regulation of APP processing. In agreement with the results of the Paul Greengard’s group, we have recently reported (Gianni et Al. J. Biol. Chem. 278, 9290, 2003) that the processing of APP is regulated by PDGF-BB, through a pathway including, downstream the PDGF-R, the non-receptor tyrosine kinase Src and the small G-protein Rac. We also demonstrated that a specific inhibitor of Src TK (PP2) inhibits Ab production in cultured cells. Considering that Abl appears to be not involved in the observed phenomena, it is possible to speculate that Gleevec is working as a Src TK inhibitor.
    In my opinion, these results open a new stimulating scenario, and many points should be now addressed. Just to mention some of these points: Why is APP processing affected but not that of Notch? Does it depend on a different intracellular localization of the two substrates of g-secretase? Does it depend on the existence of an APP-specific g-secretase regulator that is a target of TKs? Is APP itself the target of TKs? Is Src the TK inhibited by Gleevec, or is it the PDGF-R or another Src-like molecule? Are there other events, further than PDGF treatment, that induce APP cleavage through the activation of TKs? Are some of these events neuron-specific?

  2. The findings of Netzer and colleagues that Gleevec and related protein kinase inhibitors can lower formation of the amyloid-β protein (Aβ) opens a new avenue for Alzheimer's research. The investigation was prompted by previous observations that Aβ production is ATP-dependent. Gleevec, recently approved for treating chronic myelogenous leukemia, is a potent inhibitor of Abl kinase and is among the first anti-cancer drugs that target oncogenes and their protein products. Netzer et al. show that Gleevec and another Abl kinase inhibitor reduce Aβ levels in permeabilized cells, living cells, and the guinea pig brain. Evidence indicates that the drugs affect the proteolysis of the Aβ protein precursor (APP) by γ-secretase but not by β-secretase. Importantly, the processing of another γ-secretase substrate, the Notch receptor, is not affected. Concerns have been raised about γ-secretase as a therapeutic target because proteolysis of Notch is essential for proper cell differentiation, for instance, during the formation of new blood cells. Thus, selective inhibition of Aβ production that preserves the processing of Notch is desirable.

    A major question raised by this study is the identity of the protein target that leads to Aβ reduction. Presumably, the target is a kinase, but Abl kinase has been definitively ruled out: Cells from Abl kinase knockout mice generate normal levels of Aβ, and Gleevec still lowers Aβ in the absence of Abl kinase. The actual target may regulate γ-secretase activity, affecting the selectivity of the enzyme (e.g., whether it cleaves APP or Notch). In this regard, it would be interesting to know whether processing of any of the numerous other presenilin/γ-secretase substrates (e.g., CD44, Delta/Jagged) is also unaffected by Gleevec. Alternatively, the target may affect the localization of APP and whether this particular substrate will find the enzyme. A third possibility is that Gleevec interacts directly with γ-secretase, perhaps through an unidentified ATP-binding site in the protease complex, although how this might result in selective inhibition of APP cleavage vis-à-vis Notch is difficult to envision. As for the potential of Gleevec for the treatment of Alzheimer’s disease, this drug apparently does not effectively get into the brain (Gleevec had to be administered subdurally at the base of the guinea pig spinal cord). Thus, the search is on for brain-permeable compounds that work by this mechanism. In light of this study, pharmaceutical companies may be well advised to rescreen their kinase inhibitor libraries for Aβ-lowering effects in cells.

    View all comments by Michael Wolfe
  3. This seems significant. Surprised it has not been tried, and reported, on transgenic mice.

  4. I would like to bring to your attention to my poster last July. I also forwarded my proposal to the developers at Novartis at that time, detailing reasons why Gleevec may well prove to be a beneficial treatment for those with Alzheimer's disease and Down's syndrome.

    It is of interest that Dai et al. have shown entry of Gleevec into the brain to be modulated by P-glycoprotein. Perhaps we could approach treatment of Alzheimer's disease with that in mind.

    References:

    . Distribution of STI-571 to the brain is limited by P-glycoprotein-mediated efflux. J Pharmacol Exp Ther. 2003 Mar;304(3):1085-92. PubMed.

  5. Further to my comment of October 8, I note the study by Boucher et al. (1) in which they find that inactivation of LRP1 in vascular SMCs results in PDGFR overexpression.

    In view of the study by Meng et al., (2) finding that SHP-2 is necessary to inhibit PDGFR signaling, and that H2O2 inactivates protein tyrosine phosphatases, might we expect that oxidative stress in Alzheimer's disease and Down's syndrome may result in SHP-2 inactivation?

    References:

    . LRP: role in vascular wall integrity and protection from atherosclerosis. Science. 2003 Apr 11;300(5617):329-32. PubMed.

    . Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell. 2002 Feb;9(2):387-99. PubMed.

  6. It seems of interest that Liu and Burridge have found that EGF and PDGF stimulate tyrosine phosphorylation of Vav2 with subsequent increased activity of Cdc42 downstream from growth factor receptors. Might we expect decreased activity of Cdc42 following treatment with Gleevec? 

    Zhu et el have found that Cdc42 is upregulated in select neuronal populations in Alzheimer's disease.

    Etienne-Manneville and Hall find that Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity.

    Leroy et el find increased adenomatous polyposis coli immunoreactivity in reactive astrocytes in Alzheimer's disease.

    Baki et el find that PS1 and p120 bind to and mutually compete for cellular E-cadherin.

    The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.

    A number of recent findings have highlighted the similarities between neurogenesis during development and neurodegeneration during Alzheimer disease. In fact, neuronal populations that are known to degenerate in Alzheimer disease exhibit phenotypic changes characteristic of cells re-entering the cell division cycle. In this study, we extended these findings by investigating components of the cell cycle, known to trigger progression through G1 through activation of signal transduction cascades. Specifically, we found that proteins implicated in G1 transition, namely Cdc42/Rac, are upregulated in select neuronal populations in cases of Alzheimer disease in comparison to age-matched controls. Importantly, Cdc42/Rac shows considerable overlap with early cytoskeletal abnormalities suggesting that these changes are an extremely proximal event in the pathogenesis of the disease. Given the functional role of Cdc42/Rac in various cellular processes known to be perturbed in Alzheimer disease, namely cytoskeletal organization, oxidative balance, and oncogenic signaling, it is likely that increased neuronal Cdc42/Rac is highly significant in relation to the pathogenic process and contributes to neuronal degeneration. In fact, these findings suggest that Alzheimer disease is an oncogenic process.

    Cell polarity is a fundamental property of all cells. In higher eukaryotes, the small GTPase Cdc42, acting through a Par6-atypical protein kinase C (aPKC) complex, is required to establish cellular asymmetry during epithelial morphogenesis, asymmetric cell division and directed cell migration. However, little is known about what lies downstream of this complex. Here we show, through the use of primary rat astrocytes in a cell migration assay, that Par6-PKCzeta interacts directly with and regulates glycogen synthase kinase-3beta (GSK-3beta) to promote polarization of the centrosome and to control the direction of cell protrusion. Cdc42-dependent phosphorylation of GSK-3beta occurs specifically at the leading edge of migrating cells, and induces the interaction of adenomatous polyposis coli (Apc) protein with the plus ends of microtubules. The association of Apc with microtubules is essential for cell polarization. We conclude that Cdc42 regulates cell polarity through the spatial regulation of GSK-3beta and Apc. This role for Apc may contribute to its tumour-suppressor activity.

    Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are responsible for colon cancer in familial adenomatous polyposis coli and in many sporadic colorectal tumors. The product of the APC gene is also essential for normal development and is expressed in the adult brain. We have investigated the immunocytochemical localization of APC in the temporal cortex and hippocampus of normal human brain, in Alzheimer's disease (AD) and in several other neuropathological conditions. APC was expressed in neuronal cell bodies and dendrites both in control subjects and in patients with different diseases. In addition, a high APC expression was observed in a proportion of fibrillary and glial fibrillary acidic protein-positive astrocytes in AD. Furthermore, in AD the proportion of APC- positive astrocytes was higher in astrocytes associated with beta-amyloid (Abeta) deposits in senile plaques than in astrocytes not associated to Abeta deposits. APC-positive astrocytes were also observed in control cases, in diffuse Lewy body disease, in Creutzfeldt-Jacob disease, in HIV encephalitis and around cerebral infarcts. Tumoral astrocytes in pilocytic astrocytoma and in glioblastoma were also strongly APC positive. APC was not detected in cultured astroglial cells. These results indicate that APC expression is upregulated in astrocytes following their activation by several types of pathological insults and is a newly identified molecular characteristic of the reactive phenotype of astrocytes, possibly related to the control of cell proliferation. In addition, it also suggests that Abeta, and/or the inflammatory process associated with Abeta deposits, is responsible for a preferential increase of APC expression in astrocytes in AD.

    Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell-cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B. , J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328-337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell-cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell-cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.

    Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604-615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to beta- and gamma-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca(2+)-dependent cell-cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant DeltaE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell-cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo.

    References:

    . Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins. Mol Cell Biol. 2000 Oct;20(19):7160-9. PubMed.

    . Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int J Dev Neurosci. 2000 Jul-Aug;18(4-5):433-7. PubMed.

    . Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature. 2003 Feb 13;421(6924):753-6. Epub 2003 Jan 29 PubMed.

    . Increase of adenomatous polyposis coli immunoreactivity is a marker of reactive astrocytes in Alzheimer's disease and in other pathological conditions. Acta Neuropathol. 2001 Jul;102(1):1-10. PubMed.

    . p120 catenin regulates the actin cytoskeleton via Rho family GTPases. J Cell Biol. 2000 Aug 7;150(3):567-80. PubMed.

    . Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex. Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2381-6. PubMed.

References

News Citations

  1. Another Notch in the Presenilin Belt
  2. γ-Secretase Cuts Not Just Notch, but Ligands Delta and Jagged, Too
  3. Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part III

Paper Citations

  1. . Platelet-derived growth factor induces the beta-gamma-secretase-mediated cleavage of Alzheimer's amyloid precursor protein through a Src-Rac-dependent pathway. J Biol Chem. 2003 Mar 14;278(11):9290-7. PubMed.

Further Reading

Primary Papers

  1. . Gleevec inhibits beta-amyloid production but not Notch cleavage. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12444-9. PubMed.