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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 21, 2002 as doi:10.1096/fj.02-0198fje. |
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Technion-Faculty of Medicine, Eve Topf and NPF Centers for Neurodegenerative Diseases Department of Pharmacology Haifa, Israel; and
* Hebrew University, Hadassah School of Medicine Department of Pharmacology, Jerusalem, Israel
3Correspondence: Department of Pharmacology, Technion Faculty of Medicine, P.O.B. 9697, 31096 Haifa, Israel. E-mail: Youdim{at}tx.technion.ac.il
SPECIFIC AIMS
Cholinesterase (ChE) inhibitors and selective monoamine oxidase-B (MAO-B) inhibitors have been reported to have beneficial effects in Alzheimer’s disease (AD) patients. We recently developed a series of novel bifunctional anti-AD drugs—TV3326 [(N-propargyl-(3R)-aminoindan-5-yl)-ethyl methyl carbamate] and its S-isomer TV3279–in an attempt to combine the pharmacophore neuroprotective properties of the anti-Parkinson’s disease MAO-B inhibitor drug rasagiline (N-propargyl-(1R)-aminoindan) with the ChE inhibitory moiety of rivastigmine, a drug with proven efficacy in AD. TV3326 possesses ChE and MAO inhibitory activities, whereas TV3279, which also inhibits ChE, lacks MAO inhibitory activity. Similar to other ChE inhibitors, both drugs antagonized impairment in reference and working memory induced by scopolamine, a procedure for evaluating anti-AD drugs. We studied the effects of these compounds on the regulation of APP processing, using human SH-SY5Y neuroblastoma and rat PC12 cells, and the role of mitogen-activated protein (MAP) kinase in the modulation of soluble amyloid precursor protein 
(sAPP) release.
PRINCIPAL FINDINGS
1. TV3326 and TV3279 stimulate sAPP
Treatment of SH-SY5Y cells for 20 h with increasing concentrations of TV3326 or TV3279 resulted in a concentration-dependent increase in sAPP release into the conditioned medium. The maximal effect of both drugs was obtained at a concentration of 10 µM, which resulted in 
threefold increase in sAPP over the control level (Fig. 1
). Since the same immunoreactive band was detected by the monoclonal antibody 22C11 and the monoclonal antibody 6E10, which recognized epitopes in the first 16 amino acids of Aß domain, it is suggested that secreted sAPP was derived by nonamyloidogenic -secretase cleavage. PC12 cells treated for 20 h with various concentrations of TV3326 or TV3279 also showed dose-dependent release of sAPP.
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-Secretase is a zinc metalloprotease susceptible to inhibition by hydroxamic acid-based compounds. Thus, we examined the effect of the hydroxamic acid-based metalloprotease inhibitor Ro31–9790 on sAPP-induced release by TV3326 and TV3279. Ro31–9790 (100 µM) significantly inhibited the release if sAPP induced by TV3326 and TV3279. Altogether, the results clearly show that the effect of these anti-AD drugs on sAPP release is mediated via -secretase activity.
2. Inhibition of TV3326-induced sAPP release by inhibitors of PKC, mitogen-activated kinase kinase (MEK) and tyrosine kinase activity
To determine which signaling pathway mediates the TV3326-induced increase in sAPP secretion, we used various specific signaling inhibitors. sAPP release, induced by TV3326, was significantly blocked by the PKC inhibitors GF109203X (2.5 µM) or calphostin C (1 µM), the MEK inhibitors PD98059 (30 µM) and U0126 (5 µM), and the specific tyrosine kinase inhibitor genistein (20 µM). These findings indicate that TV3326 modulates the release of sAPP by PKC-, MAP kinase-, and tyrosine kinase-dependent mechanisms.
3. Activation of MAP kinase by TV3326 and TV3279
TV3326 and TV3279 dose-dependently increased immunoreactivity of the phosphorylated MAP kinase in PC12 cells but had no effect on total levels of MAP kinase proteins (Fig. 2
A, D). Activation occurred with doses as low as 0.1 µM of TV3326, with maximal activation at 1 and 10 µM TV3326. MAP kinase activation increased in the first 30 min of TV3326 addition and decreased thereafter (Fig. 2B
). Similar stimulation of MAP kinase activation was observed by TV3279.
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The synthetic, cell-permeable, noncompetitive inhibitors of upstream kinase, MEK phosphorylation, and activation, PD98059 (30 µM) and U0126 (5 µM), abolished TV3326 and TV3279-mediated MAP kinase phosphorylation (Fig. 2C
). These results indicate that MAP kinase is involved in mediating the effects of TV3326 and TV3279 on sAPP secretion.
4. Rasagiline stimulates sAPP release and MAP kinase activation
Since TV3326 and TV3279 are derivatives of the selective irreversible MAO-B inhibitor rasagiline, we examined its effects on sAPP release and MAP kinase activation. Rasagiline (10 µM) significantly increased sAPP release in SH-SY5Y neuroblastoma and PC12 cells (2.65±0.12 and 3.08±0.11-fold, respectively, n=3, P<0.05); 10 µM rasagiline stimulated (1.77±0.26-fold, n=3, P<0.05) the activation of MAP kinase.
CONCLUSIONS AND SIGNIFICANCE
ChE inhibitors and selective MAO-B inhibitors have been reported to have beneficial effects in AD patients. Recently, we have developed a series of novel bifunctional anti-AD drugs—TV3326 and its S-isomer TV3279—in an attempt to combine the pharmacophore neuroprotective properties of the anti-Parkinson’s disease MAO-B inhibitor drug rasagiline with the ChE inhibitory moiety of rivastigmine, a drug with proven efficacy in AD. The present study demonstrates that TV3326 and TV3279 promote activation of the nonamyloidogenic -secretase pathway, resulting in the formation of neuroprotective sAPP. Increased sAPP secretion was detected by monoclonal antibodies 22C11 and 6E10. Stimulation of sAPP release by TV3326 and TV3279 was blocked by the hydroxamic acid-based metalloprotease inhibitor Ro31–9790, further indicating the involvement of an -secretase.
Stimulation of -secretase activity and an increase in -secretase-cleaved sAPP might be beneficial for the treatment of AD, as proteolytic cleavage of APP within the Aß sequence precludes the formation of Aß and its accumulation into amyloid plaques.
Indeed, previous studies have shown that the proportion of APP, processed by -secretase vs. ß-secretase may affect the amount of the amyloid fragments: for instance, mutations in APP found in a Swedish familial AD pedigree map to the ß-secretase cleavage site in APP and favor ß-secretase cleavage of APP. Thus, cells expressing these mutations secrete increased amounts of Aß compared with cells expressing wild-type APP. In contrast, activation of PKC by PMA has been shown to favor -secretase, nonamyloidogenic cleavage at the expense of ß-secretase cleavage. In cells overexpressing APP, cholesterol depletion-induced sAPP secretion is accompanied by a decrease in Aß production. Moreover, transgenic mice engineered to produce high levels of Aß have decreased levels of brain Aß after PMA treatment, further suggesting that stimulation of -secretase cleavage may be a useful intervention to influence the production of nondeleterious and even beneficial sAPP and reduce the relative amounts of Aß peptides. Several studies, however, demonstrate a dissociation between sAPP release and Aß generation, suggesting there might be a more complex regulatory mechanism of these two processing events of APP.
Induced sAPP secretion might have additional advantages, since various studies strongly suggest that sAPP has potent neurotrophic and protective activities against excitotoxic and oxidative insults. Thus, sAPP stimulates neurite outgrowth, regulates synaptogenesis, has trophic effects on cerebral neurons in culture, stabilizes neuronal calcium homeostasis, and protects hippocampal and cortical neurons against the toxic effects of glutamate and Aß peptide. It has been also shown that sAPP can counteract the proapoptotic action of mutant presenilin-1 by activating the transcription factor NF-
B and that sAPP has memory-enhancing effects in normal and amnestic mice. Indeed, TV3326 and TV3279 exert potent antiapoptotic and neuroprotective activities against a variety of insults in vitro and in vivo, similar to those described for rasagiline. These compounds also prevented glutamate, NMDA- and peroxynitrite-induced neurotoxicity in neuronal cell cultures and the fall in mitochondrial membrane potential. TV3326 and TV3279 antagonized scopolamine-induced impairments in spatial memory and reduced the sequelae of closed head injury in mice and neuronal damage caused by streptozotocin in rats.
Stimulation of sAPP release is highly regulated through activation of various second messenger pathways, including PKC, MAP kinase, and tyrosine kinase cascades. This is not due to the phosphorylation of APP, but may result from protein phosphorylation that modulates the activity of -secretase or the trafficking of APP. The data presented here using several signaling inhibitors enabled us to suggest that PKC-, MAP kinase-, and tyrosine kinase-dependent pathways may be involved in the enhancement of sAPP release by TV3326 and TV3279. We further examined the effect of the drugs on the MAP kinase pathway, since several reports have indicated that MAP kinase could act as a critical mediator in the regulated shedding of different membrane proteins, including APP. In results complementary to the inhibitor studies, we show for the first time that the ChE inhibitors TV3326 and TV3279 stimulate ERK1 and ERK2 MAP kinase phosphorylation. These findings are in line with previous data in relation to the involvement of ERK activation in regulating sAPP release. Indeed, the synthetic MEK inhibitor PD98059 was shown to antagonize nerve growth factor stimulation of sAPP release and ERK in PC12 cells (Fig. 3
). Moreover, exposure to PD98059 or overexpression of a kinase-inactive MEK mutant reduced PKC-mediated effects on APP processing in a variety of cell lines. TV3326- and TV3279-induced MAP kinase activation may be also implicated in an intriguing prospect that suggests involvement of ERK activation in the central nervous system in mammalian synaptic plasticity and learning. Thus, the effect on ERK activation may be attributed in the context of our previous observations regarding the antagonistic effect of these compounds on scopolamine-induced impairments in spatial memory (Fig. 3)
.
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ChE inhibitor drugs were previously found to alter the metabolism of APP, which in turn might affect the processing of Aß deposition. Recently, it was suggested that the ChE inhibitor metrifonate promoted the secretion of sAPP by an indirect cholinomimetic effect coupled to PKC. However, the effect of TV3326 and TV3279 does not appear to result from their ChE inhibition activities, since we found that the MAO-B inhibitor rasagiline is also able to induce ERK activation and sAPP release. This was further confirmed by our recent study showing that the propargyl moiety present in TV3326, TV3279, and rasagiline is essentially required for these stimulation effects. It is the propargyl moiety that confers various neuroprotective activities and APP processing in these molecules in these molecules, since M-methylpropargyl amine has identical activities (unpublished results).
In conclusion, the results of our studies demonstrate that the novel drugs TV3326 and TV3279 can modulate MAP kinase-dependent release of the neurotrophic/neuroprotective sAPP, which in turn could affect the formation of potentially amyloidogenic derivatives. The neuroprotective effects of TV3326 and TV3279, together with their ability to induce nonamyloidogenic sAPP secretion, could clearly be of clinical importance for the treatment of AD; these drugs are currently in development for this purpose.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0198fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0198fje 
2 These two authors are each to be considered first author of this article.
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