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Neuroprotection and neurorescue against Aß toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate¹

YONA LEVITES^*,,2, TAMAR AMIT^*,2, SILVIA MANDEL^* and MOUSSA B. H. YOUDIM^*,3

^* Eve Topf and USA National Parkinson Foundation, Centers of Excellence for Neurodegenerative Diseases Research, Technion Faculty of Medicine and
Intradepartmental Unit for Biotechnology Haifa, 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

We recently demonstrated that the neuroprotective mechanism of (-)-epigallocatechin-3-gallate (EGCG) against oxidative stress (OS)-induced cell death includes stimulation of protein kinase C (PKC) and modulation of cell survival/cell cycle genes in human SH-SY5Y neuroblastoma cells. Considering these findings, we examined in the present study: the 1) neuroprotective and neurorescue effects of EGCG on ß amyloidpeptide (Aß)-induced neurotoxicity, 2) regulation of amyloid precursor protein- (APP-) processing, and 3) involvement of PKC-dependent mechanisms in the modulation of soluble APP- (sAPP) release in cell culture and in vivo.

PRINCIPAL FINDINGS

1. Stimulation of sAPP release from SH-SY5Y and PC12 cells by EGCG
Treatment of human SH-SY5Y neuroblastoma cells for 2 h with increasing concentrations of EGCG resulted in a concentration-dependent increase in sAPP released into the conditioned media. Stimulation occurred with doses as low as 1 µM EGCG; maximal stimulation was obtained at 10 µM, which resulted in a sixfold increase of sAPP over the control level. Increased sAPP secretion was detected by monoclonal antibody 22C11 and the monoclonal antibody 6E10, which recognizes epitopes in the first 16 amino acids of the Aß domain, suggesting that the sAPP released was derived by -secretase processing. We have studied the effect of EGCG on another cell line, rat PC12 cells. Treatment for 2 h with various doses of EGCG also resulted in a concentration-dependent release of sAPP; maximal stimulation was observed at 10 µM.

An hydroxamic acid-based metalloprotease inhibitor Ro31-9790 (100 µM) significantly blocked the release of sAPP induced by EGCG. These findings further indicate that the effect of EGCG on sAPP release is mediated via -secretase activity.

2. Involvement of PKC activity in EGCG-stimulated sAPP release
Down-regulation of PKC by a chronic 20 h preincubation with PMA (1 µM) blunted the increase in sAPP secretion by short-term PMA and EGCG treatments. Inhibition of PKC with the inhibitor GF109203X (2.5 µM) attenuated the effect of EGCG on sAPP release. These results suggest the involvement of a PKC-dependent pathway in EGCG-stimulated sAPP secretion.

3. Effect of EGCG on PKC activation
The effect of EGCG on phosphorylation of PKC was examined in PC12 cells, using a phospho-PKC (pan) antibody. EGCG dose-dependently induced PKC phosphorylation; the maximal effect was obtained with 1 µM EGCG, which resulted in twofold increase in phospho-PKC (pan) level.

4. Neuroprotective and neurorescue effects of EGCG against Aß toxicity
PC12 cells were preincubated with 1 µM EGCG, followed by 48 h treatment with 10 µM aggregated Aß_25–35, Aß_1–42 or Aß_1–40. PC12 cell viability was significantly reduced by all Aß peptides as measured by an apoptotic cell death detection ELISA (Fig. 1 A) or MTT reduction (Fig. 1B ). EGCG was able to significantly protect PC12 cells against Aß-induced toxicity (Fig. 1A, B ). EGCG (0.1 and 1 µM) rescued PC12 cells against the toxicity induced by Aß_25–35 (10 µM), even if added 2 h post-Aß _25–35 treatment (Fig. 1C ).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effects of EGCG against Aß-induced toxicity. A, B) Neuroprotective effect of EGCG: PC12 cells were pretreated without or with EGCG (1 µM) for 30 min, then incubated without or with Aß25–35 (10 µM), Aß1–42 (10 µM), or Aß1–40 (10 µM) for 48 h. Cell death detection ELISA (A) and MTT test (B) were applied to determine cell death. C) Neurorescue effect of EGCG: PC12 cells were exposed to Aß25–35 (10 µM) for 2 h and EGCG was added for 48 h. Levels of cell viability were measured using the MTT assay. Results are the mean ±SE (n=8). *P<0.05; **P<0.001 vs. Aß-induced cell death; P<0.05, P<0.001 vs. control, untreated cells.

5. Regulation of APP processing by EGCG in mice hippocampus
To determine the effect of EGCG on APP processing in vivo, membrane-bound APP (holoAPP) and sAPP levels in the hippocampus of EGCG-treated mice (2 mg/kg/ 3, 7, and 14 days) were assessed. Figure 2 A shows that APP levels were significantly reduced in the hippocampal membrane fraction of mice treated for 7 or 14 days with EGCG vs. those treated with vehicle. To examine the possibility that the decrease of holoAPP is the result of stimulated APP processing in the EGCG-treated mice, sAPP levels were measured in the soluble cytosolic fractions obtained from the hippocampus. sAPP was significantly increased in hippocampus obtained from mice treated for 14 days with EGCG compared with untreated control (Fig. 2A ).

View larger version (48K): [in this window] [in a new window]   Figure 2. Effect of EGCG on APP processing and PKC levels in mice hippocampus. A) Representative Western blots of levels of holoAPP in the membranes and sAPP in the cytosol obtained from hippocampus of mice treated with EGCG (2 mg/kg) for 3, 7, and 14 days, detected with 22C11 antibody. B) Representative Western blots of levels of PKC and PKC in membrane and cytosol fractions obtained from hippocampus of mice treated with EGCG (2 mg/kg) for 3, 7, and 14 days. Densitometric analysis is expressed as % of the control, untreated animals after normalizing to the levels of ß-actin. Data are expressed as the mean ±SE (n=6 mice in each group; each experiment was repeated twice). *P<0.05 and **P<0.001 vs. control.

6. Effect of EGCG on PKC levels in mice hippocampus
We studied the effect of EGCG on two PKC isoforms, PKC and PKC, in vivo. The levels of PKC and PKC were determined in membrane and cytosolic fractions obtained from the hippocampus of EGCG- and vehicle-treated mice. As seen in Fig. 2B , administration of EGCG (2 mg/kg/ 3, 7, and 14 days) significantly increased PKC levels in the membrane fractions of hippocampus compared with vehicle control. PKC levels in the membrane fractions increased only after 14 days of EGCG treatment. Administration of EGCG for 14 days significantly increased PKC and PKC levels in the cytosolic fractions of the hippocampus of EGCG-treated mice compared with vehicle control.

CONCLUSIONS AND SIGNIFICANCE

This study demonstrates that EGCG promotes the nonamyloidogenic -secretase pathway of APP processing, in both cultured cells and mice hippocampus. At low concentrations, EGCG is not only able to protect but also to rescue the sympathetic nerve pheochromocytoma PC12 cells from Aß toxicity. The protective effect of EGCG against Aß_25–35 in cultured hippocampal neurons was shown to involve the inhibition of caspase-3-like protease activity. The neuroprotective effect of EGCG may be derived from its potent antioxidant and iron-chelating properties, as Aß neurotoxicity has been reported to be mediated by free radicals and attenuated by antioxidants and free radical scavengers. Indeed, EGCG has been shown to prevent various neuronal damages induced by OS, with involvement of specific cell signaling pathways in its protective action. In addition, the nonamyloidogenic sAPP has potent neurotrophic and neuroprotective activities against excitotoxic and oxidative insults in various cellular models. Therefore, it can be suggested that sAPP derived from EGCG-mediated APP processing can serve as a neuroprotective agent against the toxic activity of Aß. In this study we observed that EGCG can dose-dependently affect APP metabolism by stimulating sAPP release from SH-SY5Y neuroblastoma and PC12 cells. Increased sAPP secretion was detected by the mAb 22C11 as well as by the mAb 6E10, which recognizes -secretase-cleaved APP. The stimulatory effect of EGCG on sAPP secretion was inhibited by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, indicating the effect was mediated via -secretase-dependent processing.

EGCG may affect APP metabolism by increasing the -secretase processing pathway and thereby could be beneficial for the treatment of AD by shifting the balance of APP processing toward a presumably nonpathogenic pathway. Thus, stimulation of -secretase cleavage may be a useful intervention to influence the production of nondeleterious and even beneficial sAPP, and, at the same time, reduce the relative amounts of Aß peptide. In mice, long-term treatment with EGCG resulted in decreases in cell-associated, full-length APP levels and increases in sAPP levels in the hippocampus. These results indicate that proteolytic processing of APP can also be regulated by treatment with EGCG in vivo.

To clarify the mechanism by which EGCG stimulates sAPP secretion, we examined the relationship between EGCG-induced sAPP release and PKC activity, whose involvement in sAPP release is well established. Inhibition of PKC by the kinase inhibitor GF109203X or down-regulation of PKC by prior chronic treatment with PMA inhibited the increase in sAPP secretion by EGCG in PC12 and SH-SY5Y cells, indicating the crucial role of PKC in mediating EGCG-induced sAPP release. These findings are consistent with previous studies demonstrating that activation of PKC and PKC-coupled receptors increases the generation of sAPP derived by -secretase cleavage both in vitro and in vivo. It is not known which isoenzyme of PKC plays a major role in modulating APP processing, but several lines of evidence suggest the involvement of PKC and PKC in APP processing. Accordingly, our present findings show that repeated administration of EGCG for 7 or 14 days caused significant increases in the protein expression of PKC isoenzymes and in the membrane and cytosolic fractions of mice hippocampus.

Based on the results of this study, a model was adopted to describe the effects of EGCG on Aß neurotoxicity and on APP processing (Fig. 3 ). The model suggests that EGCG activates PKC and PKC, leading to increased production of potentially neuroprotective, nonamyloidogenic sAPP. Since sAPP and Aß are formed by two mutually exclusive mechanisms, stimulation of the secretory processing of sAPP might prevent the formation of the amyloidogenic Aß. EGCG reduces cellular APP holoprotein, further reducing amyloidogenic processes.

View larger version (23K): [in this window] [in a new window]   Figure 3. Proposed schematic model for the neuroprotective effect and regulation of APP processing by EGCG. increased levels/activity, decreased levels/activity, sharp arrows indicate positive inputs, whereas blunt arrows are for inhibitory inputs.

In conclusion, we show here for the first time that EGCG can be neuroprotective against Aß toxicity and can regulate processing of APP via a PKC-dependent cascade.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0881fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0881fje

² Both authors are to be considered as a first author.

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This Article Full Text (PDF) All Versions of this Article: 17/8/952 02-0881fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LEVITES, Y. Articles by YOUDIM, M. B. H. Search for Related Content PubMed PubMed Citation Articles by LEVITES, Y. Articles by YOUDIM, M. B. H.