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, 455 (3), 191-4

Leptin Inhibits Glycogen Synthase kinase-3beta to Prevent Tau Phosphorylation in Neuronal Cells

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Leptin Inhibits Glycogen Synthase kinase-3beta to Prevent Tau Phosphorylation in Neuronal Cells

Steven J Greco et al. Neurosci Lett.

Abstract

We have previously demonstrated that Leptin reduces extracellular amyloid beta (Abeta) protein both in vitro and in vivo, and intracellular tau phosphorylation in vitro. Further, we have shown that these effects are dependent on activation of AMP-activated protein kinase (AMPK) in vitro. Herein, we investigated downstream effectors of AMPK signaling directly linked to tau phosphorylation. One such target, of relevance to Alzheimer's disease (AD), may be GSK-3beta, which has been shown to be inactivated by Leptin. We therefore dissected the role of GSK-3beta in mediating Leptin's ability to reduce tau phosphorylation in neuronal cells. Our data suggest that Leptin regulates tau phosphorylation through a pathway involving both AMPK and GSK-3beta. This was based on the following: Leptin and the cell-permeable AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), reduced tau phosphorylation at AD-relevant sites similarly to the GSK-3beta inhibitor, lithium chloride (LiCl). Further, this reduction of tau phosphorylation was mimicked by the downregulation of GSK-3beta, achieved using siRNA technology and antagonized by the ectopic overexpression of GSK-3beta. These studies provide further insight into Leptin's mechanism of action in suppressing AD-related pathways.

Figures

Fig. 1
Fig. 1
Enzymatic regulation of tau phosphorylation in RA-SY5Y. A. RA-SY5Y were incubated with the GSK-3β inhibitor, LiCl (10 mM), for 1 hr, or non-treated (vehicle), and phosphorylation of tau at multiple sites was measured. Treatment with Leptin for 4 h (100 nM; 1600 ng/ml) or AICAR for 1 h (2 mM) served as positive control. Whole-cell lysates were prepared and analyzed by immunoblot with phosphorylated tau-specific antibodies (pSer396, PHF-1, AT8 or pSer181). Membranes were stripped and re-probed with total tau antibody for normalization. Representative blots are shown, n=3. B. Normalized bands were analyzed by densitometry and results are presented as the mean ± SD percent change, relative to vehicle. C. Lysates from A were prepared and analyzed by ELISA for GSK-3β (pSer9). Cells incubated for 1 hr with LiCl served as positive control. Results (n=3) are presented as the mean normalized GSK-3β (pSer9) concentration (Units/mg total protein) ± SD, relative to non-treated samples. D. Cells were incubated with or without LiCl, in the presence of Leptin, AICAR or no additional treatment (vehicle), and phosphorylation of tau (pSer396) was measured by immunoblot as in A. Results (n=3) are presented as in B. *p<0.05 vs. non-treated (vehicle)
Fig. 2
Fig. 2
Loss-of-function studies show that Leptin and AICAR modulate tau phosphorylation via a GSK-3β-dependent mechanism. RA-SY5Y were transiently transfected with GSK-3β-specific siRNA, or untransfected (lane 1), and later treated with Leptin for 4 h (100 nM; 1600 ng/ml – lane 6), AICAR for 1 h (2 mM – lane 7) or no treatment (vehicle – lane 5). Cells transfected with fluorescein-conjugated control siRNA with or without Leptin and AICAR treatment (lanes 2–4) were used to assess tranfection efficiency and served as negative controls. Whole-cell lysates were prepared and analyzed by immunoblot with GSK-3β-specific (panel I) or phosphorylated tau-specific antibodies (pSer396, PHF-1, AT8 or pSer181; panel II). Membranes were stripped and re-probed with total GSK-3β (panel I) or total tau (panel II) antibodies for normalization. Representative blots are shown, n=3. Normalized tau bands were analyzed by densitometry and results (panels III–IV) are presented as the mean ± SD percent change, relative to negative control samples. *p<0.05 vs. negative control siRNA-transfected cells treated with vehicle (lane 2)
Fig. 3
Fig. 3
Gain-of-function studies show that Leptin and AICAR modulate tau phosphorylation via a GSK-3β-dependent mechanism. RA-SY5Y were transiently transfected with a GSK-3β full-length cDNA expression vector (lanes 5–7), or untransfected (lane 1), and later treated as described in the Legend of Fig. 2. Cells transfected with empty expression vector (lanes 2–4), with or without Leptin and AICAR treatment were used to assess transfection efficiency and served as negative controls. Whole-cell lysates were prepared, analyzed and normalized as described in the Legend of Fig. 2. Results (n=3) are presented as described in the Legend of Fig. 2 *p<0.05 vs. negative control siRNA-transfected cells treated with vehicle (lane 2) **p<0.05 vs. GSK-3β-overexpressing cells treated with vehicle (lane 5)

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