doi: 10.1016/j.bbrc.2009.01.041. Epub 2009 Jan 21.
Leptin regulates tau phosphorylation and amyloid through AMPK in neuronal cells
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- PMID:19166821
- PMCID: PMC2657956
- DOI: 10.1016/j.bbrc.2009.01.041
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Leptin regulates tau phosphorylation and amyloid through AMPK in neuronal cells
Steven J Greco et al. Biochem Biophys Res Commun..
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Abstract
Leptin, which serves as a lipid-modulating hormone to control metabolic energy availability, is decreased in Alzheimer's disease (AD) patients, and serum levels are inversely correlated to severity of dementia. We have previously described the effects of leptin in reducing amyloid beta protein both in vitro and in vivo, and tau phosphorylation in vitro. Herein, we systematically investigated the signaling pathways activated by leptin, leading to these molecular endpoints, to better understand its mechanism of action. Inhibition of amyloid beta production and tau phosphorylation in leptin-treated human and/or rat neuronal cultures were both dependent on activation of AMP-activated protein kinase (AMPK). Direct stimulation of AMPK with the cell-permeable activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), replicated leptin's effects and conversely, Compound C, an inhibitor of AMPK, blocked leptin's action. The data implicate that AMPK is a key regulator of both AD-related pathways.
Figures
Leptin-mediated signaling pathways regulating tau phosphorylation. RA-SY5Y were incubated with inhibitors to known downstream effectors of leptin signaling (STAT3, AMPK, PI3K, Akt, p38) in the presence of leptin (100 nM; 1600 ng/ml – lanes 3–7), or non-treated (vehicle – lane 1). Cells treated with leptin alone served as positive control (lane 2). Whole-cell lysates were prepared and analyzed by immunoblot (Panel I) 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. Normalized tau bands were analyzed by densitometry and results (panels II and III) are presented as the mean ± SD percent change, relative to non-treated samples, which were arbitrarily assigned a value of 0.* vs. non-treated (vehicle – lane 1)** vs. leptin alone (lane 2)
Phosphorylation of downstream signaling proteins activated by leptin and AICAR.A. RA-SY5Y were treated with leptin (100 nM; 1600 ng/ml), AICAR (2 mM) or non-treated (vehicle), and signaling proteins (Jak2, AMPK, p38, Akt) implicated in regulating tau phosphorylation were examined by immunoblot using phosphorylation-specific antibodies. Membranes were stripped and re-probed with α-tubulin antibody for normalization. Representative blots are shown, n=3. Normalized bands were analyzed by densitometry and results (B–E) are presented as the mean ± SD. Phosphorylation of (F) GSK-3β (pSer9) in the above cells was measured by immunoblot. Membranes were stripped and re-probed with total GSK-3β for normalization. Bands were analyzed by densitometry and results (G) are presented as the mean ± SD.* vs. non-treated
Leptin and AICAR regulate Aβ production via overlapping signaling pathways.A. RA-SY5Y were treated for 6 hrs with leptin (100 nM; 1600 ng/ml – lane 2), AICAR (2 mM – lane 5) and/or inhibitors to each of the following signaling proteins – AMPK (lanes 3 and 6) or PPARγ (lanes 4 and 7). Non-treated (vehicle – lane 1) cells or cells treated with inhibitor alone (lanes 8–9) served as control. Culture media was collected and assayed for Aβ(1–40) by ELISA. Results were normalized to total protein in cell lysates and are presented as the mean Aβ(1–40) concentration (pg/ml) ± SD.B. Cells fromA were treated with leptin, alone or in the presence of Akt inhibitor (1L6HCI). Culture media was collected and assayed for Aβ(1–40) by ELISA. Results are presented as inA.C. Cells fromA were treated with leptin alone or in the presence of PPARγ antagonist (G3335). Whole-cell lysates were prepared and analyzed by immunoblot with phosphorylated tau-specific antibodies as described in the legend of Figure 1.D. Cartoon depicting the hypothesized signaling pathways activated by leptin and AICAR in RA-SY5Y.* vs. non-treated (vehicle – lane 1)** vs. leptin alone (lane 2)# vs. AICAR alone (lane 5)
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