Abeta peptide is the major proteinateous component of the amyloid plaques found in the brains of Alzheimer's disease (AD) patients and is regarded by many as the culprit of the disorder. It is well documented that brain lipids are intricately involved in Abeta-related pathogenic pathways. An important modulator of lipid homeostasis is the pluripotent peptide leptin. Here we demonstrate leptin's ability to modify Abeta levels in vitro and in vivo. Similar to methyl-beta-cyclodextrin, leptin reduces beta-secretase activity in neuronal cells possibly by altering the lipid composition of membrane lipid rafts. This phenotype contrasts treatments with cholesterol and etomoxir, an inhibitor of carnitine-palmitoyl transferase-1. Conversely, inhibitors of acetyl CoA carboxylase and fatty acid synthase mimicked leptin's action. Leptin was also able to increase apoE-dependent Abeta uptake in vitro. Thus, leptin can modulate bidirectional Abeta kinesis, reducing its levels extracellularly. Most strikingly, chronic administration of leptin to AD-transgenic animals reduced the brain Abeta load, underlying its therapeutic potential.

We have previously reported anti-amyloidogenic effects of leptin using in vitro and in vivo models and, more recently, demonstrated the ability of leptin to reduce tau phosphorylation in neuronal cells. The present study examined the efficacy of leptin in ameliorating the Alzheimer's disease (AD)-like pathology in 6-month old CRND8 transgenic mice (TgCRND8) following 8 weeks of treatment. Leptin-treated transgenic mice showed significantly reduced levels of amyloid-β (Aβ) 1-40 in both brain extracts (52% reduction, p=0.047) and serum (55% reduction, p=0.049), as detected by ELISA, and significant reduction in amyloid burden (47% reduction, p=0.041) in the hippocampus, as detected by immunocytochemistry. The decrease in the levels of Aβ in the brain correlated with a decrease in the levels of C99 C-terminal fragments of the amyloid-β protein precursor, consistent with a role for β-secretase in mediating the effect of leptin. In addition, leptin-treated TgCRND8 mice had significantly lower levels of phosphorylated tau, as detected by AT8 and anti-tau-Ser 396 antibodies. Importantly, after 4 or 8 weeks of treatment, there was no significant increase in the levels of C-reactive protein, tumor necrosis factor-α, and cortisol in the plasma of leptin-treated TgCRND8 animals compared to saline-treated controls, indicating no inflammatory reaction. These biochemical and pathological changes were correlated with behavioral improvements, as early as after 4 weeks of treatment, as recorded by a novel object recognition test and particularly the contextual and cued fear conditioning test after 8 weeks of treatment. Leptin-treated TgCRND8 animals significantly outperformed saline-treated littermates in these behavioral tests. These findings solidly demonstrate the potential for leptin as a disease modifying therapeutic in transgenic animals of AD, driving optimism for its safety and efficacy in humans.

Peptide hormones and neurotransmitters constitute a large class of neurohumoral agents that mediate cell-cell communication in neuroendocrine systems. Their biosynthesis requires proteolytic processing of inactive protein precursors into active neuropeptides. Elucidation of the proteolytic components required for prohormone processing is important for identifying key proteases that may control the production of neuropeptides. This article compares the subtilisin-like PC1/3 and PC2 processing enzymes identified through molecular biological approaches, and several candidate processing enzymes identified biochemically, including the 'proopiomelanocortin converting enzyme' (PCE) and the 'prohormone thiol protease' (PTP), as well as others of different classes (aspartyl, cysteine, metallo, and serine proteases). A role for PTP in cellular proenkephalin processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by Ep453 that is converted intracellularly to E-64c, a selective cysteine protease inhibitor that potently inhibits PTP. A possible role for endogenous protease inhibitors in prohormone processing represents a new aspect of cellular mechanisms that may regulate neuropeptide biosynthesis. Future studies of the enzymology and molecular biology of processing enzymes and endogenous protease inhibitors will be necessary to elucidate mechanisms of prohormone processing.

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 β 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 β 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.

Leptin is a centrally-acting hormone controlling metabolic pathways. Recently, it was shown that leptin can reduce amyloid β levels both in vitro and in vivo. Herein, phosphorylation of tau was investigated following treatment of neuronal cells with leptin and insulin. Specifically, phosphorylation of tau at aa residues Ser 202 , Ser 396 and Ser 404 were monitored in retinoic-acid induced, human cell-lines: SH-SY5Y and NTera-2. Both hormones induced concentration-and timedependent reductions of tau phosphoylation, and were synergestic at suboptimum concentrations. Importantly, leptin was 300-fold more potent than insulin (IC 50 L= 46.9 nM vs IC 50 I= 13.8 µM). A central role for AMP-dependent kinase as a mediator of leptin's action is demonstrated by the ability of 5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) to decrease tau phosphorylation and, by blocking leptin in the presence of Compound C. Thus, leptin, which ameliorates both, amyloid β and tau-related pathological pathways, holds promise as a novel therapeutic for Alzheimer's disease.

Adipocyte-derived leptin appears to regulate a number of features defining Alzheimer's disease (AD) at the molecular and physiological level. One activity of leptin is the control of AMPdependent kinase (AMPK). In addition to maintaining lipid levels, AMPK regulates glycogen synthase kinase-3, which modulates tau phosphorylation. Leptin has been shown to reduce the amount of extracellular amyloid-β, both in cell culture and animal models of AD, as well as reduce tau phosphorylation in neuronal cells. Importantly, chronic administration of leptin resulted in a significant improvement in the cognitive performance of transgenic animal models of AD. In humans, weight loss often precedes the onset of dementia in AD and the level of circulating leptin is inversely proportional to the severity of dementia among AD patients. It is speculated that a deficiency in leptin levels or function may contribute to systemic and central nervous system abnormalities leading to AD, suggesting that a leptin replacement therapy may be beneficial for AD. This may be an attractive alternative to the drugs that are currently under development.

We have previously demonstrated that Leptin reduces extracellular amyloid β (Aβ) 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-3β, which has been shown to be inactivated by Leptin. We therefore dissected the role of GSK-3β in mediating Leptin’s ability to reducing tau phosphorylation in neuronal cells. Our data suggest that Leptin regulates tau phosphorylation through a pathway involving both AMPK and GSK-3β. 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-3β inhibitor, lithium chloride (LiCl). Further, this reduction of tau phosphorylation was mimicked by the downregulation of GSK-3β, achieved using siRNA technology and antagonized by the ectopic overexpression of GSK-3β. These studies provide further insight into Leptin’s mechanism of action in ameliorating AD-related pathways.

The Af3 peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific