Steven J. Greco

Leptin regulates tau phosphorylation and amyloid through AMPK in neuronal cells.

Leptin, which serves as a lipid-modulating hormone to control metabolic energy availability, is decreased in Alzheimers 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 leptins effects and conversely, Compound C, an inhibitor of AMPK, blocked leptins action. The data implicate that AMPK is a key regulator of both AD-related pathways.

Leptin reduces pathology and improves memory in a transgenic mouse model of Alzheimer's disease.

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 Alzheimers 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-beta (Abeta){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 Abeta in the brain correlated with a decrease in the levels of C99 C-terminal fragments of the amyloid-beta protein precursor, consistent with a role for beta -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-alpha, 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.

Leptin: A Novel Therapeutic Strategy for Alzheimer's Disease

Adipocyte-derived leptin appears to regulate a number of features defining Alzheimers disease (AD) at the molecular and physiological level. Leptin has been shown to reduce the amount of extracellular amyloid beta, both in cell culture and animal models, as well as to reduce tau phosphorylation in neuronal cells. Importantly, chronic administration of leptin resulted in a significant improvement in the cognitive performance of transgenic animal models. In AD, weight loss often precedes the onset of dementia and the level of circulating leptin is inversely proportional to the severity of cognitive decline. It is speculated that a deficiency in leptin levels or function may contribute to systemic and CNS abnormalities leading to disease progression. Furthermore, a leptin deficiency may aggravate insulin-controlled pathways, known to be aberrant in AD. These observations suggest that a leptin replacement therapy may be beneficial for these patients.

Leptin reduces Alzheimer’s disease-related tau phosphorylation in neuronal cells

Leptin is a centrally acting hormone controlling metabolic pathways. Recently, it was shown that leptin can reduce amyloid beta 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 amino acid residues Ser(202), Ser(396) and Ser(404) was monitored in retinoic acid induced, human cell lines: SH-SY5Y and NTera-2. Both hormones induced a concentration- and time-dependent reduction of tau phosphorylation, and were synergistic at suboptimum concentrations. Importantly, leptin was 300-fold more potent than insulin (IC(50)L=46.9 nM vs. IC(50)I=13.8 microM). A central role for AMP-dependent kinase as a mediator of leptins 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 beta and tau-related pathological pathways, holds promise as a novel therapeutic for Alzheimers disease.

Leptin Inhibits Glycogen Synthase Kinase-3β to Prevent Tau Phosphorylation in Neuronal Cells

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 Alzheimers 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 Leptins 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 Leptins mechanism of action in suppressing AD-related pathways.

Leptin Boosts Cellular Metabolism by Activating AMPK and the Sirtuins to Reduce Tau Phosphorylation and β-Amyloid in Neurons

Leptin is a pleiotropic hormone primarily secreted by adipocytes. A high density of functional Leptin receptors has been reported to be expressed in the hippocampus and other cortical regions of the brain, the physiological significance of which has not been explored extensively. Alzheimers disease (AD) is marked by impaired brain metabolism with decreased glucose utilization in those regions which often precede pathological changes. Recent epidemiological studies suggest that plasma Leptin is protective against AD. Specifically, elderly with plasma Leptin levels in the lowest quartile were found to be four times more likely to develop AD than those in the highest quartile. We have previously reported that Leptin modulates AD pathological pathways in vitro through a mechanism involving the energy sensor, AMP-activated protein kinase (AMPK). To this end, we investigated the extent to which activation of AMPK as well as another class of sensors linking energy availability to cellular metabolism, the sirtuins (SIRT), mediate Leptins biological activity. Leptin directly activated neuronal AMPK and SIRT in cell lines. Additionally, the ability of Leptin to reduce tau phosphorylation and β-amyloid production was sensitive to the AMPK and sirtuin inhibitors, compound C and nicotinamide, respectively. These findings implicate that Leptin normally acts as a signal for energy homeostasis in neurons. Perhaps Leptin deficiency in AD contributes to a neuronal imbalance in handling energy requirements, leading to higher Aβ and phospho-tau, which can be restored by replenishing low Leptin levels. This may also be a legitimate strategy for therapy.

Low Plasma Leptin in Cognitively Impaired ADNI Subjects: Gender Differences and Diagnostic and Therapeutic Potential

Analysis of data derived from the Alzheimers Disease Neuroimaging Initiative (ADNI) program showed plasma leptin levels in individuals with Mild Cognitive Impairment (MCI) or Alzheimers disease (AD) to be lower than those of subjects with normal cognition (NC). Approximately 70% of both men and women with MCI have plasma leptin levels lower than the median values of NC. Additionally, half of these subjects carry at least one apolipoprotein-E4 (APOE-ε4) allele. A subgroup of participants also had cerebrospinal fluid (CSF) leptin measured. Plasma leptin typically reflected the levels of leptin in CSF in all groups (Control/MCI/AD) in both genders. The data suggest that plasma leptin deficiency provides an indication of potential CNS leptin deficiency, further supporting the exploration of plasma leptin as a diagnostic marker for MCI or AD. The important question is whether leptin deficiency plays a role in the causation of AD and/or its progression. If this is the case, individuals with early AD or MCI with low plasma leptin may benefit from leptin replacement therapy. Thus, these data indicate that trials of leptin in low leptin MCI/early-stage AD patients should be conducted to test the hypothesis.

Chronic Leptin Supplementation Ameliorates Pathology and Improves Cognitive Performance in a Transgenic Mouse Model of Alzheimer's Disease.

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 Alzheimers 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-beta (Abeta){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 Abeta in the brain correlated with a decrease in the levels of C99 C-terminal fragments of the amyloid-beta protein precursor, consistent with a role for beta -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-alpha, 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.

Isotyping the Human TOMM40 Variable-Length Polymorphism by Gene Amplification and Restriction Digest

Recent studies have shown that the translocase of outer mitochondrial membrane 40 homolog (TOMM40) contains a polymorphic poly-T variant, the long variant of which is associated with an increase in AD incidence among APOE 3 carriers. Current methods to isotype the poly-T region rely on long PCR, subcloning and sequencing to distinguish among the allelic variants. While such methods are extremely accurate as well as quantitative in determining the number of T residues in the poly-T region, the process can be cumbersome, time consuming and expensive to employ in routine laboratories. To this end, we have developed a quick and simple method to isotype the human TOMM40 variable length polymorphism using a PCR- and restriction digest-based approach, enabling rapid genotyping of TOMM40 variants.