A common pathological hallmark of a number of neurodegenerative diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntingdon’s disease (HD) and prion disease, is the accumulation of proteins in the parenchyma of brain. While these proteins have a normal function in healthy individuals, they accumulate by unknown mechanisms contributing to disease onset. Heat shock proteins are an important group of proteins that regulate cellular protein homeostasis (proteostasis) and understanding their role in aberrant protein homeostasis may lead to the identification of new drug targets.

Receptor-mediated oscillations in cytosolic Ca2+ concentration ([Ca2+]i) could originate either directly from an autonomous Ca2+ feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca2+ feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca2+ oscillations or could just be a secondary response to the [Ca2+]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca2+]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca2+]i rise and the speed of Ca2+ wave propagation and, at high levels, abolished [Ca2+]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca2+]i oscillations and waves.

Analysis of data derived from the Alzheimer's Disease Neuroimaging Initiative (ADNI) program showed plasma leptin levels in individuals with Mild Cognitive Impairment (MCI) or Alzheimer's 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.

Adipocyte-derived leptin is a pleiotropic hormone implicated in control of lipid storage and mobilization, bone homeostasis, immune function and neuronal plasticity. Leptin has been shown to prevent accumulation of extracellular Aβ and hyperphosphorylation of tau in both cell culture and animal models. Herein an investigation was undertaken to test leptin’s ability to prevent the exacerbation/activation of AD-related pathways in neurons following their exposure to a high concentration of a variety of lipids. Specifically, cholesterol, oleic acid and/or ceramide were added to the media of cells resulting in decreased cellular viability and energy metabolism, and in increased tau phosphorylation and extracellular Aβ. Leptin increased viability, boosted cellular metabolism by activating AMP-activated protein kinase (AMPK) and the sirtuins (SIRT) and reduced tau phosphorylation and Aβ accumulation in a dose-dependent manner in response to select challenges. These findings demonstrate that leptin can attenuate the harmful effect of certain lipids that lead to exacerbation or activation of AD pathways. The study herein also provides the basis for a novel screening platform to define and identify a novel class of “metabolic” compounds addressing Alzheimer’s disease, based on a biological profile similar to leptin.

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.

The data from the initial clinical trials utilizing recombinant human leptin as an obesity therapy were published in 1998. Since then, numerous studies have been described which address dosage, safety and efficacy of leptin replacement for a variety of disorders with diverse patient groups, including pediatric and adult subjects. We review the current clinical trial data, demonstrate that leptin administration is safe for long term use in humans, and summarize reported cognitive benefits. The functions of leptin in neuroprotection and cognition have been largely overlooked. Accumulating data suggest a very significant application of leptin may be a therapy for Alzheimer's disease.

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. Alzheimer's 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 Leptin's 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.

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-beta (A beta)(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 A beta 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, 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.

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.

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-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.

Adipocyte-derived leptin appears to regulate a number of features defining Alzheimer's 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 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 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 beta and tau-related pathological pathways, holds promise as a novel therapeutic for Alzheimer's disease.

Stimulation of type-1 astrocytes, by a number of agonists, has been shown to increase cytosolic Ca2+ concentrations in an oscillatory manner. However, it is unknown how these are driven or altered by aging, injury or disease. Therefore, we characterized the signaling properties of rat type-1 astrocytes in monolayer cultures. Ca2+ responses were recorded in astrocytes stimulated with ATP or glutamate. Oscillations were evident in cultures at 3 days in vitro (DIV 3) with a peak percentage (26%) of cells responding by DIV 7. The presence or absence of serum in the culture medium did not influence this percentage. Likewise, long-term culture (DIV 30+) did not increase the oscillating cell numbers. ATP was found to have a more potent effective dose (50 microM) than glutamate (10 mM). Membrane potential was recorded with fluorescent voltage-sensitive dye (membrane potential dye for FLIPR) and was similar regardless of the culture age and intracellular Ca2+ response. This suggests that mechanisms associated with the intracellular release of Ca2+ from endogenous Ca2+ stores, rather than ion fluxes across the plasma membrane, may contribute to the oscillations in the astrocytes. In order to identify a possible pathological significance of this response, we transfected astrocytes with wild-type presenilin (PS1) as well as PS1 harboring a mutation linked to familial Alzheimer's disease (FAD). PS mutation expressing astrocytes oscillated at lower ATP and glutamate concentrations when compared to wild-type PS1 expressing astrocytes. This indicates that PS1 mutation may introduce aberrations in the intracellular Ca2+ mobilization in astrocytes contributing to the accelerated pathogenesis in FAD.

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.

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.

Dopaminergic cell therapy is a potential viable treatment for Parkinson's disease. However, lack of a well-characterized cell preparation of known phenotypic composition containing a high percentage of dopaminergic neurons, has prevented a definitive, controlled, pilot clinical trial from being conducted. We report the successful in vitro expansion of rat E12 mesencephalic progenitors to produce 5-fold the normal number of dopaminergic neurons. The expanded neurons (MAP2+) were detached, resuspended, and formed into small aggregates of 10-200 neurons containing 25-50% of dopaminergic neurons (TH+) that will likely be optimal for use in successful cell therapy. After storage in DPBS, in 0 mM Ca(2+) for up to 24 h at room temperature, aggregated cells were still 90% viable. These results demonstrate that it might be feasible to use a similar protocol to expand human dopaminergic progenitors in vitro. If successful, the requisite large numbers of dopaminergic neurons required to conduct a pilot clinical trial for Parkinson's disease will be produced in vitro. Indications are that the cells can be maintained at optimal viability for the duration of the neural transplantation procedure, under real operating conditions.

Cu(II) desferal conjugated to d(T)10 at 5′-end is shown to direct the cleavage of single stranded 19-mer target DNA d(TA1GCCCG-GCG), with a base preference G > C ⪢ A. In contrast, the corresponding Co(II)-desferal conjugate directed the cleavage specific to only G sites (G12, G16, G17 and G19). Triplex targetting on the duplex 5t́-TCCTGATAAAGGAGGAGATGAAGAAAAAATGA-3′: 3′AGGACTATTTCCTCCTCTACTTCTTTTTTACT-5′, using Cu(II) desferal conjugated to 5′-end of 3′-TTTCCTCCTCT-5′ directed the cleavage at G5.

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early‐onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild‐type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C‐terminal tail of the 170 kDa cytoplasmic linker protein (CLIP‐170) and Reed–Sternberg cells of Hodgkin's disease‐expressed intermediate filament‐associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP‐170/restin requires Ca2+. Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP‐170 immunoprecipitates. Further, PS1 and CLIP‐170 co‐localize in transfected cells and neuronal cultures.