Systematic gene expression profile of hypothalamus in calorie-restricted mice implicates the involvement of mTOR signaling in neuroprotective activity.

Calorie restriction (CR) delays aging and onset of age-related diseases in a variety of organisms from yeast to mammals. However, the molecular mechanism underlying the CR effect remains to be elucidated. It is well known that the hypothalamus is an important component of the brain neuroendocrine system for the regulation of the aging process. In this report, we have systematically examined the gene expression profiles of hypothalami from 5-, 12-, 19- and 24-month-old mice fed ad libitum or subjected to CR since weaning. Our results demonstrated that CR significantly altered the expression level of 490 genes in an age-dependent manner, with the greatest impact at middle age. Classification based on functional analysis indicated that a large number of these genes were involved in brain development and neurogenesis, including genes involved in Wingless (Wnt) and Notch signaling pathways. In addition, the expression levels of numerous genes involved in the stress and inflammatory responses, as well as apoptosis, were affected by CR. Interestingly, we found that a number of genes involved in the stress response and apoptosis were down-regulated in early but up-regulated in late stage CR. The most notable finding was that CR altered the expression of genes associated with the mammalian target of the rapamycin (mTOR) nutrient sensing pathway, which has recently been shown to be involved in the regulation of energy intake and aging. By applying rapamycin, a specific pharmacological inhibitor of mTOR signaling, we found that the inhibition of mTOR could significantly prevent neuronal apoptosis induced by Paraquat. Taken together, our results provided not only a systematic expression profile of the hypothalamic response to CR, but also revealed the linkage between CR and mTOR signaling in the neuroprotection in mice.

Fructose-derived carbohydrates from Alisma orientalis.

Nine fructose-derived carbohydrates were obtained from the methanol extract from the rhizome of Alisma orientalis. On the basis of spectroscopic analysis, their structures were determined to be alpha-D-fructofuranose (1), beta-D-fructofuranose (2), ethyl alpha-D-fructofuranoside (3), ethyl beta-D-fructofuranoside (4), 5-hydroxymethyl-furaldehyde (5), sucrose (6), raffinose (7), stachyose (8) and verbascose (9), along with two oligosaccharides of manninotriose (10) and verbascotetraose (11). Compounds 3, 4 and 7-11 were isolated from this plant for the first time. A hypothetical biosynthesis pathway among these isolated carbohydrates (1-11) was briefly introduced.

New guaiane sesquiterpenes and furanocoumarins from Notopterygium incisum.

In addition to twenty-nine known compounds, two new guaiane sesquiterpenes and two new furanocoumarins were isolated from the chloroform extract of the rhizomes of Notopterygium incisum. The new structures were elucidated by means of spectroscopic methods including 2 D NMR techniques and mass spectrometry to be 8 beta-acetoxy-4 alpha,6 alpha-dihydroxy-1 alpha,5 alpha( H)-guai-9-ene (incisumdiol A, 1), 4 alpha,6 alpha-dihydroxy-1 alpha,5 alpha( H)-guai-9-ene (incisumdiol B, 2), 5-[(2 E,5 Z)-7-hydroxy-3,7-dimethyl-2,5-octadienoxy]psoralene ( 3) and 5-[(2,5)-epoxy-3-hydroxy-3,7-dimethyl-6-octenoxy]psoralene ( 4).

Protective effects of EUK4010 on beta-amyloid(1-42) induced degeneration of neuronal cells.

EUK4010 has been identified to exhibit an inhibitory effect on beta-amyloid (Abeta)(1-42)-induced loss of neuronal cell viability. Further studies demonstrated that EUK4010 attenuated the Abeta(1-42)-induced degeneration in both cultured rat hippocampal neurons and human neuroblastoma cells, as demonstrated by typical morphological changes, cell viability and the chip-based flow cytometric assay. Gene expression analysis using DNA microarray showed that the senescence marker calcium-binding protein, regucalcin (Rgn), GABA-A receptor pi subunit (Gabrp), the huntingtin binding protein, optineurin (Optn) and a semaphorin family plexin A3 similar protein (Plex-similar) changed their expression levels significantly in cultured neurons after Abeta(1-42) treatment. In this report, we have undertaken a chemical genetic approach to study the molecular basis of Abeta(1-42) effects on the neuronal degeneration. Our results demonstrate that EUK4010 completely blocked the Abeta(1-42)-induced up-regulation of GABA-A receptor pi subunit and the semaphorin family plexin A3 similar protein, and partially attenuated the down-regulation of senescence marker calcium-binding protein, regucalcin. These observations suggest that EUK4010 may prevent or reduce the Abeta toxicity by regulating the expression of genes involved in the Abeta induced neuronal degeneration. These genes may represent a promising target for the therapeutic drug development for Alzheimer's disease (AD) and other neurological disorders. Furthermore, EUK4010 and its analogues could potentially be developed as neuronal protective agents for the treatment of these diseases.

Regulation of the expression of GABAA receptor subunits by an antiepileptic drug QYS.

It has been reported that the antiepileptic drug qingyangshenylycosides (QYS) modulated the function of GABAergic system. However, little is known about the effects of QYS on the gene expression of GABA receptors in the central nervous system (CNS). In the present study, we examined the effects of QYS on the expression of GABAA receptor subunits in different regions of the mouse brain. The results showed that treatment of QYS significantly increased the expressions of Gabra1, Gabra2 and Gabr4 and decreased the expression of Gabrg2 in inferior colliculus. Moreover, Gabrb2 expression was up-regulated and Gabra5 was down-regulated in hippocampus, while the expressions of Gabra1 and Gabrb2 were induced in cortex after QYS treatment. These data indicated that QYS had different effects on the expression of GABAA receptor subunits in different brain regions. These results may help to reveal the molecular mechanism of anticonvulsant action of QYS.

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay.

AIM: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. METHODS: Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. RESULTS: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor. CONCLUSION: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

Involvement of Scn1b and Kcna1 ion channels in audiogenic seizures and PTZ-induced epilepsy.

We have undertaken chemical genetic approach using Qingyangshenylycosides (QYS), a natural product compound, to explore the molecular mechanisms underlying different types of epilepsy models. Two animal models were used for these studies, i.e., audiogenic seizure (AGS) and pentylenetetrazol (PTZ)-induced generalized epilepsy in DBA/2J mice. We show that the latency of AGS is prolonged and the severity of seizures (the percentages of the tonus, Tonus_%) is reduced in the QYS-treated animals. These results indicate that QYS has anticonvulsant effect on the AGS model. However, we find that administration of QYS has an opposite effects on PTZ-induced generalized epilepsy. Both the latency of the generalized epilepsy and the latency of death are decreased after QYS treatment in PTZ-induced epilepsy. We examine the molecular basis of the distinct roles of QYS in these two epilepsy models by using gene expression data. Our results show that a voltage-gated sodium channel (Scn1b) and a voltage-gated potassium channel (Kcna1) are differentially expressed in AGS and PTZ-induced epilepsy models as well as in QYS-treated animals. Our results demonstrate that a chemical genetic approach may help to reveal both the molecular mechanisms of different epilepsies and the mechanism of action of the antiepileptic drugs.

Gene expression profiling reveals the mechanism of action of anticonvulsant drug QYS.

We have examined the gene expression profiling of inferior colliculus from DBA/2J mice with high-intensity noise induced audiogenic seizure (AGS). We have also tested the effects of Qingyangshenylycosides (QYS), a traditional Chinese medicine, on the audiogenic seizure, and examined how the drug affected the gene expressions in inferior colliculus. Our results demonstrated that the latency was increased and the Tonus% of AGS was decreased in the animals treated with QYS, indicating that the drug effectively prevented audiogenic seizure. Gene expression analysis using Agilent oligo microarray showed that total of 134 genes were either up- or down-regulated during AGS. QYS prevented many of the AGS induced gene expression changes. Nevertheless, some of the AGS induced genes were further enhanced or reversed by QYS treatment. Our gene expression profiling data provided important information regarding the molecular mechanisms of AGS and the mechanism of action of QYS. Further analysis of the function of these genes may help to identify therapeutic targets for epilepsy.

Establishment of a cell-based assay to screen regulators for Klotho gene promoter.

AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS: The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment. The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2 effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.