ACTA NEUROPHARMACOLOGICA

Neuron-synapse loss and dementias

ZHANG Juntian

2011, 1 (1): 1-15.

Abstract ( 6089 )

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Basic and clinical study of Alzheimer’s disease showed that senile plagues and neurofibrillary tangle are not related to the degree of cognitive impairment. Synaptic loss occur in Alzheimer’s disease and other neurodegenerative dementias which block LTP，induce neuropsychological dysfunction and memory deficit . Neuron-synapses or synaptic loss was considered as main cause of dementias. Several hidden toxins such as oligomer of Aβ and phosphorylated Tau,glutamate receptor at extrasynaptic region and shank protein can lead to synaptic loss. Therefore，strategy for AD treatment is that ① To clean oligomer by native oligomer of Aβ immunotherapy. ②To maintain the normal homeostasis of synaptic protein by mTOR(a protein kinase) and UPS. ③To activate synaptic NMDA receptors or block extrasynaptic NMDA receptors. ④To increase hippocampal neurogenesis and synaptogenesis. In our recent study，ginsenoside Rg1 and Ppt were proved to increase synaptic plasticity in both efficacy and structure as well as increase neurogenesis both in vitro and in vivo，under physiological and pathological circumstances，indicating that they are promising agents for preventing and treatment of dementias and many kinds of memory impairment.

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Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation

Stephanie Richardson Milazi Ph.D., Benjamin Rix Brooks M.D., Jean-Luc Mougeot Ph.D.

2011, 1 (1): 16-26.

Abstract ( 4971 )

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Objective: Our objective was to isolate motor neuron-like cell precursors (MNLCPs) derived from mouse embryonic stem cells (mESCs) within embryoid bodies (EBs) for the development of drug screening assays and transplantation therapies in motor neuron disorders. EBs, induced with native Shh protein (or agonists of Shh pathway) and retinoic acid, contain variable proportions of MNLCPs and undifferentiated cells. Undifferentiated cells may interfere in drug screening assays or proliferate following transplantation if not sufficiently removed from the culture. We have developed a method based on density gradient centrifugation to enrich MNLCPs.Methods: mESCs (HBG3:eGFP: HB9) were expanded and differentiated using a modified protocol by Wichterle et al. (2008). EBs containing GFP (+) MNLCPs and undifferentiated cells were gently dissociated into single cells by chemical and enzymatic treatments without trituration. MNLCPs were recovered after centrifugation of dissociated cells on Optiprep^TM 8%~20% step-gradient. The amount of GFP (+) MNLCPs was determined by flow cytometry.Results: Our results show that mESCs grown on gelatinized plates prior to EB formation, decreases the ability of mESCs to differentiate into MNLCPs. mESCs were grown on gelatin, gelatin with PMEFs, and PMEFs alone and found that growing mESCs on PMEF yielded GFP (+) MNLCPs at 54.1% ± 11.0% ( ±s；n=12) compared with gelatin 2.8% ±1.1% ( ±s；n=9). We obtained enriched fractions containing on average 87.7% ± 5.5%( ±s；n=3) GFP(+) MNLCPs by density gradient centrifugation.Conclusion: Our data support that dissociation without trituration and density gradient centrifugation can be used to significantly enrich MNLCPs retaining high viability without the use of a cell sorter. Further characterization of MNLCPs is necessary to design an appropriate assay such as physiological relevance in vitro and in vivo and phenotype such as neurite outgrowth, and the ability to form neuromuscular junctions.

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Effects of nicotine analog ZY-1 on β-amyloid production and learning and memory in transgenic Alzheimer disease mice

NIE Hui-Zhen, WANG Ze-Jian, ZHAO Wen-Juan, LU Jin-Miao, ZHANG Can, LUO Qing-He, WANG Yu-Liang, GAO Xiang, YIN Ming

2011, 1 (1): 26-31.

Abstract ( 5931 )

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Objective: To observe the effects of ZY-1, a nicotine analogue, on in vitro receptor binding and β-amyloid (Aβ) production and in vivo transgenic Alzheimer’s disease mice. Methods: Receptor binding assay was carried out in SH-EP1-α4β2 nAChR cell line highly expressing α4β2 nAChR; Western blotting was used to detect the effects of ZY-1 on amyloid protein production in the SH-EP1-α4β2 nAChR cell line transfected with APP695 plasmid; and effects of ZY-1 on learning and memory were measured in B6C3-Tg（APPswe, PSEN1dE9）85Dbo/J mice. Results: 1. Receptor binding assay showed that Ki is 21.5 n mol•L-1, suggesting specific binding of ZY-1 to α4β2 nAChR; 2. ZY-1(1 μmol•L-1) reduced intracellular production of Aβ; 3. Middle (0.50 mg•kg-1) and high dose (0.75 mg•kg-1) of ZY-1 improved learning and memory of transgenic mice in Morris water maze and platform test. Conclusion: ZY-1 might be of potential to be an AD therapeutic agent, but the druggability remains to be further confirmed.

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Involvement of Kir6.2-composing ATP-sensitive potassium channels in phencyclidine-induced negative symptoms of schizophrenia

DAI Ting, CHEN Zhong-Guo, DUAN Lei, DING Jian-Hua, FAN Yi, HU Gang

2011, 1 (1): 31-38.

Abstract ( 5255 )

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Objective: ATP-sensitive potassium (K-ATP) channels are crucial for dopaminergic and glutamatergic transmission，which has been demonstrated to be the important feature of pathophysiology in schizophrenia. We studied the effects of K-ATP channels on the phencyclidine (PCP)-induced changes in the forced swimming test (FST，an animal model of negative symptoms of schizophrenia)，using mice with knockout of the Kir6.2 (a pore-forming subunit of neuronal K-ATP channel). Methods:After repeated PCP (10 mg·kg^-1，i.p. daily for 14 days) administration，The FST were used to assess depression-related behaviors associated with negative symptoms. Striatal dopamine and dopamine turnover were measured by high pressure liquid chromatography with electrochemical detection. Cell proliferation in the subgranular zone (SGZ) was determined by bromodeoxyuridine immunohistochemistry. The expressions of total Akt and phosphorylation Akt were evaluated using Western blot. Results: After repeated PCP administration，the enhancement of immobility induced by the FST was attenuated in Kir6.2 knockout mice. Meanwhile，Kir6.2 knockout decreased striatal dopamine turnover，whereas wild-type mice exhibited an augmentation in response to PCP treatment. Furthermore，Kir6.2 knockout could prevent the inhibition of neural stem cell proliferation in the SGZ and suppressed the PCP-induced phosphorylation of Akt^Ser473. Conclusion: These results suggest a possible implication of Kir6.2-composing K-ATP channels dysfunction in the pathogenesis of negative symptoms associated with schizophrenia.

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Research on the block of cell cycle and apoptosis induction on glioma cell line U251 of a novel HDAC inhibitor named DWP0016

JIN Hui, LIU Li-Feng, LIANG Lei, DENG Wei-Ping, LIU Jian-Wen

2011, 1 (1): 38-45.

Abstract ( 4352 )

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Objective: Examine the effects of a novel HDAC inhibitor named DWP0016 on glioma cell line U251 and explore the molecular mechanisms underlying the cell cycle arrest and apoptosis induction. Methods: The anti-proliferative effects of DWP0016 were detected by MTT assays and the effects on the cell cycle arrest and apoptosis induction were examined by flow cytometry. The regulations on mRNA level of P21 and P53 were measured by Real-time PCR assay; the protein expressions of acetylation-H3，P21，P53，Pi3K，p-Pi3K，Akt and p-Akt were detected by Western blotting and measured by densitometry. Results: DWP0016 was found to inhibit the proliferation of U251 cell line at a much lower concentration（IC₅₀=0.531μmol· L^-1) than Oxaliplatin (IC₅₀=4.792μmol·L^-1). DWP0016 induced G₁ phase cell cycle arrest and apoptosis（P<0.05）while the acetylation of histone H3 was significantly up-regulated. The mRNA level and protein expression of tumor suppressor P21，P53 were up regulated whereas the protein expression of phosphorylation of Pi3K and Akt were down regulated. Conclusion: DWP0016 could effectively inhibit the proliferation of glioma cell line U251 and induce cell cycle arrest and apoptosis in U251 cells. The molecular mechanisms might relate to activate tumor suppressor P21，P53 and inhibit Pi3K/Akt cell signal pathway. All the results suggested that DWP0016 was a potent compound to be developed as an anti-glioma agent for clinic application in the future.

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Establishment about several anti-oxidant models of microplate quantification suitable for drug-screening and their stability testing

ZHANG Dan-shen

2011, 1 (1): 45-51.

Abstract ( 4974 )

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Objective: To establish a series of anti-oxidant models of microplate quantification suitable for the drug screening. Methods: By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector，the anti-oxidant microplate quantification experiment modles of anti-DPPH oxidation activity，elimination hydroxy free radical，reduction ability determination，and lipid peroxidation were established. Results: All of the anti-oxidant microplate quantification experiment modles had more common characteristices of the good stability，the fewer reagent types and quantity，simple operation and step，the good repeatability，and so on. Relatively speaking，the two anti-oxidant modles of anti-DPPH oxidation activity，reduction ability determination had more advantage in these experiment modles，and had more suitable for initially screening experiment of the anti-oxidant drug as basic research. Conclusion: These four modles of the anti-oxidant microplate quantification may be widely applied in anti-drug screening，especially high-throughput screening research.

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Cultivation and identification of neural stem cells cultivated by the methods of neurospheres and adherent monolayer cells

HAO Jun-Rong, ZHANG Li, QIN Zheng-Hong

2011, 1 (1): 51-55.

Abstract ( 5968 )

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Objective:AIM To explore two methods of culturing mouse embryo neural stem cells in vitro and identification of their differentiation． Methods: The neural stem cells were derived from the cortex tissue of mouse embryo at gestation day of 14～16 with two methods: culturing of neurosphere in floating and culturing of monolayer adherent cells. To observe the development of the neural stem cell，the expression of the stem cell marker Nestin and the neural marker MAP-2 and the glial marker GFAP was examined with immunofluorescence. Results: The cultured neural stem cells proliferated．The maintenance of stem cell property was preserved with both methods as revealed by Nestin immunofluorescence. These stem cells are able to differentiate into glial (GFAP positive) and neuronal lineage (MAP-2positive). Conclusion: With the help of growth factors and serum-free technique，the mouse neural stem cells cultivated by methods of neural stem cell spheres and adherent monolayer cells，have potential of proliferation and differentiation.

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Behavioral Pharmacology: History and Current Status

LI Jun-xu

2011, 1 (1): 55-64.

Abstract ( 6360 )

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As a branch of pharmacology，behavioral pharmacology has been stepping into its 50s. Since the publication of the hallmark paper of this field in 1955，behavioral pharmacology has seen a steady，vibrant and healthy development. This review traced back into 1950s，summarizing the major events during the history of behavioral pharmacology，and discuss the current status in this field. The paper also discussed several issues related to behavioral pharmacology research.

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