α7 neuronal nicotinic acetylcholine receptor/α7nAChR

The reduction in cell viability caused by Aβ25-35 was prevented by inhibition with 5 and 10 μM methylaconitine citrate (MLA). After being exposed to methylaconitine citrate (2.5, 5, 10, 20 μM), there was no change in cell viability. Methylaconitine citrate inhibits the increase of LC3-II levels caused by aβ25-35 therapy. In SH-SY5Y cells, methylaconitine citrate also prevents Aβ-induced autophagosome accumulation. Methylaconitine citrate therapy resulted in a decrease in MDC-labeled vacuoles, as demonstrated by flow cytometry [1].

Intraperitoneal injection of methylaconitine citrate (MLA) (6 mg/kg) alone did not stimulate climbing behavior. Using citrate methylaconitine can greatly reduce methamphetamine (METH)-induced climbing behavior, with roughly 50% of the transcriptional locus. Methylaconitine citrate does not modify basal locomotor activity or METH-induced hyperlocomotion. In the model replicated with methylaconitine citrate (250±43 fmol/mg, n=7), METH-induced total depletion of dopamine neurons was fully prevented. Methylaconitine citrate was omitted due to its direct effect on body temperature. Methylaconitine citrate did not change basal body temperature (37.0±0.5°C, n=5) or lessen METH-induced hyperthermia (38.2±0.4 °C, n=6, MLA+METH group, ns vs. METH group)[1].

Cell culture and drug treatment [1]
The human neuroblastoma cell line SH-SY5Y was cultured in RPMI-1640 supplemented with 10% FBS at 37°C. Cells at 60–70% confluence were treated with concentrations of Aβ25–35, methyllycaconitine (MLA)  , rapamycin or Aβ25–35 with or without MLA. Control cells were cultured under normal conditions.

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Cell viability assay [1]
Cells were plated in 96-well plates containing complete medium and cultured for 24 h. Then cells were treated with compounds at the indicated concentrations for specified times. After drug treatment, cell viability was measured by MTT assay. Briefly, 10 µl of the MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37°C. After removing the supernatant, 100 µL DMSO was added into each well. The absorbance was measured at 570 nm with a microplate reader. All experiments were repeated 3 times.

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Monodansylcadaverine staining (MDC) [1]
To detect autophagy in SH-SY5Y cells, cells were plated on coverslips in 6-well plates. After 24 h, cells were treated with compounds at the indicated concentrations, fixed with 4% paraformaldehyde for 15 min at room temperature, then stained with MDC (1 µg/mL in phosphate buffered saline [PBS]) at 37°C in the dark, and observed immediately with fluorescence microscopy. To quantify the number of cells with acidic vesicles, cells were seeded into 6-well plates and cultured overnight, then stained with 1 µg/mL MDC at 37°C for 15 min. After incubation, cells were washed with PBS and removed with trypsin-EDTA, resuspended, and analyzed by flow cytometry.

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Apoptosis detection by Hoechst 33258 staining [1]
Hoechst 33258 staining was used to detect apoptotic nuclei. Cells were plated in 24-well plates. After drug treatment, cells were stained with 10 µg/mL Hoechst 33258 for 15 min. After being gently washed with PBS once, cells were observed and photographed under a fluorescence microscopy.

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Apoptosis detection by flow cytometry [1]
Cells were plated in six-well plates and incubated for 24 h, exposed to desired concentrations of Aβ25–35 for 24 h, then harvested by trypsinization, and washed twice in PBS. After staining with a combination of AnnexinV/fluorescein isothiocyanate (FITC) and propidium iodide (PI), cells were immediately analyzed by flow cytometry.

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Immunocytochemistry [1]
Immunocytochemical staining was performed as described. Briefly, cells were seeded on cover slips over night. After drug treatment, cells were fixed for 30 min in 4% paraformaldehyde. After blocking, cells were incubated with primary antibody (anti-LC3) overnight at 4°C. After being washed with PBS, cells were incubated with PE-labeled secondary antibodies (1∶500; Invitrogen) at room temperature for 1 h, then counterstained with 4–6-diamidino-2-phenylindole (DAPI) for 10 minutes. Images were obtained by laser scanning microscopy.

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Western blot analysis [1]
After treatment, cells were collected and washed gently with PBS twice, then lysed with protein lysis buffer (1% SDS in 25 mM Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 1% cocktail protease inhibitor). Samples were centrifuged at 12,000 g for 15 min at 4°C, and supernatants were collected. The concentration of the protein was determined by Coomassie brilliant blue protein assay. Equal amounts of protein (50 µg) were resolved by SDS-PAGE and transferred onto nitrocellulose membrane, which was blocked with 5% non-fat dry milk in TBS for 1 h at room temperature, and then incubated with primary antibodies (1∶1000) overnight at 4°C. Membranes were washed and treated with appropriate secondary antibodies for 1 h at room temperature. The immunocomplexes were detected with an enhanced chemiluminescence plus kit.

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Electron microscopy (EM) [1]
Cells were postfixed with 2% osmium tetroxide, followed by an increasing gradient dehydration step with ethanol and propylene oxide. Cells were then embedded in LX-112 medium (Ladd), and sections were cut ultrathin (90 nm), placed on uncoated copper grids, and stained with 0.2% lead citrate and 1% uranyl acetate. Images were examined under a JEOL-1010 electron microscope (JEOL) at 80 kV.

In a previous study, we demonstrated that in rat striatal synaptosomes, methamphetamine (METH)-induced reactive oxygen species (ROS) production was prevented by methyllycaconitine (MLA), a specific antagonist of alpha7 neuronal nicotinic acetylcholine receptors (alpha7 nAChR). The aim of this study was to test the influence of MLA on acute METH effects and neurotoxicity in mice, using both in vivo and in vitro models. MLA inhibited METH-induced climbing behavior by 50%. Acute effects after 30-min preincubation with 1 microM METH also included a decrease in striatal synaptosome dopamine (DA) uptake, which was prevented by MLA. METH-induced neurotoxicity was assessed in vivo in terms of loss of striatal dopaminergic terminals (73%) and of tyrosine hydroxylase levels (by 90%) at 72 h post-treatment, which was significantly attenuated by MLA. Microglial activation [measured as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide binding] was also present at 24 h post-treatment and was fully prevented by MLA, tending to confirm its neuroprotective activity. MLA had no effect on METH-induced hyperthermia. Additionally, flow cytometry assays showed that METH-induced ROS generation occurs inside synaptosomes from mouse striatum. This effect implied release of vesicular DA and was calcium-, neuronal nitric-oxide synthase-, and protein kinase C-dependent. MLA and alpha-bungarotoxin, but not dihydro-beta-erythroidine (an antagonist that blocks nAChR-containing beta2 subunits), fully prevented METH-induced ROS production without affecting vesicular DA uptake. The importance of this study lies not only in the neuroprotective effect elicited by the blockade of the alpha7 nicotinic receptors by MLA but also in that it proposes a new mechanism with which to study METH-induced acute and long-term effects. [2]

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Methyllycaconitine (MLA) is reported to be a selective antagonist for the nicotinic acetylcholine receptor alpha7 subtype and has been found in animal behavioral studies to reduce nicotine self-administration and attenuate nicotine withdrawal symptoms. While MLA crosses the blood-brain barrier (BBB), no studies have assessed brain uptake in animals subjected to chronic nicotine exposure. Given that chronic nicotine administration has been reported to alter BBB parameters that may affect the kinetic BBB passage of MLA, we evaluated MLA brain uptake in naive and S-(-)nicotine-exposed rats (4.5 mg/kg/day for 28 days; osmotic minipumps) using in situ rat brain perfusions. Our results demonstrate that in situ(3)H-MLA brain uptake rates in naive animals approximate to intravenous kinetic data (K(in), 3.24 +/- 0.71 x 10(-4) mL/s/g). However, 28-day nicotine exposure diminished (3)H-MLA brain uptake by approximately 60% (K(in), 1.29 +/- 0.4 x 10(-4) mL/s/g). This reduction was not related to nicotine-induced (3)H-MLA brain efflux or BBB transport alterations. Similar experiments also demonstrated that the passive permeation of (14)C-thiourea was diminished approximately 24% after chronic nicotine exposure. Therefore, it appears that chronic nicotine exposure diminishes the blood-brain passive diffusion of compounds with very low extraction rates (i.e. permeability-limited compounds). These findings imply that the pharmacokinetics of neuropharmaceutical agents that are permeability limited may need to be re-evaluated in individuals exposed to nicotine. [3]

[1]. Methyllycaconitine alleviates amyloid-β peptides-induced cytotoxicity in SH-SY5Y cells. PLoS One. 2014 Oct 31;9(10):e111536.

[2]. Methyllycaconitine prevents methamphetamine-induced effects in mouse striatum: involvement of alpha7 nicotinic receptors. J Pharmacol Exp Ther. 2005 Nov;315(2):658-67.

[3]. Chronic nicotine exposure alters blood-brain barrier permeability and diminishes brain uptake of methyllycaconitine. J Neurochem. 2005 Jul;94(1):37-44.

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer's disease. In this study, we investigated whether Aβ25-35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ25-35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD. [1]

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Several signaling pathways regulate the autophagy process with the mTOR pathway playing a key role. We paid attention to the downstream targets. 4E-binding protein 1 and p70S6K are directly phosphorylated by activated mTORC1 to negatively regulate autophagy. In our previous study, we showed that Aβ could induce autophagy in PC12 cells through an mTOR-dependent pathway. Here, we also found that Aβ induced autophagy in SH-SY5Y cells via mTOR signaling as evidenced by the downregulation of phosphorylated p70S6K levels. Moreover, Aβ-decreased p70S6K phosphorylation was attenuated by administration of MLA. The upregulation of mTOR signaling by MLA may inhibit Aβ-induced autophagy and contribute to its protective effect against Aβ-related cytotoxicity. In conclusion, we showed that Aβ25–35 inhibited SH-SY5Y cell growth and induced autophagy. Furthermore, MLA could provide neuroprotection against the cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy via an mTOR pathway. MLA may be a safe and promising drug candidate for treatment of AD.[1]

C43H58N2O17

874.923834323883

874.373

351344-10-0

45073440

White to yellow solid powder

6

18

15

62

1610

14

CCN1C[C@@]2(CC[C@@H]([C@@]34[C@@H]2[C@@H]([C@@]([C@H]31)([C@]5(C[C@@H]([C@H]6C[C@@H]4[C@@H]5[C@H]6OC)OC)O)O)OC)OC)COC(=O)C7=CC=CC=C7N8C(=O)C[C@@H](C8=O)C.C(C(=O)O)C(CC(=O)O)(C(=O)O)O

INBLZNJHDLEWPS-OULUNZSJSA-N

InChI=1S/C37H50N2O10.C6H8O7/c1-7-38-17-34(18-49-32(42)20-10-8-9-11-23(20)39-26(40)14-19(2)31(39)41)13-12-25(46-4)36-22-15-21-24(45-3)16-35(43,27(22)28(21)47-5)37(44,33(36)38)30(48-6)29(34)36;7-3(8)1-6(13,5(11)12)2-4(9)10/h8-11,19,21-22,24-25,27-30,33,43-44H,7,12-18H2,1-6H3;13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)/t19-,21+,22+,24-,25-,27+,28-,29+,30-,33-,34-,35+,36-,37+;/m0./s1

[(1S,2R,3R,4S,5R,6S,8R,9S,10S,13S,16S,17R,18S)-11-ethyl-8,9-dihydroxy-4,6,16,18-tetramethoxy-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecan-13-yl]methyl 2-[(3S)-3-methyl-2,5-dioxopyrrolidin-1-yl]benzoate;2-hydroxypropane-1,2,3-tricarboxylic acid

Methyllycaconitine (citrate); 351344-10-0; 20-ethyl-1alpha,6beta,14alpha,16beta-tetramethoxy-4-[[[2-[(3S)-3-methyl-2,5-dioxo-1-pyrrolidinyl]benzoyl]oxy]methyl]-aconitane-7,8-diol,2-hydroxy-1,2,3-propanetricarboxylate; [(1S,2R,3R,4S,5R,6S,8R,9S,10S,13S,16S,17R,18S)-11-ethyl-8,9-dihydroxy-4,6,16,18-tetramethoxy-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecan-13-yl]methyl 2-[(3S)-3-methyl-2,5-dioxopyrrolidin-1-yl]benzoate;2-hydroxypropane-1,2,3-tricarboxylic acid; MLA; G12422

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~142.87 mM)
H2O : ~2.18 mg/mL (~2.49 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)