The Mediation Of Trisialoganglioside 1b In The Neuritogenetic Stimulatory Effect Of Botulinum Neurotoxin Serotype-A Heavy Chain In Neuro-2a Cell Line

Objective:To determine whether Neuro-2a cell line that can be used as a model for the study of botulinum neurotoxin serotype-A heavy chain(Bo NT/A HC) in vitro.To observe the effects of mino-doses Bo NT/A HC on neurites outgrowth of neuro-2a cells and exploring the mechanisms of its neuritogenetic role as well.To observe the intervene effect of GT1 b on neuritogenetic action of Bo NT/A HC when increasing its expression by exogenous adding GT1 b into cultures or inhibiting its function by adding TVL into culture medium.Methods:1. Synaptic vesicle protein 2(SV2) and trisialoganglioside 1b(GT1b) on the membrane of Neuro-2a cells were detected by immunofluorescence staining and laser confocal microscopy. Briefly, Neuro-2a cells after growing several days in plates were fixed by 4% paraformaldehyde, penetrated by 0.1% Triton X-100, blocking with 10%normal sera, incubated with either SV2 or GT1 b primary antibodies, and then followed with the secondary antibodies incubation which were conjugated with fluorescent dyes in dark environment. The cells were observed under laser confocal microscopy, images were obtained at the same time.2. The neurites outgrowth after exposure of Bo NT/A HC was assessed by measuring the length of neuronal processes, the number of neurites and the percentage of cells with neurites to total cells on image based on phase-contrast microscope. Summarily, adding different concentrations(0.01, 0.1, 1, 10nmol/L) of Bo NT/A HC into cell culture supernatant after 4 hours of cell plating. As a control, the equals of DMEM was applied.With different period of Bo NT/A HC treatment, the cells and the neuronal processes were observed under phase-contrast microscope and the 4-5 images were taken randomly in one well(8wells/group). The length and number of neuronal processes, and the percentage of cells with neurites to total cell number on image were measured.3. The phosphorylation of intracellular signal proteins, including Akt/PKB and ERK1/2 after different time of Bo NT/A HC treatment were assessed using In Cell Western and regular SDS-PAGE and Western Blot. 1) In Cell Western: the cells were fixed,penetrated, primary and secondary antibody incubation like immunofluorescent staining,and then phosphor Akt and phosphor-ERK1/2 were detected by the two-color infrared laser imaging system supported by LI-COR Odyssey In Cell Western is one of the high sensitive methods to detected both function and location of target protein at one time. 2) Regular SDS-PAGE and WB: whole cell protein was extracted by RIPE buffer, appropriate protein was uploaded onto SDS-PAGE. Afetr transferring of protein onto PVDF membrane, either anti-phospho-Akt or anti-phospho-ERK1/2 was probed, protein bands were visualized by ECL.4. For the intervening effect of GT1 b on the neuritogenetic role of Bo NT/A HC,exogenous GT1 b or Triticum vulgaris Lectin(TVL, lectin) was added into cell cultures when cells plating, which was presumed to increase the membranous content of GT1 b or decrease the GT1 b function. the length, number of neuronal processes after following Bo NT/A HC treatment was measuredunder phase contrast microscope. Meanwhile, the phosphorylation of Akt and ERK1/2 were also be evaluated based on SDS-PAGE and Western Blot as the same as the method-3.Results:1. Both the hign affinity SV2 and low affinity GT1 b receptors for Bo NT/A HC are expressed on the membrane of neuro-2a cells. The results indicated that neuro-2a cell line can be used as one of the cell model for the study of Bo NT/A and Bo NT/A HC in vitro.2. Bo NT/A HC promote the outgrowth of neuro-2a cell neuronal processes. After exposure to mino-doses of Bo NT/A HC with different time, the length and number ofneurites, the percentage of cell with processes to total cells on images are increased compared to those in controls. The stimulatory role of Bo NT/A HC in nurites outgrowth showed a dose-linear response. However, when the concentration of Bo NT/A HC reached to 10 nmol/L, the its promoting effect on neurites growth attenuated obviously..3. The results of In Cell Western and regular SDS-PAGE/WB displayed that the phosphoylation of Akt/PKB and ERK1/2 increased while adding 0.1nmol/L of Bo NT/A HC into cell cultures. The peak of phospho-Akt and phospho-ERK appeared at 1 hour and2 hours respectively after Bo NT/A HC treatment. The result hinted that increased phosphorylation of either Akt or ERK1/2 was possibly involved in the mechanisms of Bo NT/A HC to stimulating neurites outgrowth in vitro.4. Exogenous adding GT1 b into cell cultures before 4 hour of 0.1 nmol/L of Bo NT/A HC treatment accelerated the phosphorylation of both Akt and ERK1/2, and brought the peak of phosphor-Akt and ERK1/2 forward to 30 min and 90 min. Meanwhile,when TVL was added into cell cultures before Bo NT/A HC, the phosphorylation of Akt and ERK1/2 were decreased than Bo NT/A HC only group. Also, the neurites growth was inhibited by TVL pre-treatment before Bo NT/A HC, including the length of neurites, the number of processes, and the percentage of cells with neurites. All together, the data gave a clue that the phosphorylation of Akt and ERK1/2 are the major intracellualar signals involved in the mechanism of Bo NT/A HC to promoting neurites outgrowth.Conclusion:Neuro-2a cells express the essential machineries of Bo NT/A HC actions, including the high affinity binding receptor(SV2) and low affinity binding receptor(GT1b). The cell line can be used as one of the cell models for Bo NT/A HC study in vitro.Mino-doses of Bo NT/A HC has the stimulatory effect on the neurites outgrowth of Neuro-2a cells in vitro. The increase of membranous GT1 b magnify the effect of Bo NT/A HC on neuritogenesis vice versa.Bo NT/A HC accelerate the phosphorylation of Akt and ERK1/2. The activation of Akt and ERK1/2 are possibly contributed to the mechanism of Bo NT/A HC in promoting theregeneration of neuronal processes.