Aggravated effects of coexisting marginal thiamine deficits and zinc excess on SN56 neuronal cells.

Objectives: Zinc excitotoxicity and thiamine pyrophosphate deficiency (TD) are known pathogenic signals contributing to mechanism of different encephalopathies through inhibition of enzymes responsible for energy metabolism such as pyruvate dehydrogenase, aconitase or ketoglutarate dehydrogenase. The aim of this work was to investigate whether subclinical Zn excess and TD, frequent in aging brain, may combine yielding overt neuronal impairment.Results: Clonal SN56 cholinergic neuronal cells of septal origin were used as the model of brain cholinergic neurons, which are particularly susceptible to neurodegeneration in the course of Alzheimer's disease, hypoxia and other dementia-linked brain pathologies. Neither subtoxic concentration of Zn (0.10 mM) nor mild 20-25% TD deficits alone caused significant negative changes in cultured cholinergic neurons viability and their acetyl-CoA/acetylcholine metabolism. However, cells with mild TD accumulated Zn in excess, which impaired their energy metabolism causing a loss of neurons viability and their function as neurotransmitters. These negative effects of Zn were aggravated by amprolium which is an inhibitor of thiamine intracellular transport.Conclusion: Our data indicate that TD may amplify otherwise non-harmful border-line Zn excitotoxic signals yielding progress of neurodegeneration.

Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3ß enzyme, ß-amyloid and tau protein levels.

Cadmium is a neurotoxic compound which induces cognitive alterations similar to those produced by Alzheimer's disease (AD). However, the mechanism through which cadmium induces this effect remains unknown. In this regard, we described in a previous work that cadmium blocks cholinergic transmission and induces a more pronounced cell death on cholinergic neurons from basal forebrain which is partially mediated by AChE overexpression. Degeneration of basal forebrain cholinergic neurons, as happens in AD, results in memory deficits attributable to the loss of cholinergic modulation of hippocampal synaptic circuits. Moreover, cadmium has been described to activate GSK-3ß, induce Aß protein production and tau filament formation, which have been related to a selective loss of basal forebrain cholinergic neurons and development of AD. The present study is aimed at researching the mechanisms of cell death induced by cadmium on basal forebrain cholinergic neurons. For this purpose, we evaluated, in SN56 cholinergic mourine septal cell line from basal forebrain region, the cadmium toxic effects on neuronal viability through muscarinic M1 receptor, AChE splice variants, GSK-3ß enzyme, Aß and tau proteins. This study proves that cadmium induces cell death on cholinergic neurons through blockade of M1 receptor, overexpression of AChE-S and GSK-3ß, down-regulation of AChE-R and increase in Aß and total and phosphorylated tau protein levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on cholinergic neurons and suggest that cadmium could mediate these mechanisms by M1R blockade through AChE splices altered expression.

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells.

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways.

Ubiquitin C-terminal hydrolase L1 interacts with choline transporter in cholinergic cells.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme, which is highly expressed in neuronal cells. Previous studies have indicated that UCHL1 is involved in cognitive function, neurodegenerative diseases, and neuromuscular junction development. Acetylcholine (Ach) is a critical neurotransmitter in these functions. Yet, the effect of UCHL1 on the cholinergic system has not been reported. In this study, using a cholinergic neuronal cell line, SN56, as an invitro model, we detected the physical interaction of UCHL1 and high affinity choline transporter (CHT), which is a key protein regulating Ach re-synthesis. Reduction of UCHL1 by siRNA gene knockdown significantly increased poly-ubiquitinated CHT and decreased native CHT protein level, but did not affect CHT mRNA expression. Biotinylation assay showed that UCHL1 is localized only in the cytosol of the cells and that the gene knockdown of UCHL1 significantly reduced cytosolic CHT but had no significant effect on membrane CHT level. These data provide novel and potentially important evidence that UCHL1 may play a role in the regulation of cholinergic function by affecting CHT ubiquitination and degradation.

Effect of low concentration Aβ1-42 monomer/oligomers and CORM-2 on livability of SN56 cells / 中华行为医学与脑科学杂志

Objective To observe the effect of low concentration Aβ1-42 monomer/oligomers and CORM-2 in different concentration on livability of SN56 cells. Methods SN56 cells were cultured in the 96-well plate with uniform concentration, and were divided into control group, Aβ1-42 group, Aβ1-42 + CORM-2 50μM group, and Aβ1-42 + CORM-2 100 μM group. Three lines of cells in Aβ1-42 group were cultured in the surroundings of 10nM,100nM and 1 μM Aβ1-42monomer/oligomers, respectively. Aβ1-42 + CORM-2 50μM group and Aβ1-42 + CORM-2 100μM group had the same culture condition as group Aβ1-42 ,except contain 50μM, and 100μM CORM-2, respectively. Control group didnt have any effect factor. Three days later,the livability of different groups was compared with MTT method. Results The livability of group Aβ1-42 with the increasing concentration of Aβ1-42 was (79.73 ±0.94)% ,(67.99 ±0.79)% ,(60.42 ±0.62)% , respectively. The higher the concentration of Aβ1-42 was,the lower the livability of SN56 cell was. The livability of group Aβ1-42 + CORM-2 50μM/100μM was( 75.15±0.096)%,(63.20 ±0.17)%, (55.33 ±0.19)%; (73.20 ±0.27)%, (64.34 ±0.11 )%, (54.17 ±0.12)% , respectively. Both were lower than group Aβ1-42. And different CORM-2 concentration had discrepancy in the ability of decreasing the cell livability. Conclusion Low concentration of Aβ1-42 can reduce the livability of SN56 cells, and higher concentration has more significant effect; CORM-2 in different concentration both can decrease the livability of SN56 cells,and there is a discrepancy in the intensity.