Phenotype-Dependent Interactions between N -acetyl-L-Aspartate and Acetyl-CoA in Septal SN56 Cholinergic Cells Exposed to an Excess of Zinc

Abstract

Pyruvate dehydrogenase reaction utilizing glucose-derived pyruvate is an almost exclusive source of acetyl-CoA in different cell mitochondrial compartments of the brain. In neuronal mitochondria, the largest fraction of acetyl-CoA is utilized for energy production and the much smaller one for N-acetyl-L-aspartate (NAA) synthesis. Cholinergic neurons, unlike others, require additional amounts of acetyl-CoA for acetylcholine synthesis. Therefore, several neurotoxic signals, which inhibit pyruvate dehydrogenase, generate deeper shortages of acetyl-CoA and greater mortality of cholinergic neurons than noncholinergic ones. NAA is considered to be a marker of neuronal energy status in neuropathic brains. However, there is no data on putative differential fractional distribution of the acetyl-CoA pool between energy producing and NAA or acetylcholine synthesizing pathways in noncholinergic and cholinergic neurons, respectively. Therefore, the aim of this study was to investigate whether zinc-excess, a common excitotoxic signal, may evoke differential effects on the NAA metabolism in neuronal cells with low and high expression of the cholinergic phenotype. Differentiated SN56 neuronal cells, displaying a high activity of choline acetyltransferase and rates of acetylcholine synthesis, contained lower levels of acetyl-CoA and NAA, being more susceptible to ZnCl₂ exposition that the nondifferentiated SN56 or differentiated dopaminergic SHSY5Y neuronal and astroglial C6 cells. Differentiated SN56 accumulated greater amounts of Zn^2 + from extracellular space than the other ones, and displayed a stronger suppression of pyruvate dehydrogenase complex activity and acetyl-CoA, NAA, ATP, acetylcholine levels, and loss of viability. These data indicate that the acetyl-CoA synthesizing system in neurons constitutes functional unity with energy generating and NAA or acetylcholine pathways of its utilization, which are uniformly affected by neurotoxic conditions.

Keywords

acetyl-CoA ATP cholinergic neurons neurotoxicity zinc

INTRODUCTION

N-acetyl-L-aspartate (NAA) is an amino acid derivative present in high concentrations in the mammalian brain [1, 2]. Its average level in the human brain varies from 8 to 12 μmol/g of tissue [3, 4]. NAA is synthesized exclusively in neurons from acetyl-CoA and L-aspartate by acetyl-CoA: L-aspartate N-acetyltransferase (aspartate N-acetyltransferase, Asp-NAT; EC 2.3.1.17) [4 –6]. The highest relative activities of this enzyme were found in mitochondrial membranes [2 , 8]. NAA transported to oligodendrocytes serves as a source of acetyl units for lipid synthesis necessary for myelin formation in the developing brain [2 , 9–12].

Decreases in NAA levels were observed in magnetic resonance studies of brains, in a number of neurological disorders, including Alzheimer’s disease (AD), Huntington’s disease, multiple sclerosis, schizophrenia, stroke, and mechanical brain injury [2 , 13–18]. There is a speculation that the significant correlation between decreases in cerebrospinal fluid amyloid-β (Aβ) levels and NAA in AD brains reflects the disruption of their mitochondrial energy metabolism [19, 20]. Pyruvate-derived acetyl-CoA is a key precursor-substrate for both energy production and NAA synthesis in neuronal mitochondria [1 , 22]. There are also suggestions that NAA might not only be a marker of neuronal viability but also a direct indicator of acetyl-CoA availability in the mitochondrial compartment [6, 23].

The loss of cognitive functions in AD, is tightly correlated with respective decreases in the activities of pyruvate dehydrogenase complex (PDHC) and other enzymes of energy metabolism, in the affected brains areas [20]. These alterations are also accompanied by preferential, early impairment of cholinergic neurons in the brain basal ganglia and septum of AD victims [24, 25]. The unique feature of cholinergic neurons is that, unlike noncholinergic ones, they utilize additional amounts of acetyl-CoA for acetylcholine (ACh) synthesis [21, 22]. Therefore, neurodegenerative, PDHC-suppressing conditions may generate deeper, shortages of acetyl-CoA in the former [20 , 26]. In fact, different neurotoxic signals were found to suppress not only energy production but also synthesis of transmitter ACh [21 , 26]. Recent studies of old Tg2576 mice revealed the inhibition of pyruvate utilization and suppression of acetyl-CoA and NAA levels in their brains[27, 28].

However, no direct NAA-acetyl-CoA interactions in the neuronal compartment have been investigated. It is also not known whether neurotoxic inputs may differentially affect NAA synthesis in cholinergic and noncholinergic neurons.

The redistribution of Zn^2 + released from excessively stimulated gluzinergic terminals to postsynaptic neurons is one of the earliest neurotoxic events taking place in AD brains [29 –33]. In such conditions, levels of Zn^2 + in the synaptic cleft were reported to rise to concentrations as high as 0.3 mmol/L [30]. Our studies revealed that Zn overload inhibited PDHC yielding depression of acetyl-CoA, ACh, and the viability of cultured cholinergic SN56 neuronal cells[22 , 35].

Therefore, ZnCl₂ was employed here to generate an early neurotoxic signal for evoking and monitoring putative differential interactions between acetyl-CoA provision and NAA, ATP, and ACh synthesis taking place in the cells with low, high, or no expression of the cholinergic phenotype. The presented data support our hypothesis that ACh synthesis in cholinergic neurons may contribute to their preferential susceptibility to Zn-evoked neurotoxicity [23 , 34].

MATERIALS AND METHODS

Materials

Unless otherwise specified, biochemicals and cell culture growth media were obtained from Sigma–Aldrich (Poznan, Poland), the C18-RP HPLC column was from Shim-Pol (Warsaw, Poland), [L-U-¹⁴C]-aspartic acid 0.1 mCi/ml from Perkin–Elmer (Boston, MA, USA), and cell culture disposables derived from Sarstedt (Stare Babice, Poland).

Cell cultures

The cholinergic neuroblastoma SN56.B5.G4 [36] (donation from Dr. JK Blusztajn), dopaminergic neuroblastoma SHSY5Y [37], and C6 astroglioma cells (Sigma-Aldrich, Poznań, Poland) were seeded at a density of 40,000 cells/ cm² on 75 cm² Falcon vessels, grown 48 h in Dulbecco’s modified Eagle’s medium (DMEM) and differentiated with 0.5 mmol/L dibutyryl-cAMP (db-cAMP) and 0.001 mmol/L retinoic acid (RA) as described earlier [35, 38]. Then, the media were replaced by experimental DMEM without differentiating factors and without or with ZnCl₂ added, as indicated and the culture was continued for a subsequent 24 h. The cells were harvested, and used for enzyme and metabolite assays as described elsewhere [26 , 38].

Morphology and cell counts

The morphology of the control and Zn-exposed cells was assessed by inverted light microscope (Zeiss, Axiovert 25, Germany) with a digital camera (Olympus, DP10, Tokyo, Japan) at 400×magnification. The total number of cells was estimated in a Fuchs-Rosenthal hemocytometer at 200×magnification under inverted light microscope [39].

Cell viability assays

The cells viability was assessed by fractional (S)-lactate:NAD⁺ oxidoreductase (lactate dehydrogenase, LDH, EC 1.1.1.27) release method [40, 41], and by trypan blue exclusion assays [42].

Whole cell calcium and zinc quantitative assays

The harvested cells were collected by centrifugation, deproteinized with 4% HClO₄, and neutralized supernatants were used for cation assays. Whole cell Ca level was measured by arsenazo III spectrophotometric method [43]. The total Zn content in the cells was determined by the fluorimetric method using N-(6-Methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) [44]. Subcellular Zn distribution was assessed using digitonin solubilization followed by centrifugation through silica oil layer [45].

Subcellular distribution of free Zn^2 +

To examine subcellular in situ distribution of free Zn^2 +, NC/DCs were cultured on cover slips for 48 h in DMEM-10% serum medium followed by 30 min exposition to ZnCl₂. Intracellular localization of Zn^2 + was assessed after staining with TSQ, using inverted fluorescence microscope (Olympus IX83, Tokyo, Japan) [46].

N-acetyl-L-aspartate assay

The modified HPLC method was employed for NAA determinations [48].

Acetyl-CoA assay

The deproteinized extracts of whole cells were treated with maleic anhydride solution in ethyl ether for 2 h to remove free CoA and subjected to cycling reaction using phosphotransacetylase coupled with citrate synthase, as described elsewhere [49].

ATP assay

ATP levels were assayed by the luciferin –luciferase luminescent method [50].

Acetylcholine assay

Cellular ACh content was assessed using acetylcholinesterase-choline oxidase, coupled with peroxidase –luminol, detection system [51].

Aspartate N-acetyltransferase activity assay

Asp-NAT activity was assessed by the modified radiometric method [52]. The assay medium in a final volume of 0.2 mL contained: 20 mmol/L potassium phosphate, 20 mmol/L Na-HEPES (pH 7.1), 1 mmol/L MgCl₂, 0.05 mmol/L L-aspartate, 0.001 mmol/L L-[U-¹⁴C]-aspartate (0.004 μCi/sample), 0.2 mmol/L acetyl-CoA and 0.2% Triton X-100 solubilized cell extract (0.1 mg protein per sample). After 15 min incubation at 30°C reaction was stopped by heating at 80°C for 5 min, and NAA radioactivity was measured after isolation by solid phase extraction [52].

Aspartate N-acetyltransferase protein quantification

Quantification of Asp-NAT protein was carried out using commercially available ELISA kits (Cusabio Biotech, Wuhan, China) according to the manufacturer’s instructions.

Pyruvate dehydrogenase complex activity assay

PDHC activity was assayed measuring acetyl-CoA formed by the citrate synthase coupled method followed by citrate quantitation using citratelyase [53].

Choline acetyltransferase activity assay

Choline acetyltransferase (acetyl-CoA: choline-O-acetyltransferase, ChAT, EC 2.3.1.6) activity was assessed by the radiometric method using[1-14C]acetyl-CoA and choline as substrates [54].

Protein assay

Protein was assayed by the method of Bradford [55] with human immunoglobulin as a standard.

Statistics

Statistical analyses were carried out by the Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons. For comparisons between the two groups, the Mann-Whitney U test was used. p < 0.05 was considered statistically significant.

RESULTS

Effect of differentiation and ZnCl₂ on SN56 cell morphology

The SN56 cells grown for 48 h in a differentiating medium displayed more mature phenotype (SN56DC) than the nondifferentiated ones (SN56NC), forming numerous extensions and synapse-like connections (Fig. 1A, C). In SN56DC, the addition of 0.15 mmol/L ZnCl₂ to the medium caused a distinct loss of intercellular connections, vacuolar degeneration in the cytoplasm, and a decrease in cell density (Fig. 1D). In the same conditions, SN56NC displayed much smaller alterations (Fig. 1B).

Extracellular ZnCl₂ up to 0.1 mmol/L altered neither the morphology (not shown) nor any metabolic parameter of NC and DC due to its binding with the serum proteins present in the culture media (Figs. 4–7) [35, 56]. Therefore, kinetic analyses for the concentration-dependent effects of ZnCl₂ on the SN56 cells were performed after subtracting 0.1 mmol/L value from its concentration added to the medium (Figs. 2A, 4–6).

Effect of extracellular ZnCl₂ on its intracellular distribution and Ca contents in SN56

In the control conditions, the intracellular levels of total Zn ([Zn_in]) were similar in neuronal SN56DC and NC, being equal to about 1.8 nmol/mg protein (Fig. 2A). Extracellular ZnCl₂ in concentrations higher than 0.1 mmol/L, caused progressive elevation of its intracellular accumulation (Fig. 2A). Maximal accumulation of Zn in SN56DC, was estimated to be 2 times higher than in SN56NC (Table 1, Fig. 2B). In control conditions, mitochondrial fraction of Zn constituted about 1% of its whole SN56 cell content (Fig. 2C). The addition of 0.15 mmol/L ZnCl₂ brought about its 3.9–and 5.7–fold increases in cytoplasmic, and 59–and 99–fold elevations in mitochondrial levels in the SN56NC and DC, respectively (Fig. 2C).

Extracellular 0.15 mmol/L ZnCl₂ increased the content of intracellular Ca, from about 24 nmol/mg protein in the controls to 33.7 and 43.4 nmol/mg of protein in the SN56NC and SN56DC, respectively (Fig. 2D).

TSQ histochemistry of the control SN56NC/DC, displayed no significant Zn^2 +-TSQ complex fluorescence (Fig. 3A, B). A short, 30– min exposure of SN56NC/DC to 0.1–0.2 mmol/L ZnCl₂ in a serum-free medium resulted in its accumulation in granular structures within the cytoplasmic compartment (Fig. 3C-F). At either ZnCl₂ concentration, the fluorescence was brighter in the SN56DC than in the corresponding SN56NC (Figs. 3C, D, E, F).

Effect of ZnCl₂ on SN56 cell viability

Extracellular ZnCl₂ (above 0.10 mmol/L) caused concentration-dependent decreases of viable cell fraction, measured with trypan blue exclusion and LDH release assays (Fig. 4A,C). These alterations were always more distinct in the SN56DC than in the SN56NC (Fig. 4A,C) and also evidenced by the lower [IC₅₀] and higher linear regression slopes respective values for extracellular ZnCl₂ (Table 1).

On the other hand, the SN56NC and SN56DC viability data, when plotted against respective [Zn_in], appeared to be fully superimposable, yielding a single correlation plots (Fig. 4B, D). Note that points of the plot presenting highest mortality and [Zn_in] corresponded to the SN56DC (Fig. 4B, D).

Effect of ZnCl₂ on SN56 PDHC activity, and acetyl-CoA/ATP levels

PDHC is the enzyme complex providing the bulk of acetyl-CoA for ACh and NAA synthesis in neurons [1 , 57]. The levels of extracellular ZnCl₂ above 0.10 mmol/L caused concentration-dependent suppression of PDHC activity, and acetyl-CoA/ATP contents in SN56 cells. These effects were stronger in SN56DC than in SN56NC, as evidenced by deeper suppressions of these parameters by the same concentrations of extracellular ZnCl₂ and lower [IC₅₀] and higher slopes of linear regression values in the former (Fig. 5A, C, E, Table 1). On the other hand, PDHC activities, as well as acetyl-CoA and ATP levels in SN56 cells of both phenotypes, plotted against [Zn_in] were fully super imposable, forming single straight line correlation plots (Fig. 5B, D, F). Note that the upper segments of these plots that are formed by the values of PDHC, acetyl-CoA and ATP against [Zn_in] belong to the SN56DC (Fig. 5B, D, F).

Effects of ZnCl₂ on SN56 parameters of N-acetyl-L-aspartate metabolism

In the control conditions, SN56DC displayed 26% higher level Asp-NAT protein, similar activity ofAsp-NAT but 24% lower content of NAA than SN56NC, respectively (Fig. 6A-C). ZnCl₂ in 0.15 mmol/L concentration brought about moderate 33% decreases in Asp-NAT protein content, in both cell phenotypes (Fig. 6A). In the same conditions Asp-NAT activity was suppressed by 40% and 60% in SN56NC and DC, respectively (Fig. 6B). In the SN56NC, 0.11 mmol/L ZnCl₂ resulted in no significant change, whereas in the SN56DC it brought about a 48% decrease of the NAA level (Fig. 6C). Higher concentrations of added ZnCl₂ caused further suppressions of NAA intracellular levels both in the SN56DC and NC, down to 31% and 21% of the control values, respectively (Fig. 6C). The [IC₅₀] value for extracellular ZnCl₂ against NAA content in SN56DC was not significantly lower, but slope of linear regression appeared to be over two times higher than in the NC, respectively (Table 1). On the other hand, a single plot of NAA levels against [Zn_in] could be drawn from the SN56DC/NC data (Figs. 2A, 6C, D). NAA levels in Zn-treated SN56NC/DC displayed highly significant, direct correlations with PDHC activities, and acetyl-CoA levels (Fig. 6E, F).

Effects of ZnCl₂ on cholinergic markers

The differentiation caused more than two-fold increases of ChAT activity and ACh content in the SN56 cells (Figs. 7A, B). The addition of 0.15 mmol/L ZnCl₂ caused 36% and 26% inhibition of ChAT activities, and non-proportionally greater, 72% and 78% decreases in ACh contents in the SN56NC and DC, respectively (Fig. 7A, B). Note that the absolute values of Zn-cholino-suppressive effects in the SN56DC were 2.3 times greater than in the SN56NC (Fig. 7). The SHSY5YDC dopaminergic neuroblastoma cells displayed very low (0.034±0.006 nmol/min/mg protein) ChAT activity, as compared with SN56DC (Fig. 7), was not suppressed by Zn (not shown). C6DC astrocytoma contained no detectable ChAT activity.

Effects of intracellular Zn on different clonal cell lines

Dopaminergic SHSY5YDC and astrocytoma C6DC required higher concentrations than SN56DC of extracellular ZnCl₂ for its intracellular accumulation to the level of 5 nmol/mg protein (Table 2). In such standardized conditions [Zn_in] exerted comparable inhibition of PDHC activities, and suppression of acetyl-CoA and NAA contents in all neuronal cell groups (Table 2). However, loss of viability of SN56NC/DC was several times greater than that of SHSY5YDC (Table 2). Also, the mortality rate of Zn-exposed C6DC, which synthesized neither NAA nor ACh, was lower than SN56, despite comparable relative suppression of acetyl-CoA content (Table 2).

DISCUSSION

SN56DC, with high expression of cholinergic phenotype, consumed greater fraction of pyruvate-derived acetyl-CoA for ACh synthesis than the SN56NC (Figs. 1, 5C, 7) [22, 38]. These relative shortages of acetyl-CoA in the SN56DC yielded lower levels of NAA and their greater susceptibility than in SN56NC to various neurotoxic conditions (Figs. 2, 4, 5C, 6C, 7B, 8) [22 , 58].

The presented data prove that the higher rate of Zn accumulation in SN56DC is an important factor contributing to their greater susceptibility to this compared to nondifferentiated cholinergic or noncholinergic neuronal or astroglial cells (Figs. 2, 4, Table 2) [22, 38]. Two times higher maximal Zn accumulation in SN56DC than in NC, may result from increased expression of NMDA receptors, ZnT3, or voltage-gated Ca channels taking place during SN56 differentiation (Table 1, Figs. 2, 8) [29 , 59–62]. Therefore, at the same concentrations, extracellular ZnCl₂ brought about greater losses of SN56DC than SN56NC viability (Table 1, Figs. 1, 4). The same mechanism could cause Ca overload in the Zn-exposed SN56DC; it could be an independent factor aggravating the Zn-mediated detrimental effects (Figs. 2D, 4, 5) [61, 62].

The [Zn_in] exerts direct inhibitory effects on PDHC and other energy-linked mitochondrial enzymes [32 , 35]. Under physiologic conditions, such effects do not take place due to very low Zn levels in SN56NC/DC mitochondrial compartments (Figs. 2C, 3) [63]. However, an excitotoxic increase of extracellular ZnCl₂ may cause its extremely high accumulation in the mitochondrial compartment (Fig. 2C) [33 , 60]. Higher accumulation of Zn in SN56DC than in SN56NC mitochondria could be responsible for greater inhibition of PDHC, acetyl-CoA, and ATP levels, yielding earlier loss of viability of the former (Table 1, Figs. 2A, C, 4, 5, 8) [32 , 65]. On the other hand, there are single-plot interdependencies for all the tested metabolic parameters in SN56NC and DC against [Zn_in] (Figs. 4–6). They indicate that particular steps of acetyl-CoA metabolism display similar sensitivities to direct Zn effects, which appear to be independent of the expression of cholinergic phenotype (Fig. 4, 5) [22 , 64]. This is confirmed by the fact that standardized, 5 nmol/mg protein [Zn_in] exerted similar suppression of PDHC, acetyl-CoA and NAA in SN56NC/DC and SHSY5YDC, as well as acetyl-CoA in astroglial C6DC (Table 2). On the other hand, it induced a greater loss of SN56 viability, compared with SHSY5Y and C6DC (Table 2). This indicates that ACh synthesis in the former could make them more susceptible to acetyl-CoA deficits (Fig. 7, Table 2) [34, 35]. These findings remain in accord with our past reports which demonstrated greater susceptibility of SN56DC than SN56NC to cytotoxic signals such as Aβ, NOO^-, or Al excess [23 , 65]; they proved that it was due to higher rate of acetyl-CoA utilization for ACh synthesis, yielding its relative deficits for energy production, appearing in SN56DC in neurotoxic conditions [22 , 66].

We demonstrate here for the first time that cholinergic SN56 neuroblastoma and dopaminergic SHSY5Y cells synthesize and accumulate NAA levels similar to those found in different brain preparations (Fig. 6) [1–3 , 67–70]. Therefore, they may decently reflect the quantitative relationships between acetyl-CoA and NAA metabolism taking place in brain neurons in vivo (Table 2, Fig. 6A-C) [27, 28].

The NAA synthesis by Asp-NAT takes place in neuronal mitochondria, where concentrations of acetyl-CoA in SN56NC and DC were estimated to be equal to about 13 and 8 μmol/L, respectively [2 , 72]. The Km for acetyl-CoA in Asp-NAT reaction was about 58 μmol/L [73]. Thus, the calculated rate of Asp-NAT reaction in situ in the SN56DC mitochondria, would be equal to 71% of that in the SN56NC. Compatible with that remains the level of NAA in SN56DC equal to 76% of that in SN56NC (Table 2, Fig. 6C). Hence, lower levels of NAA in highly cholinergic SN56DC, in respect to low cholinergic SN56NC or dopaminergic SHSY5YDC, may result from the competition for acetyl-CoA with systems transporting acetyl units to sites of ACh synthesis in the cytoplasm (Table 2, Figs. 5D, 6–8) [1 , 58]. Therefore, an increased level of Asp-NAT protein in the SN56DC may reflect the existence of an adaptive, albeit insufficient,compensatory mechanism aiming at maintaining a stable rate of NAA synthesis despite decreased acetyl-CoA availability (Figs. 5D, 6) [9, 58]. The C6DC cells were more resistant to [Zn_in] in spite of lower levels of acetyl-CoA than in SN56, as they utilized it neither for ACh nor for NAA synthesis (Table 2).

Highly significant correlations between Zn-evoked decreases of NAA and acetyl-CoA levels/PDHC activities, in the SN56 septal cholinergic cells remain in accord with neuroimaging studies showing overlapping between regional reductions in F¹⁸-deoxyglucose uptake and NAA levels in brains of AD patients (Table 1, Figs. 5, 6) [2 , 74–76]. The results presented here indicate that suppression of NAA in AD brains may be an early indicator of Zn-evoked shortages of acetyl-CoA, both in noncholinergic and cholinergic neurons (Table 2, Figs. 5C, 6C)[2 , 76].

The decrease of ChAT activity in the brain regions affected by AD pathology is an established marker of cholinergic neurons network loss, which correlates with cognitive functions deficits of the patients [24, 25]. However, a Zn-evoked decrease of ChAT activity in the SN56 cells appeared to be disproportionally smaller than corresponding reductions in their ACh contents (Fig. 7). This may result from coexistent Zn-induced, instant shortages of acetyl-CoA in the cytoplasmic compartment, which brought about immediate reductions of ChAT reaction rate, and a steady state-adjusted ACh level (Figs. 5C, 7) [34 , 57]. These alterations would precede structural disintegration of the neurons marked by loses in the ChAT activity (Fig. 1, 5, 7) [32 –35].

This report provides the first direct evidence that excitotoxicity-induced suppression in neuronal NAA level reflects the existence of its multiple causal correlations with decreased PDHC activity, acetyl-CoA, and ATP levels and viability of SN56 cells (Figs. 4–6). The data remain in accord with neuroimaging studies revealing NAA deficits in AD brains, thereby justifying the thesis that early shortages of acetyl-CoA in the brain may be the direct signal triggering AD neurodegeneration with preferential impairment of cholinergic neurons (Fig. 8) [22, 34]. The existence of multiple, reciprocal correlations between these parameters indicates that the acetyl-CoA generating system in neurons forms the functional unity with multiple pathways of its utilization.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the Ministry of Research and Higher Education MN001-0059/08, National Science Center 2015/17/N/NZ3/01428 projects and the Medical University of Gdańsk fund ST-57. The authors are grateful to Dr. J.K. Blusztajn for the SN56 cells.

Authors’ disclosures available online ().

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Phenotype-Dependent Interactions between N -acetyl-L-Aspartate and Acetyl-CoA in Septal SN56 Cholinergic Cells Exposed to an Excess of Zinc

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Materials

Cell cultures

Morphology and cell counts

Cell viability assays

Whole cell calcium and zinc quantitative assays

Subcellular distribution of free Zn2 +

N-acetyl-L-aspartate assay

Acetyl-CoA assay

ATP assay

Acetylcholine assay

Aspartate N-acetyltransferase activity assay

Aspartate N-acetyltransferase protein quantification

Pyruvate dehydrogenase complex activity assay

Choline acetyltransferase activity assay

Protein assay

Statistics

RESULTS

Effect of differentiation and ZnCl2 on SN56 cell morphology

Effect of extracellular ZnCl2 on its intracellular distribution and Ca contents in SN56

Effect of ZnCl2 on SN56 cell viability

Effect of ZnCl2 on SN56 PDHC activity, and acetyl-CoA/ATP levels

Effects of ZnCl2 on SN56 parameters of N-acetyl-L-aspartate metabolism

Effects of ZnCl2 on cholinergic markers

Effects of intracellular Zn on different clonal cell lines

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Subcellular distribution of free Zn^2 +

Effect of differentiation and ZnCl₂ on SN56 cell morphology

Effect of extracellular ZnCl₂ on its intracellular distribution and Ca contents in SN56

Effect of ZnCl₂ on SN56 cell viability

Effect of ZnCl₂ on SN56 PDHC activity, and acetyl-CoA/ATP levels

Effects of ZnCl₂ on SN56 parameters of N-acetyl-L-aspartate metabolism

Effects of ZnCl₂ on cholinergic markers