Phenotype dependent differential effects of interleukin-1β and amyloid-β on viability and cholinergic phenotype of T17 neuroblastoma cells

Neurochemistry International 47 (2005) 466–473 www.elsevier.com/locate/neuint

Phenotype dependent differential effects of interleukin-1b and amyloid-b on viability and cholinergic phenotype of T17 neuroblastoma cells Hanna Bielarczyk a, Agnieszka Jankowska-Kulawy a, Sylwia Gul a, Tadeusz Pawełczyk b, Andrzej Szutowicz a,* a

Department of Laboratory Medicine, Medical University of Gdan´sk, De˛binki 7 str., 80-211 Gdan´sk, Poland b Department of Molecular Medicine, Medical University of Gdan´sk, Poland Received 20 May 2005; received in revised form 23 June 2005; accepted 30 June 2005 Available online 24 August 2005

Abstract Amyloid-b accumulation in brains of Alzheimer’s disease (AD) victims is accompanied by glial inflammatory reactions and preferential loss of cholinergic neurons. Therefore, the aim of this study was to find out whether proinflamatory cytokine interleukin 1b (IL1b) modifies effects of amyloid-b (Ab) on viability and cholinergic phenotype of septum derived T17 cholinergic neuroblastoma cells. In nondifferentiated T17 cells (NC) Ab(25-35) (1 mg/ml) caused no changes in choline acetyltransferase (ChAT) activity, acetylcholine (ACh) release, subcellular distribution of acetyl-CoA, but doubled content of trypan blue positive cells. IL1b (10 ng/ml) increased ACh release (125%) but did not change other parameters of NC. In the presence of Ab IL1b also increased ChAT activity (47%), ACh release (100%) but had no effect on acetyl-CoA distribution and cell viability. Differentiation with retinoic acid and dibutyryl cyclic AMP caused over two-fold increase of ChAT activity and ACh content, four-fold increase of ACh release and about 50% decrease of acetyl-CoA level in the mitochondria. In differentiated cells (DC), Ab decreased ChAT activity (31%), ACh release (47%) and content of acetyl-CoA (80%) in cell cytoplasmic compartment, whereas IL1b elevated ChAT activity (54%) and ACh release (32%). IL1b totally reversed Ab-evoked inhibition of ChAT activity and ACh release and restored control level of cytoplasmic acetyl-CoA but increased fraction of nonviable cells to 25%. Thus, IL1b could compensate Ab-evoked cholinergic deficits through the restoration of adequate expression of ChAT and provision of acetyl-CoA to cytoplasmic compartment in cholinergic neurons that survive under such pathologic conditions. These data indicate that IL1b posses independent cholinotrophic and cholinotoxic activities that may modify Ab effects on cholinergic neurons. # 2005 Elsevier Ltd. All rights reserved. Keywords: Cholinergic neuroblastoma; Acetyl-CoA; Interleukin 1b; Amyloid-b; Cholinotoxicity

1. Introduction Preferential loss of cholinergic neurons in the brain takes place in several neurodegenerative diseases such as Alzheimer’s disease (AD), aluminum/dialysis encephalopathy, thiamine deficiency, as well as various hypoxemic/ ischemic brain injuries. Post mortem studies in brains of AD victims showed that the extent of loss of cholinergic markers in septum, hippocampus and parietal cortex correlated well * Corresponding author. Tel.: +48 58 349 2770; fax: +48 58 349 2784. E-mail address: [email protected] (A. Szutowicz). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.06.010

with an impairment of cognitive functions found immediately before their death (Pappas et al., 2000). Similar loss of cholinergic innervation was found in brains of transgenic mice overexpressing amyloid precursor protein (Van Dam et al., 2005). Several toxic signals, such as oxygen or nitrosyl free radicals, excitotoxic stimulation, amyloid-b (Ab), aluminum and proinflamatory cytokines were suggested to contribute to the mechanisms of neuronal degeneration in these pathologies (Guan et al., 2003; Hynd et al., 2004; Szutowicz, 2001 for review). However, sources of particular vulnerability of cholinergic neurons in these pathologic conditions remain unclear. It has been suggested that it

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might result from the fact that cholinergic neurons unlike other ones utilize choline and acetyl-CoA not only for structural lipids and energy production, but also for acetylcholine (ACh) synthesis (Szutowicz, 2001; Szutowicz et al., 1998, 2000; Wurtman, 1992). Interleukin 1b (IL1b) exerts proinflamatory and cytotoxic effects in several tissues. In the brain, microglial cells are the main source of this cytokine both under physiologic and pathologic conditions. Levels of IL1b rose in human brains in postischaemic period, Alzheimer’s disease and aluminum intoxication as well as in various animal models of neurodegeneration (McGeer and McGeer, 2003; Rothwell and Luheshi, 2000). However, reports evidencing causal relationships between increased IL1b levels and preferential loss of cholinergic neurons in these pathologies are scarce (Giovannini et al., 2002). It is known that different groups of brain cholinergic neurons display a differential susceptibility to similar harmful signals (Szutowicz, 2001). Cell culture studies also demonstrated that neuronal cells with high expression of cholinergic phenotype were more sensitive to excess of NO, aluminum and Ab than those with low or no expression of cholinergic functions (Szutowicz, 2001 for review). In addition, Ab increased level of IL1b in rat brain, what might aggravate their detrimental effects on cholinergic neurons (Giovannini et al., 2002). Also ILs themselves may evoke excitotoxic activity in the brain and increase Ab production. Thus, reciprocal interactions between IL1b and other neurotoxins may aggravate or attenuate neurotoxic effects of each of them (Rothwell and Luheshi, 2000). For instance, IL1b and IL-6 decreased ACh release from the myenteric plexus (Kelles et al., 2000). In primary neuronal cultures and PC12 cells IL1b-enhanced expression and activity of acetylcholinesterase, which could be responsible for cholinergic hypofunction (Li et al., 2000). Lipopolysachride injection into basal forebrain caused IL-dependent loss of cholinergic neurons (Willard et al., 1999). Other data showed no differences in ACh content in rat brains after treatment with IL1b (Casamenti et al., 1999). These data are inconsistent with the finding showing differential regional, phenotype dependent susceptibility of cholinergic neurons to neurotoxins activating production of IL1b (Giovannini et al., 2002; Rothwell and Luheshi, 2000; Szutowicz, 2001). Different ILs themselves are also able to induce excessive production of NO and Ab that would trigger vicious cycle of neurodegeneration (Matsuoka et al., 1999; Rothwell and Luheshi, 2000). Our recent data indicate that other cytokine, nerve growth factor (NGF) may modify cholinergic transmission and cell sensitivity to neurotoxic signals depending on the expression of their cholinergic phenotype (Szutowicz et al., 2004). It rose the assumption that the effect of IL1b on cholinergic neurons may also depend on the degree of their differentiation. The aim of this study was to find out whether and how IL1b affects acetylcholine metabolism and viability of cholinergic cells, derived from neurotoxin-sensitive septal

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region of mouse brain, depending on the degree of their differentiation (Hammond et al., 1990). Presented data indicate that IL1b might differentially modify effects of Ab on neurotransmission and viability of cholinergic neurons depending on their phenotypic properties.

2. Materials and methods Unless otherwise specified biochemicals were obtained from Sigma–Aldrich (Poznan´, Poland), Amyloid-b(25-35) was from Bachem (Heidelberg, Germany), acetyl-CoA [1-14C-acetyl] 4 mCi/mmol was from Perkin-Elmer (Boston, MA, USA), Cell cultures growth media and components were provided by Gibco Life Technologies (Warsaw, Poland), cell culture disposables derived from Sarstedt (Stare Babice, Poland). SN56.B5.G4 cholinergic murine neuroblastoma stably transfected with rat trkA cDNA (T17) and geneticine resistance gene and expressing TrkA receptors were used in this study (gift from Dr J.K. Blusztajn, Boston MA, USA). The cells (passage 42–48) were plated at the density of 4.2
104 cells/cm2 and grown for 3 days to subconfluency in Dulbecco’s modified Eagle medium (DMEM) containing 2 mM L-glutamine, 2500 IU streptomycin, 0.6 mg geneticine per 1 ml and 10% fetal bovine serum at 37 8C in atmosphere 5% CO2, 95% air. Cells were differentiated by 3 day culture in the presence of 1 mM dibutyryl cAMP (cAMP) and 0.001 mM all-trans-retinoic acid (RA). Differentiation was demonstrated by increased ChAT activity, ACh content and morphologic maturation of the cells (Szutowicz et al., 2000, 2004) (see Section 3). Differentiated (DC) and nondifferentiated (NC) cells were harvested and replated for experimental passage in the same growth medium containing no differentiating agents. Interleukins and Ab were added as indicated in Results. In such conditions DC-controls maintained differentiated phenotype, gained in the preceding passage, for 3 days. (Bielarczyk et al., 2003a). After this time, cells were harvested into 10 ml of ice cold HEPES buffered 0.9% NaCl (pH 7.4), washed twice by centrifugation at 200
g for 10 min with the same solution and suspended in 0.32 M sucrose containing 10 mM HEPES buffer (pH 7.4) and 0.1 mM EDTA to obtain the protein concentration 10.0 mg/ ml. Immediately after collection, the cells were used for Trypan blue exclusion assay and for metabolic studies. For enzyme assays, samples were kept frozen at 20 8C for 2–7 days. 2.1. Trypan blue exclusion assay Cell suspension was mixed with equal volume of 0.4% isotonic trypan blue solution. Total cell number and fraction of nonviable, dye accumulating cells were counted after 2 min in Fuchs-Rosenthal haemocytometer under light microscope (Wang et al., 2001).

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2.2. Metabolic studies Incubation medium contained in a final volume of 1.0 ml 2.5 mM pyruvate, 2.5 mM L-malate, 90 mM NaCl, 30 mM KCl, 20 mM NaHEPES (pH 7.4), 1.5 mM Na-phosphate, 0.01 mM choline chloride, 0.015 mM eserine sulfate, 0.02 mM EDTA, 32 mM sucrose and 1–2 mg of cell protein. Incubation was started by the addition of cell suspension and continued for 30 min at 37 8C with shaking at 100 cycles per min. For assay of acetyl-CoA content in cell mitochondria 0.5 ml of incubation medium was mixed with equal volume of ice cold lysing solution containing 1.4 mg digitonin/ml in 125 mM KCl with 20 mM NaHEPES buffer (pH 7.4) and 3 mM EDTA. Lysate was transferred on 0.5 ml of silicone oil mixture (AR 20 and AR200, 1:2). After 30 s mitochondrial fraction was separated from the soluble one by centrifugation for 40 s at 12,000
g. After removal of soluble fraction and silicon oils, mitochondrial pellet was deproteinized by suspension in a small volume of 5 mM HCl and incubation in a boiling bath for 1 min. For determination of total acetyl-CoA content, 0.3 ml of incubation medium was centrifuged at 5000
g for 2 min. Supernatant was removed and cell pellet was deproteinized as described above. 2.3. Acetyl-CoA assay Deproteinized extracts of whole cells and mitochondria were treated with maleic anhydride solution in ethyl aether for 2 h to remove CoA-SH. Cycling reaction was carried for 60 min in 0.1 ml of medium containing 1.9 mM acetyl phosphate, 1.2 mM oxaloacetate, 0.72 IU phosphotransacetylase and 0.12 IU citrate synthase. Cycling reaction was stopped by heating samples at 95 8C for 6 min and citrate formed was determined (Szutowicz and Bielarczyk, 1987). Cytoplasmic acetyl-CoA level was calculated by subtraction of mitochondrial acetyl-CoA from total acetyl-CoA content (Szutowicz and Bielarczyk, 1987).

O-acetyl transferase, EC 2.3.1.6, ChAT) activity was assessed by the radiometric method using [1-14C]acetylCoA as a substrate (Fonnum, 1975). 2.6. Protein assay Protein was assayed by the method of Bradford (1976) with human immunoglobulin as a standard. 2.7. Statistical analysis Statistical analyses were carried out by one-way ANOVA with Bonferroni multiple comparison post hoc test. When comparisons were being made between two treatments a non-paired Student’s t-test was performed and p < 0.05 was considered significant.

3. Results 3.1. Effect of differentiation on cholinergic cells Differentiation with cAMP and RA for 3 days and subsequent replating in the medium free of these compounds for another 3 days yielded cells with ChAT activity, ACh content and ACh release being 130, 150 and 285%, respectively, higher than in nondifferentiated cells (NC) (Fig. 1; Table 1). DC contained 48% less acetyl-CoA in mitochondria than NC (Table 2). They also displayed some decrease of viability (Fig. 2) as well as increased formation of extensions and synapse like connections when compared with NC (Figs. 3A and 4A). After 3 day culture, the density of NC controls was about 10
104 cells/cm2 and that of DC about 7
104 cells/cm2, respectively. Additions of IL1b and Ab caused no significant changes in cell density (not shown).

2.4. Acetylcholine assay For ACh assay, 0.2 ml incubation medium was centrifuged for 3 min at 10,000
g. Obtained pellet and supernatant were used for assays of intracellular and released ACh, respectively. ACh was determined by HPLC method with an enzymatic reactor containing acetylcholinesterase (EC 3.1.1.7) and choline oxidase (EC 1.1.3.17) and electrochemical detector by use of commercial kit (Bioanalytical Systems, West Lafayette, IN, USA) (Pedersen et al., 1995). 2.5. Enzyme assays Immediately before the assay samples were thawed and diluted to desired protein concentration in 0.2% (v/v) Triton X-100. Choline acetyltransferase (acetyl-CoA: choline

Fig. 1. Effect of interleukin 1b (IL1b, 10 and 100 ng/ml) and amyloidb(25-35) (Ab, 1 mg/ml) on ChAT activity in nondifferentiated and differentiated cholinergic T17 neuroblastoma cells. Data are means  S.E.M. from 4 to 10 experiments. Significantly different from respective: control, * p < 0.05, **p < 0.001; IL1b 10 ng/ml alone, yp < 0.001; Ab 1 mg/ml alone, zp < 0.01 by one-way ANOVA with Bonferroni post hoc test. Significantly different from respective NC, §p < 0.05, by unpaired t-test.

H. Bielarczyk et al. / Neurochemistry International 47 (2005) 466–473 Table 1 Effect of IL1b and Ab on acetylcholine metabolism in T17 cells Conditions

Acetylcholine content (pmols/mg protein)

Acetylcholine release (pmols/(min mg protein))

Nondifferentiated cells Control IL1b 10 ng/ml Ab(25-35) 1 mg/ml IL1b 10 ng/ml + Ab(25-35) 1 mg/ml

42  6 42  6 48  6 49  5

0.63  0.08 1.42  0.22* 0.53  0.08 1.06  0.13

Differentiated Control IL1b 10 ng/ml Ab(25-35) 1 mg/ml IL1b 10 ng/ml + Ab(25-35) 1 mg/ml

107  6z 96  6z 92  14z 109  14z

2.43  0.11z 3.20  0.31z 1.30  0.06*z 2.93  0.16yz

Data are means  S.E.M. from four duplicate experiments. Significantly different from: respective control *p < 0.05; Ab(25-35) alone, yp < 0.05, by one-way ANOVA with Bonferroni post hoc test; nondifferentiated cells, z p < 0.01, by unpaired t-test.

3.2. Effect of IL1b and Ab on cholinergic phenotype and viability of NC There are suggestions that Ab-evoked increase of IL1b content may be responsible for cholinergic hypofunction in the brain (Giovannini et al., 2002). However, presented data show that neither low (10 ng/ml) nor high (100 ng/ml) concentrations of IL1b significantly changed ChAT activity and ACh content in NC (Fig. 1; Table 1). Moreover, IL1b in low concentration stimulated ACh release by 125% (Table 1). In NC, Ab (1.0 mg/ml) alone caused no changes in their ChAT activity, ACh content/release, acetyl-CoA level and compartmentation but increased number of nonviable cells from 6 to 12% (Figs. 1 and 2; Tables 1 and 2). In the presence of Ab, low concentration of IL1b retained its ability to increase ACh release (100%) and ChAT activity (34%) (Fig. 1; Table 1). Combined addition of Ab and high concentration of IL1b tended to suppress ChAT activity and increased fraction of nonviable NC to 15% (Figs. 1 and 2A).

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3.3. Effect of Ab and IL1b on cholinergic phenotype and viability of DC It has been shown that Ab caused greater decrease in pyruvate utilization and acetyl-CoA content in SN56 DC than in NC (Bielarczyk et al., 2003a; Szutowicz et al., 2004). Therefore, one could expect that IL1b/Ab-evoked changes of the cholinergic phenotype will affect acetyl-CoA distribution in the cells. Differentiation caused about 48% decrease of acetyl-CoA content in cell mitochondria (Table 2). In DC Ab resulted in 69% decrease of whole cell acetyl-CoA content. This decrease was confined to cytoplasmic compartment where 80% decrease of acetylCoA level was observed (Table 2). IL1b alone brought about no significant shifts in acetyl-CoA distribution, but totally overcame Ab-evoked suppression of this metabolite in cytoplasmic compartment (Table 2). In DC low IL1b concentration caused 54% increase, whereas its high concentration tended to inhibit ChAT activity (Fig. 1). No significant increases in ACh content/ release and cell impairment were observed under these conditions (Table 1; Fig. 2). In DC Ab alone caused about 31 and 47% decreases in ChAT activity and ACh release and increase of nonviable cell content to 17%, respectively (Tables 1 and 2; Fig. 1). No change in the DC transmitter level was observed (Table 1). In addition, Ab abolished activation of ChAT by low concentration of IL1( (Fig. 1). On the other hand, IL1b reversed Ab-evoked inhibition of ACh release and ChAT activity (Table 1; Fig. 1). Combined addition of Ab with low and high IL1b concentrations increased DC mortality to 20 and 25%, respectively (Fig. 2). 3.4. Effect of Ab and IL1b on cell morphology Ab changed shape of NC from angular to more round one (Figs. 3A and B). Addition of low concentration IL1b increased number of cells with long branched extensions and synapse-like connections (Fig. 3C). They disappeared after

Table 2 Effect of IL1b and Ab on acetyl-CoA distribution in subcellular compartments of T17 cells Conditions

pmols/mg whole cell protein Total acetyl-CoA

Mitochondrial acetyl-CoA

Cytoplasmic acetyl-CoA

Nondifferentiated cells Control IL1b 10 ng/ml Ab(25-35) 1 mg/ml IL1b 10 ng/ml + Ab(25-35) 1 mg/ml

36.0  3.4 41.8  3.9 31.0  3.5 43.3  6.0

15.9  2.1 14.1  4.0 14.1  3.3 13.0  2.5

20.1  3.9 27.7  3.9 16.9  3.5 20.3  1.3

Differentiated cells Control IL1b 10 ng/ml Ab(25-35) 1 mg/ml IL1b 10 ng/ml + Ab(25-35) 1 mg/ml

36.9  5.6 23.8  4.4z 11.4  3.8*z 37.7  6.1y

8.3  2.0z 8.7  1.6 7.7  3.4 8.2  2.1

28.6  3.5 16.6  3.3 5.8  1.0*z 29.3  5.6y

Data are means  S.E.M. from four duplicate experiments. Significantly different from: respective control, *p < 0.05; Ab(25-35) alone, yp < 0.05, by one-way ANOVA with Bonferroni post hoc test; nondifferentiated cells, zp < 0.05 by unpaired t-test.

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round-shaped cells (Fig. 4A and B). Low concentration of IL1b resulted in no apparent changes in DC mortality (Fig. 2), but partial loss of morphological differentiation was observed under these conditions (Fig. 4C) despite of increased ChAT activity (Fig. 1). Combined addition of Ab and IL1b increased fraction of round shaped cells with nondifferentiated morphology (Fig. 4D and F).

4. Discussion Fig. 2. Effect of interleukin 1b (IL1b, 10 and 100 ng/ml) and amyloidb(25-35) (Ab, 1 mg/ml) on trypan blue exclusion ability of nondifferentiated and differentiated cholinergic T17 neuroblastoma cells. Data are means  S.E.M. from 4 to 10 experiments. Significantly different from respective: control, *p < 0.05, by one-way ANOVA with Bonferroni post hoc test; from respective NC, §p < 0.05, by unpaired t-test.

combined application of high concentration of IL1b (Fig. 3E) or after combined addition of Ab and IL1b (Fig. 3D and F). On the other hand, in DC Ab caused loss of most extensions, synapse-like connections and appearance of

The increases of ChAT activity, ACh content and release, morphological maturation and decrease in acetyl-CoA content in mitochondria of cAMP/RA treated T17 cells shown here remain in accord with our earlier results (Tables 1 and 2; Figs. 1, 3 and 4) (Bielarczyk et al., 2003a; Pedersen et al., 1995; Szutowicz, 2001). It is estimated that ChAT activity in mature cholinergic neurons may be as high as 10 nmols/(min mg) protein (Bielarczyk et al., 2003b). Therefore, DC with their mature morphology and higher ChAT activity should be considered as more proper model for AD pathology than NC (Table 1; Figs. 3 and 4).

Fig. 3. Representative photomicrographs showing effects of Ab(25-35) and IL1b on nondifferentiated T17 cell morphology. Cells were grown for two 3 day cultures without differentiating agents. (A) Control; (B) Ab 1 mg/ml; (C) IL1b 10 ng/ml; (D) IL1b 10 ng/ml and Ab 1 mg/ml; (E) IL1b 100 ng/ml; (F) IL1b 100 ng/ml and Ab 1 mg/ml. Photos present typical morphology and not density of the cells in each particular conditions.

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Fig. 4. Representative photomicrographs showing effects of Ab(25-35) and IL1b on differentiated T17 cell morphology. Cells were grown for 3 days in the presence of cAMP/RA, replated and subcultured for 3 days in the medium free of differentiating agents: (A) control; (B) Ab 1 mg/ml; (C) IL1b 10 ng/ml; (D) IL1b 10 ng/ml and Ab 1 mg/ml; (E) IL1b 100 ng/ml; (F) IL1b 100 ng/ml and Ab 1 mg/ml. Photos present typical morphology and not density of the cells in each particular conditions.

These results also allow the conclusion that differential effects of IL1b on cell viability and cholinergic phenotype, discussed below, are at least in part, dependent on the differentiation-evoked changes in acetyl-CoA availability in mitochondrial and cytoplasmic compartments (Table 2) (Szutowicz, 2001). It is known that Ab inhibits different steps of acetyl-CoA metabolism including PDH and respiratory chain (Table 2) (Bielarczyk et al., 2003a; Hoshi et al., 1997). These observations were compatible with findings showing the suppression of ChAT expression, ACh content and release in the brain, primary neuronal cultures and cholinergic neuroblastoma cells by Ab (Table 1; Fig. 1) (Hoshi et al., 1997; Szutowicz, 2001). Presented data demonstrate that cholinergic NC and DC displayed a differential sensitivity to Ab (Tables 1 and 2; Figs. 1 and 2). Thus, relatively small increase in nonviable NC, as well as no change in ChAT activity and ACh release, in the presence of Ab could be due to the fact that these cells utilize smaller fraction of their acetyl-CoA for the transmitter synthesis (Table 1; Figs. 1 and 2). Thanks to this they contained more acetyl-CoA in

mitochondria available for energy production (Table 2). On the other hand, in DC lower content of acetyl-CoA in mitochondrial compartment and its higher utilization for ACh synthesis, could make them more prone to cytotoxic influences of Ab (Fig. 2; Table 2). Additional factors such as increased expression of voltage dependent Ca channels, rise in Ca content in mitochondria and p75 receptors expression in plasma membranes of DC could also augment their vulnerability to Ab (Kushmerick et al., 2001; Szutowicz et al., 2004). Suppression of ChAT expression and ACh release in DC could be caused by the Ab-evoked decrease of acetyl-CoA supply to cytoplasmic compartment presumably due to inhibition of PDH activity in mitochondria (Bielarczyk et al., 2003a; Hoshi et al., 1997). In summary, variations in subcellular distribution of acetyl-CoA between NC and DC may be, at least in part, responsible for their differential reactions to Ab. Few reports reveal that IL1b causes hypofunction of the cholinergic neurons in the brain (Casamenti et al., 1999). They demonstrated the decrease of ChAT expression and activity as well as ACh content in cholinergic neurons and

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Table 3 Specificity of activatory effect of IL1b on choline acetyltransferase activity in differentiated T17 cells Addition (ng/ml)

Choline acetyltransferase (percent of control value)

Control IL1b 10 IL6 10 IL1b 10 + IL6 10 TNFa 0.01 1L1b 10 + TNFa 0.01 NGF 100

100  5 154  13 * 108  8 88  10y 65  12 * 62  6y 57  4 *

Data are means  S.E.M. from 3 to 10 experiments calculated as percentage of DC control (Fig. 1). Significantly different from: control, *p < 0.05; IL1b alone, yp < 0.01.

extracellular space of brains treated with IL1b (Casamenti et al., 1999; Giovannini et al., 2002; Stoll et al., 2000). There is even less data suggesting the existence of indirect positive cholinotrophic activities of IL1b through the increase of NGF level in the brain (Stoll et al., 2000). However, one of such effects, the activation of AChE expression by IL1b led rather to the cholinergic hypofunction due to faster breakdown of ACh released into synaptic cleft (Li et al., 2000). Our report presents the first observation that low IL1b concentrations may exert a specific direct positive cholinotrophic effect on cholinergic neurons of septal origin. This effect may be caused by IL1b-dependent activation of mitogen-associated protein kinase (MAPK) (Rothwell and Luheshi, 2000), which in turn might activate cAMP response element binding protein (CREB) in the cholinergic locus yielding increase of ChAT expression and activity (Szutowicz et al., 2004). Specificity of this effect is demonstrated by the fact that low concentrations of other cytokines such as IL6, TNFa or NGF either did not change or suppressed ChAT activity (Table 3). Moreover, IL1b-evoked ChAT activation was inhibited completely by IL6 or TNFa. It indicates that these cytokines may either block IL1b receptor with high affinity, or suppress intracellular signal transduction through their own specific receptors (Table 3). On the other hand, the reversal of this activation by high concentration of IL1b may be caused by activation of cell death signaling pathways by receptor saturated with this ligand. The disparity of our findings with other reports, may result from differences in real cytokine concentrations used in our and other experimental models (Fig. 1) (Casamenti et al., 1999; Giovannini et al., 2002; Kelles et al., 2000). Lack of significant effect of IL1b on ChAT activity in NC indicates that in these conditions the respective promoter site(s) of the cholinergic locus, was inaccessible for activating signals of IL1b (Fig. 1). In turn, activation of cAMP response (CRE) and/or retinoic acid response elements could make respective site of the cholinergic locus promoter in DC available for IL1b signaling, yielding the increase of ChAT activity (Fig. 1). Similar, phenotypedependent changes of sensitivity of the ChAT gene promoter

to differentiating stimuli took place in SN56 cells treated with nerve growth and ciliary neurotrophic factors (Berse et al., 1999; Szutowicz et al., 2004). On the other hand, lack of effect of high IL1b concentration on cholinergic phenotype at simultaneous decrease in NC and DC viability indicate that these two effects are likely to be mediated by independent receptors or/and mechanisms of signal transduction (Fig. 2). Reversal of Ab-induced decrease of acetyl-CoA concentration in cytoplasmic compartment of DC by low concentration of IL1b can explain its capacity to overcome the suppression in ChAT activity and ACh release (Tables 1 and 2; Fig. 1). In addition, lack of IL1b effect on mitochondrial acetyl-CoA could be responsible for the increase in fraction of impaired cells (Fig. 2). Presented data indicate that, acetyl-CoA was a common point for opposite actions of Ab and IL1b. Inhibitory effects of Ab may result from its ability to increase free radical level through activation of high conductance Ca-channels and p75 receptors (Kaplan and Miller, 2000; Szutowicz et al., 2004; Ueda et al., 1997). These changes lead to the PDH inhibition, the decrease of acetyl-CoA content and suppression of cholinergic metabolism (Table 1; Fig. 1) (Hoshi et al., 1997; Bielarczyk et al., 2003a). However, mechanism by which IL1b alleviates Ab-evoked decrease of cytoplasmic acetyl-CoA remains unknown. On the other hand, activating effects of low concentrations of IL1b on ChAT activity in NC, that appear only in the presence of Ab, indicate that the latter is presumably capable of mimicking facilitating effects of cAMP/RA on regulatory sites in the cholinergic locus activated by the cytokine-signaling pathway(s) (Fig. 1). Fact that IL1b-evoked changes in ChAT/ACh displayed no correlation with cell viability and morphological differentiation indicate that these parameters of cholinergic cells are regulated by independent mechanisms (Figs. 1–4). Activation by IL1b ACh release (Table 1) and ChAT activity (Fig. 1) in DC and NC could be a part of the mechanism compensating deficits of cholinergic transmission caused by loss of cholinergic neurons under neurodegenerative inflammatory conditions (Szelenyi, 2001). It could be achieved through restoration by IL1b the cytoplasmic acetyl-CoA pool and ChAT expression in neurons that avoided degeneration (Fig. 1; Table 2). However, it could not prevent facilitation of cholinergic neurons death since the increase of cholinergic functions simultaneously made them more vulnerable to Ab and other neurotoxic insults.

Acknowledgements Authors thank Dr. J.K. Blusztajn for samples of T17 cells. Work was supported by Ministry of Scientific Research and Information Technology projects: 6P05A 010 20 and 2P05A 26 26 and Medical University of Gdan´sk found St-57.

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