Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures

BRAIN RESEARCH ELSEVIER

Brain Research 740 (1996) 323-328

Research report

Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures Nancy Calvo a Julia Reiriz b, Esther P&ez-Navarro ~, Jordi Alberch ''' ~ D~7~artament de Biologia Cel.hdar i Anatomia Patol?)gica, Facultat de Medicina, Uniz ersitat de Barcelona, Fundaci/~ Clhm'. Ca,~anoca 143, 08036 Barcelona, Spain t~ Departament d'h!/ermeria Fonamental i Mbdic-Quir{~rgic, Escola Uni;'ersitiwia d'b!fermeria, Uni; ersitat de Barcelona. Feixa l.lar~a .~ , n, 08907 L'Hospitalet de Llohregat, Barcelona, Spain

Accepted 16 July 1996

Abstract

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyltransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 /~M QUIN but not by 40 # M KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sarg]-Substance P. The survival of neuron specflic enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sat"'~, Met(O,)l Il-Substance P (NK l receptor agonist), [Nle m]-Neurokinin A (NK 2 receptor agonist) or [Me-Phe 7]-Neurokinin B (NK3 receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons. Kevwords: Kainic acid: Substance P: Neurokinin; Neostriatum: Neuroprotection: Rat: Choline acetyltransferase activitv: Acetylcholinesterase

1. Introduction

Tachykinins (TKs) constitute a group of naturally occurring peptides that are widely distributed and active in both the central nervous system and peripheral tissues [31]. The endogenous mammalian TKs (substance P (SP), neurokinin A ( N K A ) and neurokinin B (NKB)) act as preferred, but not exclusive, endogenous ligands of the three classes of neurokinin receptor subtypes, NK~, N K > and NK3, respectively [17,40]. TKs are regarded as neurotransmitters or neuromodulators [31,35], but they may also have neurotrophic-like actions. SP enhances neural growth in vitro [22,34] and counteracts the anatomical, biochemical and behavioral effects of neurotoxic damage to monoamine neurons [23,33]. Furthermore, these peptides may play a role in the mechanisms of neurodegenerative diseases [4], as TKs can completely reverse the neurotoxic effect of the

" Corresponding author. Fax: (34) (3) 4(12-1907.

/3-amyloid protein on hippocampal cells in vitro [46] and in vivo [28]. Moreover, some peptidergic SP antagonists have a neurotoxic action in rat brain [20,45] and spinal cord [39]. TKs are altered in several neurodegenerative disorders which affect the striatum, such as Parkinson's and Huntington's disease [16]. These peptides serve as neurotransmitters in the striato-nigral pathway [21,24]. Moderate levels of the three subtypes of neurokinin receptors have been found in the neostriatum [7,41]. TKs have a special interaction with striatal cholinergic interneurons [14]. SPcontaining terminals make synapses on the somatodendritic tree of these neurons [6,32], where TK receptors have been localized {13,25]. A functional effect of TKs on striatal cholinergic neurons has also been described. TK receptor agonists induce endogenous acetylcholine (ACh) release during striatal postnatal development [37] and in the adult [1,2,18]. Furthermore, they differentially regulate ACh release in a model for Parkinson's disease [I,2].

0006-899:~/96 /$15.00 Copyright cg~) 1996 Elsevier Science B.V. All rights reserved. PII S000~-8tm3196)00879-

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N. Cah,o et al. / Brain Research 740 (1996) 323-328

Cholinergic activity is also affected after intrastriatal quinolinic acid (QUIN) injections [3,38], used as a model for Huntington's disease [8]. These latter results showed the presence of two functional populations of striatal cholinergic neurons which respond differentially to QUIN injury, with a higher sparing of cholinergic neurons stimulated by TKs [3]. All these observations suggest an important role for TKs in the regulation of striatal circuits. The goal of the present study was to evaluate the protective effect of TKs on striatal cholinergic neurons from damage induced by excitatory amino acids. Choline acetyltransferase (CHAT) activity was examined in striatal cell cultures after treatment with QUIN (NMDA receptor agonist) or kainic acid (KA, non-NMDA receptor agonist) alone or in the presence of TKs. The effect of SP on the survival of total population of neurons and acetylcholinesterase (AChE)-positive cells after QUIN excitotoxicity, was also studied. Our results show that TKs are able to protect cholinergic neurons against QUIN but not KA induced damage.

2. Materials and methods

was stopped by adding 0.05% trypsin inhibitor. Tissue pieces were washed twice with PBS-G-A containing 1 m g / m l bovine serum albumin (BSA) and dissociated by gentle trituration with a fire-polished Pasteur pipette in the same solution. Finally, cells were resuspended in a modified L-15 medium [19] including 50 U / m l penicillin and 5 0 / x g / m l streptomycin and supplemented with: 1 0 / x g / m l insulin, 20 /xg/ml transferrin, 100 /xg/ml putrescine, 20 nM progesterone and 30 nM sodium selenite. Cells were counted in a hemocytometer using trypan blue as criterion for viability and distributed (5 × 10 4 viable cells per well) in 96-well plates (Costar) previously coated with 5 /xg/ml poly-D-lysine at least during 2 h at 37°C. Cultures were grown in the presence of 5% heat-inactivated horse serum and 0.5% heat-inactivated fetal calf serum and maintained at 37°C in a 95% air/5% CO 2 humidified atmosphere. Culture medium was partially replaced every 3 - 4 days. QUIN, KA, SP acetate salt (SP), [Sarg]-Substance P (Sar-SP: SP analog resistant to peptidases), [Sar ~), Met(O~)~J]-Substance P (SSP; NK I receptor agonist), [Nlet°]--Neurokinin A (NNKA; NK 2 receptor agonist) or [Me-PheT]-Neurokinin B (NNKB; NK 3 receptor agonist) were added to cultures 11 days after plating and maintained for 3 days without changing medium.

2.1. Materials 2.3. Choline aceO'ltransferase actil:iO' determination Leibovitz's L-15 medium was obtained from BioWhittaker and sera, penicillin and streptomycin from Gibco. [Sat 9, Met(O2)ll]-Substance P, [NleJ°]-Neurokinin A, [Me-PheV]-Neurokinin B and (5R, 10S)-(+)-5-methyl10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) were obtained from Peninsula Laboratories and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was from Research Biochemicals International. DNAse type 1 and unlabelled acetyl-coenzyme A was purchased from Boehringer Mannheim. [1-14C]Acetyl-coenzyme A (55 m C i / m m o l ) was from Amersham. 3-Heptanone and sodium tetraphenylborate were from Merck. Prediluted rabbit anti-neuron specific enolase was supplied by DAKO and Vectastain Elite ABC Kit was from Vector Laboratories. Scintillation liquid (Optiphase Hi'Safe) was from LKB. All other chemical reagents were obtained from Sigma Chemical Co.

2.2. Cell culture Pregnant Sprague-Dawley rats (Charles River, France) were used in these experiments. Neostriata from embryonic age E17-E18 fetuses were dissected under a stereomicroscope in ice cold Leibovitz's L-15 medium following the method of Hefti et al. [19] with some modifications. Tissue was dissociated by enzymatic treatment with 0.05% trypsin and 0.01% DNAse type I in Dulbecco's phosphate-buffered saline, containing 0.5 mM MgC12, 33 mM glucose, 100 U / m l penicillin and 1 0 0 / x g / m l streptomycin (PBS-G-A) for 10 min at 37°C. The dissociation

Three days after treatment, culture medium was removed and 25 /xl of 10 mM sodium phosphate buffer containing 10 mM EDTA and 0.05% Triton X-100, pH 7.4, were added. Plates were frozen and stored at - 8 0 ° C until assayed. ChAT activity was measured using the method of Fonnum [9] with some modifications. The final incubation volume contained 45 mM sodium phosphate buffer, pH 7.4, 9 mM choline chloride, 270 mM NaC1, 9 mM EDTA, 25 p,M [taC]acetyl-coenzyme A (5 mCi/mmol). In order to inhibit endogenous AChE activity, 0.1 mM eserine hemisulfate was added. After 1 h at 37°C the reaction was stopped by adding 125 /xl of 10 mM sodium phosphate buffer (pH 7.4) at 4°C. The total content of the wells was placed on 300 p,l of 3-Heptanone containing 20 m g / m l sodium tetraphenylborate. Each well was washed twice with 125 /xl of the same buffer and ACh finally extracted by centrifugation (5 rain at 14 000 × g) at 4°C. The radioactivity was measured in 200 /xl of the upper phase using 10 ml of the scintillation liquid.

2.4. Acetylcholinesterase cytochemistr3, After treatment, culture medium was removed and cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (2 h at room temperature) and rinsed twice with Tris-buffered saline (TBS) (Tris 0,05 M, pH 7.4, 0.25 M NaC1). AChE was stained following the method of Hefti et al. [19]. To evaluate the specificity of staining 0.1 mM eserine hemisulfate was employed as an AChE inhibitor.

N. Cah'o et al. / Brain Resear~'h 740 (1996,1 323 328

u')

io0 125

.~>

,~~ 100 ~o O

¢~ ~¢~--

0

cO

%

325

60

/ / / /

t-

/ / / /

/ / / /

/// /// /// /// ///

40 20

A

i C

5~0

100 , 150 i 2 0,0 Concentration

SP

QUIN

KA

QUIN

2 5,0 3 0,0 (uM)

Fig. 1. Effect of QUIN (circles) and KA (squares) on ChAT activity of striatal cell cultures. Excitotoxins were added to cultures 11 days after plating and their effects were evaluated 3 days after treatment. ChAT activity was calculated as cpm per well and expressed as percentage of the control values. Points represent the mean+ S.E.M. of at least three different experiments (four replicates in each experiment).

2.5. hnmunocymchemisto,

Cultures were fixed as described above for 1 h. After three rinses with TBS, cells were preincubated with the same buffer containing 0.4% Triton X-100, 1% BSA and 4% normal goat serum for 2 h at room temperature. Rabbit antiserum against the neuron specific enolase (NSE) was employed to identify neurons by incubating 1 h at room temperature. After rinses with TBS, cells were then incubated with a biotinylated goat anti-rabbit IgG antibody (1:200) for I h and visualized alter binding of the avidinbiotin-peroxidase complex with 3,3'-diaminobenzidine as chromogen.

+

SP

SP

Fig. 2. Effect of SP (20 p,M) against toxicity produced by QtIIN or KA on striatal cholinergic activity. Excitatory amino acid analogs and/or SP were added to cultures 11 days after plating and their effects were evaluated 3 days after treatment, ChAT activity was calculated as cpm per culture well and expressed as percentage of the conll'Cq values. Bars represent the mean_+.S.E.M. of at least three diflerent experiments (four replicates in each experiment). Statistical analysis was performed using the ANOVA test with subsequent comparisons by Student's t-test with control values. ~ P < 0.05.

3.2. Influence of SP on QU1N or RVI to.vicitv

SP was added to cultures simultaneously with QUIN or KA, in order to examine its possible protective effect on striatal cholinergic cells. SP (20 ktM) blocked the toxic effect induced by QUIN on ChAT activity (Fig. 2). In contrast, this TK was not able to protect striatal cholinergic activity from KA toxicity.

125I

3. Results

3. I. E.Ooct 0/ excitatory amino acid analogs on striatal cholinergic neurons

KA

+

.~,?, e"

In order to examine the effect of QUIN and KA on cholinergic function, ChAT activity was examined. Both excitatory amino acid agonists induced a dose dependent decrease in ChAT activity. However, KA was more potent than QUIN (Fig. 1). In further experiments, to study the protective effect of TKs against excitotoxicity, we used the concentration of QUIN or KA (125 # M and 40 /~M, respectively) that produced a 30% decrease in ChAT activity. To determine the specificity of the toxic effect, selective antagonists were used. QUIN (125 /xM) toxicity was prevented by. I p.M MK-801; a non competitive NMDA receptor antagonist. On the other hand, the toxicity induced by 40 /,M KA was blocked by 100 /xM CNQX; a competitive non-NMDA receptor antagonist (data not shown).

75

C

SP

Sar-P

QUIN

QUIN +

SP

QUIN 4-

QUIN +

SP-BC Sar-P

Fig. 3. Effect of 2 p-M SP or 1 p-M Sar-P on QUIN (125 P-M) induced toxicity on striatal cholinergic activity. In some experiments, bacitracm (2 m g / m l ) and captopril (2 mM) were simultaneously added with SP (SP-BC). Treatment was initiated 1 I days after plating and eftects were evaluated 3 days after treatment. ChAT activity was calculated as cpm per culture well and expressed as percentage of the control wdues. Bars represent the mean + S.E.M. of at least three diflerent experiments (four replicates in each experiment). Statistical analysis was performed using the ANOVA test with subsequent comparisons bv Student's t test with control vahles. P < 0.05.

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N. Cah o et al. / Brain Research 740 (1996) 323-328

hanced C h A T activity per cell. To distinguish between these two possibilities we used AChE staining as a cholinergic marker [19]. In our experimental conditions, ACHEpositive cells represented approximately 0.7% of the total cell number. QUIN addition produced a significant decrease in AChE-positive cells, which was completely blocked by SP (Table 1).

150

3.4. Characterization o f TK receptors" intJoh'ed in the neuroprotectil,e effect C

SSP NNKA NNKB QUIN QUIN QUIN QUIN ÷

÷

÷

SSP NNKA NNKB

Fig. 4. Differential effect of selective neurokinin receptor agonists on QUIN (125 #M) toxicity. SSP (NK 1 receptor agonist), NNKA (NK 2 receptor agonist) or NNKB (NK 3 receptor agonist) were assayed at 10 p~M. QUIN and/or neurokinin receptor agonists were added to cultures 1l days after plating and their effects were evaluated 3 days after treatment. ChAT activity was calculated as cpm per culture well and expressed as percentage of the control values. Bars represent the mean _+ S.E.M. of at least three experiments (four replicates in each experiment). Statistical analysis was performed using the ANOVA test with subsequent comparisons by Student's t-test with control values. * P < 0.05.

It has been reported that SP is degraded by peptidases in the medium [12]. Assays adding peptidase inbibitors (2 m g / m l bacitracin and 2 m M captopril) to prevent the peptide breakdown or a SP-analog resistant to peptidases (Sar-SP) were performed. The presence of peptidase inhibitors did not change ChAT activity in control, QUIN- or SP-treated cultures (data not shown). However, SP was able to protect cholinergic cells at a lower concentration (2 /xM). Sar-SP (1 p~M) also blocked the toxic effect of QUIN on C h A T activity (Fig. 3). 3.3. Effect o f SP on striatal neuron surHual

SP (20 /xM) did not modify the number of NSE-positive cells in culture (Table 1). However, QUIN (125 /xM) treatment produced a significant decrease in total cell number and this effect was not reversed when SP was simultaneously added to cultures. The protective effect of SP on striatal cholinergic activity from damage induced by QUIN (Fig. 2) could be a consequence of increased cholinergic cell survival or en-

Since SP acts as the preferred, but not exclusive, endogenous ligand of the NK 1 neurokinin receptor subtype, the differential effects of more selective agonists on cholinergic function were studied. SSP (NKI receptor agonist), N N K A (NK 2 receptor agonist) or NNKB (NK~ receptor agonist) were used in these experiments. SSP (10 ~ M ) treatment alone did not modify ChAT activity with respect to control cultures. However, the same concentration of N N K A or NNKB increased the enzyme activity. All of them were able to protect cholinergic neurons against QUIN toxicity, and maintained the level of ChAT activity even in the presence of QUIN (Fig. 4).

4. Discussion In the present paper, we have studied the possible protective role of TKs against excitatory amino acid-induced injury in striatal cultures. Our results show that TKs are able to protect striatal cholinergic neurons against QUIN, but not K A toxic effects. QUIN or K A treatment induced a dose dependent decrease in ChAT activity in cultured striatal neurons. Our results show that striatal cholinergic cells are relatively resistant to injury by QUIN, while exhibiting greater vulnerability to KA. These results confirm previous reports showing that N M D A agonists have a weaker toxic effect than K A both in vitro [26,27] and in vivo [36]. SP blocked the decrease in ChAT activity induced by the N M D A receptor agonist, QUIN, but not by the nonN M D A receptor agonist, KA. The differential effect of SP against the different excitatory amino acids could be due to the excitatory amino acid receptor stimulated ( N M D A or non-NMDA), as they activate different pathways of toxic-

Table 1 Effect of SP on striatal neuron survival NSE(+) cells AChE(+)cells

Control

SP

QUIN

QUIN + SP

40000 ± 1028 305_+ 17.5

37787 4- 863 282_+ 20

35237 + 799 * 227_+ 12.5 '

34750 _+ 1145 * 295+_ 22.5

Striatal cultures were incubated with 125 /xM QUIN and/or 20 /xM SP for 3 days begining 11 days after plating. Cells were stained for NSE or AChE and counted using 100 × magnification on a network of 1 × 1 cm. Counts were made in four randomly selected squares and expressed as cells/era2. Values represent the mean + S.E.M. of four independent experiments with triplicate wells tbr each culturing condition. Statistical analysis was pedk~rmedusing the ANOVA test with subsequent comparisons by Student's t-test. * P < 0.05 compared with conlrol values.

N. Cah,o et al. / Brain Research 740 (1996) 323-328

ity [11]. Present results show that SP prevented the decrease in AChE-positive cells produced by QUIN, but did not counteract its effect on total cell number. About half of striatal aspiny interneurons showing immunoreactivity for the SP receptor are cholinergic neurons and the others correspond to somatostatinergic cells [25,43]. Since AChE is a marker of both populations of interneurons in the striatum [5], our results suggest that SP may selectively act on a subpopulation of striatal cells, including cholinergic neurons. We have also characterized the type of TK receptor involved in the neuroprotective effect. Since SP acts as preferring but not exclusive NK~ receptor agonist, we used more specific agonists for NK t, NK 2 or NK 3 receptors. Our results show that the three subtypes of neurokinin receptors are involved in the protection of striatal cholinergic neurons. SSP, NNKA and NNKB were able to block the decrease in ChAT activity induced by QUIN. However, NK 2 and NK 3 receptor agonists increased ChAT activity even in the presence of QUIN. These results are in agreement with functional studies about the regulation of cholinergic neurons by TKs, showing that NK 2 and NK 3 receptor agonists are mainly involved in the induction of ACh release in striatal slices [1,2]. In a previous paper, we suggested a possible protective effect of TKs on cholinergic neurons against excitotoxicity in the neostriatum. Studying ACh release in striatal slices, we observed that TKs-stimulated cholinergic cells were more resistant to QUIN toxicity [3]. The present data reinforce this hypothesis, showing that TKs may play an important role in the maintenance and protection of cholinergic neurons in the neostriatum. The mechanism of protection of TKs against excitotoxicity is unknown. However, several possibilities may be considered. It has been suggested that neuronal activity can increase cell survival during development, hut it also plays a crucial role in the maintenance of synaptic connections in the adult [29]. In fact, the degree of damage of catecholaminergic systems can be reduced by increasing activity [23,33]. Furthermore, depolarization greatly increases the survival of neurons in different neuronal culture systems [10,30]. TKs can increase cholinergic activity in the adult [1,2,18] as well as in the developing neostriatum [37]. Thus, the neuroprotective effect of TKs may be related to an increase in neuronal activity. It has been suggested that activity-dependent regulation of neurotrophic factor expression might be a mechanism by which synaptic stimulation would regulate the survival of postsynaptic neurons [15]. Glial cells could also mediate the neuroprotective effect. It is known that astrocytes in culture synthesize a variety of neurotrophic factors and neuropeptides [42] and express TK receptors [44]. These observations suggest that the activation of TK receptors may regulate the synthesis of neurotrophic factors by astrocytes. In conclusion, our results show a neuroprotective effect

327

of TKs on striatal cholinergic cells, and suggest that these peptides could be useful in the development of therapeutic approaches to neurodegenerative disorders affecting cholinergic neurons.

Acknowledgements We gratefully acknowledge Dr. C.F. Dreyfus and Dr. W.J. Friedman for useful comments on the manuscript. We would also like to thank Anna Orozco for her excellent technical assistance. This work was supported by grants from DGCYT (Ministerio de Educaci6n y Ciencia, Spain).

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