Protective effect of nerve growth factor against glutamate-induced neurotoxicity in cultured cortical neurons

Protective effect of nerve growth factor against glutamate-induced neurotoxicity in cultured cortical neurons

296 Brain Research, f~32 (1993) 296 3(~2 ,~', 1993 Elsevier Science Publishers B.V. All rights reserved 1t006-S993/93/$116.0{~ BRES 19596 Protectiv...

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296

Brain Research, f~32 (1993) 296 3(~2 ,~', 1993 Elsevier Science Publishers B.V. All rights reserved 1t006-S993/93/$116.0{~

BRES 19596

Protective effect of nerve growth factor against glutamate-induced neurotoxicity in cultured cortical neurons ** Shun Shimohama a , , Nobuo Ogawa b Yutaka Tamura c, Akinori Akaike ~, Tetsuya Tsukahara b, Hiroo Iwata b, Jun Kimura a Department of Neurology, Faculty of Medicine, Kyoto Unicersity, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606, Japan, h Department of Cerebrocascular Surgery, National Cardiovascular Center, Suita, Osaka 565, Japan, ~ Department of Neuropharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama Unicersity, Fukuyama 729-02, Japan (Accepted 17 August 1993)

Key words: Recombinant h u m a n nerve growth factor; Glutamate; Neurotoxicity; Culture: Protection

The effect of recombinant h u m a n nerve growth factor (hNGF) and mouse N G F on cultured rat cortical neurons was examined. The D N A fragment coding the h u m a n N G F gene was isolated and inserted downstream from the SV40 promoter in a plasmid containing the dihydrofolate reductase cDNA, and this plasmid was introduced into Chinese hamster ovary (CHO) cells to establish cells producing recombinant hNGF. The recombinant h N G F protein secreted by C H O cells was confirmed to be biologically active in an assay using PC12 cells. Brief exposure of cortical cells to glutamate followed by incubation with glutamate-free medium reduced cell viability by 60-70% when compared with the control culture. Simultaneous addition of recombinant h N G F or mouse N G F to rat cortical cultures with glutamate did not affect this reduction of cell viability. However, 24 h pretreatment of rat cortical cultures with recombinant h N G F or mouse N G F resulted in a significant reduction of glutamate-induced neuronal damage. Mouse N G F also protected cortical neurons against N-methyl-D-aspartate (NMDA)- and kainate-induced neuronal damage. These findings suggest that N G F can protect cortical neurons against glutamate-induced neurotoxicity.

INTRODUCTION Growth factors play an important role in the development and maintenance of neural networks 15. Nerve growth factor (NGF) is the most thoroughly studied of the growth factors; it promotes the survival and outgrowth of peripheral sensory and sympathetic neurons 3t as well as central cholinergic neurons 16. Beyond their role in normal brain development, growth factors may have an important influence on the response of the brain to injury and to neurodegenerative disorders 1°'14' 18,42. For example, NGF can prevent damage to cholinergic neurons associated with brain lesions in animal models of neurodegenerative disorders 2A8'27'29'3°'45'5°. Such observations have led to various proposals to use neuronal growth factors for the treatment of neurodegenerative disorders tS. However, the actions of N G F have not been determined in brain regions such as the hippocampus and cerebral cortex, which are particu-

larly vulnerable to both acute (e.g. stroke) and chronic (e.g. Alzheimer's disease) neurodegenerative disorders. Cheng and Mattson 5'6 recently demonstrated that NGF can protect cultured rat hippocampal and human cortical neurons against hypoglycemic damage, suggesting that NGF may have a protective role in this brain region. It has been postulated that the neurotoxicity of glutamate plays an important role in the pathogenesis of neuronal loss from the cerebral cortex s. Recent studies using cultured cortical neurons have demonstrated that brief exposure to glutamate produces delayed degeneration of mature neurons 7. The objective of the present study was to investigate the effect of N G F on the process of neuronal damage in the central nervous system (CNS). Accordingly the effect of recombinant hNGF on glutamate-induced neuronal cell death was assessed using cultured neurons from the rat cerebral cortex.

* Corresponding author. Fax: (81) (75) 761-9780. ** This work was presented in part at the XVlth International Symposium on Cerebral Blood Flow and Metabolism. Sendal, Japan, May 24. 1993.

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297 MATERIALS AND METHODS Plasmid construction and transfection The DNA fragment coding the human NGF gene from nucleotides 9117 to 9894 reported by Ullrich et al? I was obtained by the polymerase chain reaction method 43 using genomic DNA prepared from human blood cells as a template. After confirmation of its nucleotide sequence, the fragment was inserted downstream from the SV40 promoter in a pGEM3-derived plasmid bearing the dihydrofolate reductase (dhfr) gene that was prepared from plasmid pSV2dhfr 31. Then 30 /zg of DNA from the resultant plasmid (phNGF4) was used for the transfection of 106 CHO-dhfr cells(CHO DUKX BII) 52 by the method of Chen and Okayama 4, and dhfr ÷ stable colonies and CHO cells resistant to methotrexate (MTX) were obtained by the method of Kingston26

NGF protein assay One day culture medium of MTX-resistant cells was used as the recombinant hNGF source for this study, and the number of cells in the culture dish was counted with a carter counter after suspending the cells in phosphate-buffered saline. The recombinant hNGF protein content of the medium was measured with a sandwich enzymelinked immunosorbent assay (sandwich ELISA) 13 using an anti-mouse NGF monoclonal antibody (Boehringer Mannheim), an anti-mouse

NGF monoclonal antibody conjugated with 13-galactosidase (Boehringer Mannheim), and mouse NGF (Sigma) as the standard. Bioassay of NGF was also performed with PC12 cells as described by Stephani et aLas.

Measurement of neurotoxiciy Dissociated murine cortical cell cultures were prepared by the method of Dichter 11, with some modifications~. In brief, the whole cerebral cortex was removed from fetal rats at 16-18 days of gestation. The tissue was minced using scalpel blades, and filtered through a stainless steel mesh (150 mesh). Then the resultant single-cell suspension was plated on plastic coverslips which were placed in Falcon 60 mm dishes (3-4.5× 106 cells/dish) containing Eagle's minimal essential medium (MEM, Eagle's Salts) supplemented with 10% heat-inactivated fetal bovine serum (1-9 days after plating) or 10% heat-inactivated horse serum (10-14 days after plating), glutamine (2 mM), glucose (total 11 mM), sodium bicarbonate (24 mM), and HEPES (10 mM). Cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere. After 8 days, the growth of nonneuronal cells was halted by the addition of 10 -5 M cytosine arabinoside. Only mature (10-14 days) cultures were used in the study. Glutamate-induced neurotoxicity was quantitatively assessed by the examination of cultures under Hoffman modulation microscopy at ×400. Experiments were carried out in Eagle's MEM at 37°C.

A

B

C

D

Fig. 1. Biological activity of recombinant hNGF. The biological activity ot recombinant hNGF was confirmed by its promotion of neurite outgrowth from PC12 cells. PC 12 cells were cultured in the absence (A) or in the presence of 33 ng mouse NGF/ml (B). The cells were also cultured in the conditioned medium obtained from recombinant CHO cells transfected with human NGF cDNA (approximately 7.4 ng mouse NGF equivalents/ml) (C), and in the medium from CHO cells that had not been transfected with hNGF cDNA (D). Bar = 25 ~m.

298 After drug exposure, cell cultures were stained with 1.5% Trypan blue for 10 min at room temperature, fixed with isotonic formalin (pH 7.0, 2-4°C), and then rinsed with physiological saline. Cells that stained with the Trypan blue were regarded as non-viable. At least 200 cells were counted to determine the viability of each culture. In each experiment, 5-6 coverslips were used to obtain the mean + S.D. of cell viability. Recombinant hNGF and mouse NGF were diluted with Eagle's MEM immediately before the experiments. The final concentrations of recombinant hNGF added to the cultures were approximately 3.3 and 10 ng mouse NGF equivalents/ml. Glutamate-induced neurotoxicity was determined by exposure to 1 mM glutamate for 10 min, followed by incubation in glutamate-free medium for 1 h. The protective effect of recombinant hNGF and mouse NGF against glutamate neurotoxicity was determined by the simultaneous addition or 24 h pretreatment of recombinant hNGF or mouse NGF to rat cortical cultures with glutamate. To determine whether the protective effect of NGF is specific for excitotoxicity induced by activation of particular subtypes of glutamate receptor, specific glutamate receptor agonists were also used as previously described I. In brief, cultures were treated with N-methyl-o-aspartate (NMDA) (1 mM) was added in the Mg2*-free medium for 10 min, followed by incubation in NMDA-free medium for 1 h. Kainate (1 mM) was added in the Mg2+-containing medium for 1 h without further incubation. The protective effect of mouse NGF against NMDA- or kainate-induced neurotoxicity was determined by the pretreatment of rat cortical cultures with mouse NGF for 24 h before the administration of NMDA or kainate.

Drugs Eagle's minimum essential medium was purchased from Nissui Pharmaceutical Co. (Tokyo, Japan); kainate and monosodium Lglutamate were purchased from Nakalai Tesque (Kyoto, Japan); NMDA was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent grade and were obtained commercially.

Statistical analysis Data were expressed as the mean+S.D. The statistical significance of differences was determined by Dunnett's two-tailed test. RESULTS

Establishment of CHO cells producing recombinant hNGF To establish a r e c o m b i n a n t h N G F - p r o d u c i n g cell line, p l a s m i d p h N G F 4 b e a r i n g b o t h the hNGF a n d dhfr g e n e s was i n t r o d u c e d into C H O - d h f r - cells, a n d eight colonies o f d h f r + cells w e r e o b t a i n e d . T o amplify t h e i n t e g r a t e d p l a s m i d in t h e g e n o m e , t h e s e eight colonies w e r e c u l t u r e d in m e d i u m s u p p l e m e n t e d with i n c r e a s i n g a m o u n t s of M T X , a p o t e n t i n h i b i t o r o f dhfr, a n d o n e colony which was r e s i s t a n t to 0.02 m M M T X was selected. R e c o m b i n a n t h N G F p r o t e i n s e c r e t i o n into t h e c u l t u r e m e d i u m o f t h e r e s i s t a n t cell line ( H I C A 4 - 0 2 ) was d e t e c t e d by t h e sandwich E L I S A , a n d its p r o d u c t i o n was a p p r o x i m a t e l y 370 ng m o u s e N G F e q u i v a l e n t s / 1 0 6 c e l l s / d a y . In contrast, no N G F was d e t e c t e d in t h e c u l t u r e m e d i u m o f a M T X - r e s i s t a n t cell line ( V C A 4 - 0 2 ) e s t a b l i s h e d by similar t r a n s f e c t i o n with a p l a s m i d D N A b e a r i n g t h e dhfr gene but not t h e

hNGF gene. To c o n f i r m t h a t the r e c o m b i n a n t h N G F

protein

s e c r e t e d by H1CA4-02 cells was biologically active, the c o n d i t i o n e d c u l t u r e m e d i u m of H1CA4-02 cells was a d d e d into c u l t u r e s of PC12 cells. PC12 cells c u l t u r e d in the p r e s e n c e of H I C A 4 - 0 2 m e d i u m at a final conc e n t r a t i o n of a p p r o x i m a t e l y 7.4 ng m o u s e N G F equival e n t s / m l e x t e n d e d thin fibers as they did when cult u r e d in the p r e s e n c e of 33 ng of m o u s e N G F , w h e r e a s PC12 cells c u l t u r e d with m e d i u m from VCA4-02 cells did not (Fig. 1). Since t h e s e results strongly s u g g e s t e d that the r e c o m b i n a n t h N G F p r o t e i n s e c r e t e d by H I C A 4 - 0 2 cells had significantly biological activity, the r e c o m b i n a n t h N G F - c o n t a i n i n g culture m e d i u m was used for the following studies.

Protective effect of recombinant hNGF against glutamate neurotoxicity In o u r previous study, a m a r k e d r e d u c t i o n of cell viability was i n d u c e d by exposing c u l t u r e s to l m M g l u t a m a t e for 10 rain followed by i n c u b a t i o n in glutam a t e - f r e e m e d i u m for at least l h ~. T h e r e f o r e , we assessed the cytotoxic effects of g l u t a m a t e by 10 min of e x p o s u r e followed by g l u t a m a t e - f r e e m e d i u m for 1 h. E x p o s u r e s to g l u t a m a t e i n d u c e d a significant reduction in cell viability, a n d s i m u l t a n e o u s a d d i t i o n of m o u s e N G F or r e c o m b i n a n t h N G F to rat cortical c u l t u r e s with g l u t a m a t e d i d n o t affect this r e d u c t i o n of cell viability. I n c u b a t i o n of cortical c u l t u r e s with medium containing mouse NGF or recombinant hNGF did not affect t h e i r viability w h e n c o m p a r e d to c o n t r o l cultures. H o w e v e r , 24-h p r e t r e a t m e n t of rat cortical c u l t u r e s with m o u s e N G F or r e c o m b i n a n t h N G F res u l t e d in a d r a m a t i c r e d u c t i o n in g l u t a m a t e - i n d u c e d n e u r o n a l d a m a g e . T h e p r o t e c t i v e effect of r e c o m b i n a n t h N G F against g l u t a m a t e toxicity was c o n c e n t r a t i o n - d e p e n d e n t , a n d high c o n c e n t r a t i o n s d e m o n s t r a t e d m o r e p r o t e c t i v e effect than low c o n c e n t r a t i o n s ( T a b l e l). Fig. 2 shows r e p r e s e n t a t i v e e x a m p l e s of the effects of glutam a t e a n d N G F . G l u t a m a t e t r e a t m e n t m a r k e d l y inc r e a s e d the n u m b e r of cells s t a i n e d by T r y p a n blue (Fig. 2B) c o m p a r e d with u n t r e a t e d cells (Fig. 2A). P r e t r e a t m e n t of r e c o m b i n a n t h N G F or m o u s e N G F to t h e m e d i u m with g l u t a m a t e r e d u c e d the n u m b e r of cells s t a i n e d by T r y p a n b l u e (Fig. 2C a n d 2D). In a d d i t i o n , we e x a m i n e d the effect of m o u s e N G F on n e u r o t o x i c i t y i n d u c e d by N M D A or kainate. A 10-min e x p o s u r e of t h e cells to 1 m M N M D A , followed by 1 h i n c u b a t i o n , or 1 h e x p o s u r e to 1 m M kainate, m a r k e d l y i n c r e a s e d the n u m b e r of cells s t a i n e d by T r y p a n blue. This result is c o n s i s t e n t with the observation o f o u r previous study 1. T w e n t y - f o u r h p r e i n c u b a tion of rat cortical c u l t u r e s with m o u s e N G F r e s u l t e d in a significant r e d u c t i o n in N M D A - or k a i n a t e - i n d u c e d n e u r o n a l d a m a g e ( T a b l e I).

299 DISCUSSION The recombinant hNGF produced in this study stimulated neurite outgrowth from PC12 cells with an activity as high as that of authentic mouse NGF. The specific biological activity of recombinant mouse NGF and hNGF produced by E. coli 2° and S. cerevisiae 23, respectively, are reported to be only 1/200-1/1,000 of that of mouse NGF isolated from the submaxillary gland. In contrast, the activity of our recombinant hNGF preparation was comparable to that of authentic mouse NGF. Iwane et al. 22 have also succeeded in producing substantial amounts of biologically active hNGF from CHO cells by recombinant DNA techniques. These findings suggest that CHO cells produce recombinant hNGF protein as the native form with the correct tertiary structure 44. The present study indicated that recombinant hNGF and mouse NGF can protect cortical neurons against glutamate toxicity. Thus, in addition to its actions on

peripheral neurons and central cholinergic neurons, NGF can also directly influence cultured cerebral cortical neurons which probably include both cholinergic and non-cholinergic neurons. Previous studies have failed to detect a trophic effect or neurite outgrowth promotion in cortical neurons by NGF 34'35, but the present study clearly showed that NGF affords these neurons considerable protection against glutamate-induced damage. Our present findings were in line with the reports that NGF can protect rat hippocampal and human cortical neurons against hypoglycemic damage 5'6. Thus, NGF may affect a much broader population of neuron in the CNS than has been previously appreciated. Although we could not completely exclude the possibility that the in vitro neuroprotective effect shown in this study could be due to synergic action of several, as yet unknown, growth factors which might be produced by recombinant CHO cells, it is likely that NGF has a neuroprotective effect against glutamate-induced neuronal cell death, since we did

A

B

C

D

j Fig. 2. Effects of recombinant hNGF on glutamate-induced neurotoxicity. Cultures were photographed after Trypan blue staining followed by formalin fixation. A: untreated cells (control). B: glutamate-treated cells. C: cells pretreated for 24 h with recombinant hNGF (approximately 10 ng mouse NGF equivalents/ml), followed by glutamate treatment. D: cells pretreated for 24 h with 100 ng/ml mouse NGF, followed by glutamate treatment. In B-D, cultures were exposed to 1 mM glutamate, followed by incubation with normal glutamate-free solution. Bar = 50 Izm.

3OO "FABLE 1

( N M D A ) subclass of g l u t a m a t e receptors have been

Ef]~'cts of recombinant hNGF and mouse NGF on glutamate-induced toxicity for cultured rat cortk al neurons

shown to protect cultured cortical n e u r o n s from gluta-

Treatment

n

Viability(%)

5 5

90.6_+1.2 29.6+6.0

5 5

33.4_+2.3 31.5 _+2.0

Simultaneous application with glutamate

Control (untreated) Glutamate (1 mM) u Glutamate (1 raM)h + mouse NGF ~ high-dose recombinant hNGF ~ Pretreatment (24 h)

mate cytotoxicity '~'17, suggesting that the N M D A receptor is the p r e d o m i n a n t m e d i a t o r of g l u t a m a t e - i n d u c e d toxicity in these n e u r o n s . M a t t s o n et a[ ~" revealed that N G F p r e v e n t e d the hypoglycemia-induced elevation of intracellular calcium, and p o s t u l a t e d that N G F can stabilize the calcium homeostasis of central n e u r o n s a n d thereby protect t h e m against e n v i r o n m e n t a l insuits. Iacopino et al. 21 recently showed that exogenously delivered r e c o m b i n a n t h N G F increases the neu-

Control (untreated) mouse NGF d alone high-dose recombinant hNGF d alone

5 5 5

90.6 _+1.2 91.5 _+0.9 91.7_+0.8

Control (untreated) Glutamate (1 mM) b Glutamate (1 raM) ~ + mouse NGF ~ high-dose recombinant hNGF ~ low-dose recombinant hNGF ~"

5 5

89.1 _+3.6 41.8_+2.9

5 5 5

55.6_+3.9 a 72.7_+4.0a 54.6_+4.7a

5 5 5

86.0+ 1.2 83.3 _+2.3 45.8_+1.0

ously shown that cholecystokinin prevents N M D A rec e p t o r - m e d i a t e d cytotoxicity without reducing calcium influx through inhibition of nitric oxide tk~rmation by

5 5

66.6_+1.1 ~' 35.7 _+1.0

N M D A receptor activation 49, suggesting a n o t h e r possi-

5

60.0_+1.4 a

ronal level of calcium b i n d i n g p r o t e i n (calbindin-D28k). In addition, Eichler et al. 12 have f o u n d that the N G F d e p e n d e n c e of n e u r o n s is inversely related to the intracellular calcium level. These results suggest that N G F may f u n c t i o n as a n e u r o p r o t e c t i v e factor by e n h a n c i n g the sequestration of calcium and thus retard calciumm e d i a t e d n e u r o d e g e n e r a t i o n . However, we have previ-

Control (untreated) mouse NGF f alone NMDA (1 raM) g NMDA(I raM) ~+ mouse NGF i kainate ( ! mM) h Kainate (1 mM) h + mouse NGF i

~' P < 0.01, Dunnett's two-tailed test vs. glutamate, NMDA or kainate treatment. b Cultures were treated with glutamate for 10 min followed by incubation with glutamate-free medium for 1 h. Simultaneous application of mouse NGF (100 ng/ml) or recombinant hNGF (approximately 10 ng mouse NGF equivalents/ml) with 1 mM glutamate followed by 1 h of incubation in glutamatefree medium did not affect viability when compared to glutamate treatment. d Preincubation with mouse NGF (100 ng/ml) or recombinant hNGF (approximately 10 ng mouse NGF equivalents/ml) for 24 h did not affect viability when compared to control cultures. ~ Preincubation with NGF (100 ng/ml) or recombinant hNGF (approximately 10 and 3.3 ng mouse NGF equivalents/ml) from 24 h prior to glutamate application had a significant protective effect against glutamate-induced neuronal damage. Preincubation with mouse NGF (100 ng/ml) for 24 h did not affect viability when compared to control cultures. g Cultures were treated with NMDA for 10 rain followed by incubation with NMDA-free medium for 1 h. h Cultures were treated with kainate for 1 h. Preincubation with mouse NGF (100 ng/ml) from 24 h prior to NMDA or kainate application had a significant protective effect against NMDA- or kainate-induced neuronal damage.

not transfect c D N A which may p r o d u c e p r o t e i n factors other t h a n N G F a n d the c o n d i t i o n e d m e d i u m of C H O cells without N G F c D N A had n o trophic effect on P C 12 ceils. T h e m e c h a n i s m by which N G F protects c u l t u r e d cortical n e u r o n s against g l u t a m a t e - i n d u c e d neurotoxicity is not fully u n d e r s t o o d at present. Competitive or n o n - c o m p e t i t i v e blockers of the N-methyl-D-aspartate

ble m e c h a n i s m of N G F - i n d u c e d protection. T h e present study also d e m o n s t r a t e s that N G F p r e t r e a t m e n t might p r e v e n t g l u t a m a t e neurotoxicity m e d i a t e d not only by N M D A receptors b u t also by k a i n a t e receptors t. T h e precise m e c h a n i s m is yet to be defined. T h e r e are several reports indicating that N G F receptor expression can be i n d u c e d in h i p p o c a m p a l n e u r o n s by e n v i r o n m e n t a l d a m a g e 4°'53 a n d in cortical n e u r o n s by old age a n d A l z h e i m e r ' s disease 37, suggesting that neurons within the h i p p o c a m p u s and cortex exhibit plasticity of N G F receptor expression. Moreover, the protective effect of N G F n e e d s to be r e - e v a l u a t e d in light of the recent discovery of protein kinases of the trk gene family, the p r o t e i n products of which form f u n c t i o n a l receptors for N G F , b r a i n - d e r i v e d n e u r o t r o p h i c factor, n e u r o t r o p h i n - 3 , n e u r o t r o p h i n - 4 , a n d n e u r o t r o p h i n 5 3'24'25'27'28'33'38'39"46'47. R e c e n t data indicate that the p r o d u c t of the trk gene, p140 trk, is a critical compon e n t of the N G F receptor. H o l t z m a n et al. has recently shown that the distribution of p140 irk m R N A is limited in the adult rat basal f o r e b r a i n and c a u d a t e - p u t a m e n m a g n o c e l l u l a r n e u r o n s , a n d failed to detect neuronal p o p u l a t i o n s in the cortex that c o n t a i n e d p140 "k m R N A 19. T h e r e f o r e , f u r t h e r studies are r e q u i r e d to investigate the m o l e c u l a r m e c h a n i s m of N G F - i n d u c e d p r o t e c t i o n against g l u t a m a t e toxicity in c u l t u r e d rat cortical n e u r o n s . In conclusion, we f o u n d that r e c o m b i n a n t h N G F protected rat cortical n e u r o n s from g l u t a m a t e - i n d u c e d toxicity. This observation suggests that h u m a n N G F may potentially be useful for the t r e a t m e n t of n e u r o d e -

301

generative changes due to cerebral ischemia and Alzheimer's disease. Acknowledgements. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (03224105, 04258211, 05251209, 05261206), for General Scientific Research (05670557) from the Ministry of Education, Science and Culture, Japan, and by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

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