Nerve growth factor (NGF) regulates tachykinin gene expression and biosynthesis in rat sensory neurons during early postnatal development

Neuropeptides (1993) 24,351-357 0 Longman Group UK Ltd 1993

Nerve Growth Factor (NGF) Regulates Tachykinin Gene Expression and Biosynthesis in Rat Sensory Neurons During Early Postnatal Development H. VEDDER*, H.-U. AFFOLTERt and U. OlTENS Max Planck Institute of Psychiatry, Clinical Institute, Department of Neuroendocrinology, Kraepelinstr, Z-70, W-8000 Munich 40, Germany, tH.-U. Affolter, Bioserve/Biotechnologies, 337 Paint Branch Drive, College Park, MD 20742, USA and SU. Otten, Institute of Physiology, University of Base/, Vesalianum, Vesalgasse 7, CH-4051 Base/, Switzerland (Reprint requests to HV)

Abstract-The regulatory effects of nerve growth factor (NGF) on tachykinin biosynthesis in rat primary sensory neurons during the period of postnatal development were examined under in vivo and in vitro conditions. Administration of NGF to neonatal rats led to a significant increase in protein levels of substance P (SP) and neurokinin A (NKA) in trigeminal and dorsal root ganglia (DRG). In addition, Northern blot analysis revealed that preprotachykinin mRNA was upregulated in sensory ganglia of neonatal animals after treatment with NGF. Using a well-defined in vitro system for neonatal rat DRG and trigeminal ganglia neurons, we found that addition of NGF induced SP and NKA protein levels in a dose-dependent manner. Furthermore, preprotachykinin mRNA was markedly increased in cultured DRG and trigeminal ganglia neurons in the presence of NGF. Thus, our results clearly demonstrate that NGF regulates tachykinin gene expression and biosynthesis both in vivo and in vitro during the developmental period of rat sensory neurons.

Introduction

Nerve growth factor (NGF) is a polypeptide essential for the survival and differentiation of sympathetic neurons and the majority of neural Date received 26 October 1992 Date accepted 6 January 1993 Comspondence and proofs to: Dr. H. Vedder, Max Planck Institute of Psychiatry, Clinical Institute, Department of Neuroendocrinology, Kraepelinstr. 2-10, W-8000 Munich40, Germany

crest-derived sensory neurons.‘5*3oMoreover, NGF plays a physiological role in the mammalian central nervous system (CNS) as a trophic agent for cholinergic neurons in the basal forebrain.14J8 In addition to these neurotrophic effects, recent data point to a modulatory role- of NGF in iniknmatorv and immune reactions.22 Although the trophic actibns of NGF on the survival of sensory and sympathetic neurons have been characterized in some detail, up to now its effects on the biosynthesis of neuropep-

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352 tides in primary sensory neurons have been studied only to some extent.L2J7J1 Application of NGF and NGF antibodies showed that NGF is involved in the modulation of substance P (SP) levels in sensory ganglia in vivo: Retrogradely transported NGF markedly increased the SP content in corresponding dorsal root ganglia (DRG) of adult rats.* In NGF-depleted animals, the levels of neuropeptides, including SP, were reduced in the DRG and the dorsal spinal cord.’ On the other hand, injection of NGF led to an increase in SP levels in these tissues.*J2,23The finding that NGF can prevent the neurotoxic effects of capsaicin on DRG neurons in developing rats24 strongly supports the view that this growth factor exerts trophic actions on the type of sensory neurons that are involved in nociceptive and inflammatory reactions.z1*3’ A first in vitro study on DRG by Lindsay and Harmar” indicated that NGF upregulates the neuropeptides SP and calcitonin gene-related peptide (CGRP) in adult rat DRG neurons, which are not dependent on NGF for their survival. In the present study, we used a combined in vivo and in vitro approach to show that NGF induces the biosynthesis of both tachykinins, SP and neurokinin A (NRA), not only in neonatal DRG but also in trigeminal sensory neurons during the period of early postnatal development. Materials and methods

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detail elsewhem3r After plating, neurons were cultivated in Leibovitz L15 medium (GibcoBRL, Basel, Switzerland) with additives31 and 100 @ml NGF. Non-neuronal cell proliferation was effectively suppressed by addition of cytosine arabinoside (5x10-6 M) (Serva, Heidelberg, FRG) to the culture medium.” To study the dose-dependent effects of NGF, 8-12-day-old cultures were preincubated in NGF-free medium for 2 d. Then the medium was changed and the treatment groups were exposed to NGF in different concentrations for 72 h. Radioimmunoassay

ofpeptides

Radioimmunoassays (RIAs) were performed as described previously.7J1,31 Briefly, peptides were extracted from dissected tissues in 2 N acetic acid by ultrasonication, lyophilized and resuspended in RIA buffer. After incubation with the SP and NKA antibody for 24 h at 4°C }1z51]-SPlabelled with the chloramine-T method according to Powell et al25and >L251]-NKAtracer (Amersham, Zurich, Switzerland) were added and the solutions were incubated for an additional 24 h at 4°C. Non-bound radioactivity was absorbed to a charcoal solution (for details see: Vedder and Gtten31 After centrifugal separation, pellet and supematant fractions were counted in a gamma counter. Peptide concentrations were calculated from known concentrations of synthetic standards run in the same assay (Bachem, Bubendorf, Switzerland).

In vivo experiments 4-day-old rat pups (Fuellinsdorf albino rats) were used for the in vivo experiments. Rats were injected subcutaneously with 20 pl of either saline alone (controls) or NGF in saline (2 p&g body weight) daily for 5 d. On postnatal day 10, rats were killed by decapitation and the tissues were dissected and immediately frozen on dry ice for determination of tachykinin protein and mRNA. In vitro experiments DRG and trigeminal ganglia were microdissected from neonatal rats (l-day-old Fuellinsdorf albino rats), enzymatically (0.01% collagenase }type V, Sigma, Deisenhofen, FRG]/O.4% trypsin )Amimed, Basel, Switzerland]) and mechanically dissociated and plated into pretreated 24-well dishes (Nunc, Gibco/BRL, Basel, Switzerland) as described in

Detection of tachykinin mRiVA For measurement of tachykinin mRNA, cultured neurons were removed from plastic dishes with a rubber policeman, pelleted and immediately frozen on dry ice. Cells for mRNA determination were treated with either 25 @ml NGF (control) or 200 &ml NGF for 72 h. Isolation of total cellular RNA from frozen tissue preparations andneuronal cultures was performed according to the protocol of Chirgwin et al4with modifications as described by Affolter and Reisine.’ Quantification, blotting and hybridization of filters was done according to Eiden et aP with a radiolabelled antisense mRNA probe derived from a human SP cDNA fragment containing the coding region for SP and showing 98% sequence homology to rat SP RNA (Affolter, unpublished). Lanes for markers contained radioactively labelled restriction

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fragments (Taq 1) of Xl74 RF DNA (fragments smaller than 406 bases not resolved). Sfatistical evaluation All quantitative data are expressed as mean&tandard deviation. Statistical comparisons of treatment groups and controls were made with Student’s t-test. P < 0.05 was considered significant. Preparation of NGF NGF was purified according to Bocchini and Angelett? with modifications as described by Weskamp and Otten. 32The purity of NGF preparations was controlled by silver staining of protein gels and concentrations were determined by ELISA with specific antibodies.

Results Effects of NGF administration on the tachykinin content of sensory ganglia in vivo Figure 1 shows the effects of NGF treatment on tachykinin (SP and NRA) content in lumbar and trigeminal sensory ganglia of lo-day-old rats after treatment with NGF for 5 d. Determination of tachykinin proteins in DRG revealed a significant increase both in SP from 96 to 173 pg SP/ganglion (180%) (Fig. 1A) (P < O.OS),and in NRA from 34 to 75 pgNKA/ganglion (220%) (Fig. 1B) (P < O.OS), afterNGF treatment. In addition, the tachykinin content in trigeminal ganglia was also significantly increased, from 86 to 105 pg SP/ganglion (Fig. 1C) (P < 0.05) and from 5 1 to 80 pg NRA/ganglion (Fig. 1D) (P < 0.05) after NGF treatment, reaching levels C

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Fig. 1 Effects of NGF on biosynthesis of tachykinins (SP (A, C), NIL4 (Ei, D) in primary sensory ganglia (dorsal root (A, B) and trigeminal ganglia (C, D) in vivo (2 pg/g body weight NGF in saline or saline alone (control); treatment from postnatal days 4 to 9) (P < 0.05, Student’s t-test).

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after NGF treatment (Fig. 3) (P < 0.05). These in vitro data strongly support the concept that NGF acts on the biosynthesis of tachykinins in sensory neurons via a direct cellular mechanism. Effects ofNGF on tachykinin gene expression in vivo and in vitro

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To determine whether the demonstrated increase in the tachykinin content of neuronal ganglia after NGF treatment was due to a higher transcriptional activation of the tachykinin gene, we studied mRNA levels of tachykinins in neuronal tissues in vivo and in neurons grown under more defined in vitro conditions. Northern blot analysis of tachykinin mRNA from cervical DRG of control animals and rats treated with NGF revealed a markedly higher expression ofthe tachykinin gene in animals that had received NGF (Fig. 4). Further characterization of this in vivo effect with neurons of neonatal rats grown in vitro and incubated with small (25 ngiml) and large (200 r&ml) amounts of NGF clearly supported the view of a direct action of NGF on neuronal tachykinin gene expression in trigeminal cells (A) as well as in cultured DRG neurons (B) (Fig. 5).

Discussion

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Dose-dependency of NGF effects on tachykinin biosynthesis (SP (A), NKA (B) in primary sensory ganglia (dorsal root ganglia) in vitro (P < 0.05, Student’s t-test).

NGF is a trophic molecule that is involved in the survival of peripheral and central neuronal cells.1sJ~30In the study reported here we further

of about 120% (SP) and more than 150% (NRA) compared to untreated controls. Eflects of NGF on tachykinin biosynthesis in primary sensory neurons in vitro To examine whetherNGF affects tachykinin biosynthesis by a direct action on neuronal cells, we studied the effects of NGF on SP and NRA protein levels in sensory neurons grown under in vitro conditions. Figure 2 shows that NGF dose-dependently increased the tachykinins SP (A) and NRA (B) in cultures of rat DRG sensory neurons (P < 0.05). Furthermore, determination of the NRA content of cultures derived from trigeminal sensory ganglia also revealed a significant dose-dependent increase

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Fig. 3 Dose-dependency of NGF effects on tachykinin biosynthesis (NKA) in trigeminal ganglia in vitro (P < 0.05, Student’s t-test).

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Fig. 4 Effects of NGF on tachykinin mRNA in cervical dorsal root ganglia in vivo. (CON: control). CON: saline-treated; NGF: NGF-treated (as described in Fig. 1); L: rat liver RNA; M: markers.

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Fig. 5 Effects of NGF on tachykinin mRNA in trigeminal ganglia (A) and dorsal root ganglia sensory neurons (B) in vitro. CON: control treatment __-_. . __ . (25 ngNGF/ml); NGF: NGF-treated (200 ng NCiF/ml); M: markers.

extend the importance of NGF in the physiology of sensory neurons by demonstrating that NGF regulates gene expression and biosynthesis of the tachykinins SP and NKA during the period of early postnatal development and differentiation. With a combined in vivo and in vitro approach we show that NGF increases not only the biosynthesis of SP but also the production of the second preprotachykininderived peptide, NRA. Under in vivo conditions, NGF significantly stimulated the production of the tachykinins SP and NKA in DRG and trigeminal sensory ganglia. These results are in line with other studies suggesting an involvement of NGF in the regulation of the In vitro characterization of tachykinin SP. 12~17~18~*1,23 the effects of NGF revealed a strict dose-dependent increase in SP and NKA proteins pointing to a direct cellular action of NGF on tachykinin biosynthesis. Both tachykinins are derived from a common SP/NKA precursor gene and its mRNA, preprotachykinin mRNA, although their processing results in varying amounts of peptides in different tissues*O which is also reflected by the different protein data for both tachykinins in our study. Using Northern blot methods for the detection of preprotachykinin mRNA, we show that NGF treatment results in increased concentrations of specific mRNA in sensory spinal and trigeminal ganglia under in vivo conditions. Our finding that NGF upregulates tachykinin protein levels as well as tachykinin mRNA strongly suggests that NGF acts at the level of gene expression. Using an in vitro approach, we further support this assumption and demonstrate that NGF affects tachykinin gene expression in the sensory neuron most likely without involvement of intercellular or organismal factors. These data are in accordance with a recent study by Gilchrist et al6 suggesting a direct genomic action of NGF at the bovine preprotachykinin gene via specific NGFresponsive sequences in the 5’ region of the gene. Additional experiments are necessary to examine the intracellular pathways involved in the actions of NGF on the biosynthesis of tachykinins in more detail. Effects of NGF on target cells directly depend on initial binding of the molecule to two types of cell surface receptors with a low (I&c~O-~M) and a high (L: lo-” M) affinity for NGF.*‘J9 There is evidence that NGF activates a protein kinase9 and exerts its cellular actions via the phosphoinositol pathway.3

356 Other data show that high-affinity NGF receptors are functionally coupled to a receptor-enclosed tyrosine kinase (gp140uk).13~19 Using the polymerase chain method, we detected high levels of this tyrosine kinase in the DRG of neonatal rats, which suggests that NGF modulates tachykinin biosynthesis via this intracellular pathway (Ehrhard and Gtten, manuscript submitted). In sympathetic and cortical neurons, depolarizing stimuli influence levels of tachykinins, pointing to another possible mechanism of the regulation of tachykinin biosynthesis. 1o,26 Factors such as the thyroid status of the organ&mu or other growth factor@ may also affect tachykinin levels, indicating a complex regulation of the tachykinin gene, possibly in a tissue-specific manner. In summary, we show that the tachykinins SP and NRA are increased after NGF treatment both in vivo and under more defmed in vitro conditions. Using Northern blot techniques with a specific tachykinin probe, we further demonstrate an increase in preprotachykinin-mRNA levels in sensory ganglia under both conditions. These results strongly suggest that elevated protein levels are the result of an increased activation of the tachykinin gene. Therefore, our data further support the important role of NGF for sensory ganglia showing that NGF regulates SP and NKA biosynthesis already during the postnatal period of development. Acknowledgements We thank G. We& and P. Ehrhard for preparation of NGF. The assistance of Ms. M. Craig in the preparation of this manuscript is gratefully acknowledged. The work was supported by the Swiss National Foundation (grants 31-25690.88 and 3 l-29954.90), the Gerd Zbinden-Doerenkamp Foundation and the FFVFF, Zurich, Switzerland.

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