glands versus the constitutive release from other tissues. Key words: ... release of NGF have now begun to be analysed using cultures .... IOS HPferens. 4.7 k - w.
The EMBO Journal vol.3 no. 13 pp.3183 -3189, 1984
Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues
R.Heumann, S.Korsching, J.Scott' and H.Thoenen Max-Planck-lnstitute for Psychiatry, Department of Neurochemistry, D-8033 Martinsried, FRG, and 1Clinical Research Centre, Molecular Medicine Research Group, Harrow, Middlesex HAI 3UJ, UK Communicated by H.Thoenen
We have developed a sensitive assay for the quantification of nerve growth factor mRNA (mRNANGF) in various tissues of the mouse using in vitro transcribed RNANGF. Probes of both polarities were used to determine the specificity of the hybridization signals obtained. Comparison of NGF levels with its mRNA revealed that both were correlated with the density of sympathetic innervation. Thus, vas deferens contained high levels of both NGF and mRNANGF, whereas skeletal muscle levels were barely detectable, indicating that in peripheral tissues NGF levels are primarily regulated by the quantity of mRNANGF and not by the rate of processing of NGF precursor to NGF. However, although superior cervical ganglia contained the highest levels of NGF, its mRNA was barely detectable. Thus, the high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. The quantity of NGF found in the submandibular glands of female animals was three orders of magnitude higher than expected from their mRNA levels. This observation is discussed in the context of the difference between the mechanism of storage and exocytosis of exocrine glands versus the constitutive release from other tissues. Key words: mRNA/nerve growth factor/retrograde axonal transport/SP6/sympathetic innervation Introduction The function of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating neurons was until only recently based on indirect evidence (Thoenen and Barde, 1982; Schwab and Thoenen, 1983; Bradshaw, 1983; Darling et al., 1983). The development of a very sensitive enzyme immunoassay (detection limit 0.01 fmol per assay) permitted the reliable determination of NGF levels in peripheral fields of innervation and the corresponding sympathetic ganglia. These investigations revealed a correlation between the density of sympathetic innervation and the levels of NGF in target tissues (Korsching and Thoenen, 1983a) and provided direct evidence for the retrograde axonal transport of endogenous NGF (Korsching and Thoenen, 1983b). Furthermore, the regulation of the synthesis and release of NGF have now begun to be analysed using cultures of rat iris (Barth et al., 1984). However, the information is still very fragmentary, and an essential step towards the better understanding of how NGF synthesis is controlled would be the quantification of its mRNA. The prerequisite to this goal was the availability of cDNA probes hybridizing not only to the coding region of mature NGF but also to its precursor (Scott et al., 1983; Ullrich et al., 1983). This is of particular importance because antibodies against ,BNGF do not recogIRL Press Limited, Oxford, England.
nize its precursor(s) (Schwab et al., 1976) to the extent that they could be used for a quantitative determination of both precursor and (3NGF. Thus, the relationship between (3NGF and its mRNA must be established, in order to decide whether NGF levels are determined by the processing from its precursor(s) or by regulation of the quantity of mRNANGF available. Although sympathetic ganglia contain by far the highest NGF levels of all tissues investigated (Thoenen et al., 1983; Korsching and Thoenen, 1983a) it remains to be established whether these high levels result from retrograde axonal transport or local synthesis in sympathetic ganglia. The production of probes hybridizing to the coding region of jNGF and its precursor has now allowed us to quantitate mRNANGF in peripheral tissues in order to answer the above questions. The observations indicate that the levels of NGF in peripheral tissues are primarily determined by the level of mRNANGF and not by the rate of processing of NGF precursor to NGF and that the very high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. Results Description of the cDNANGF used The mouse cDNANGF insert used here for most of the experiments starts 14 bases downstream to the proposed translation initiation site (Scott et al., 1983) and ends at an internal PstI site located 10 bases downstream to the translation termination codon (Figure 1). Thus the bases coding for five amino acids of the putative leader sequence are lacking and are replaced by part of the C-tail added during the cloning procedure. To separate the DNA sequences coding selectively for the NGF precursor protein versus those coding for mature ,3NGF protein, the cDNANGF was cleaved by HhaI giving rise to fragments of 545 and 372 bases, respectively. The identity of the isolated fragments was verified by hybridization with chemically synthesized oligonucleotides corresponding to bases 974-992 (18-mer) and 498-519 (21-mer) (Scott et al., 1983) and the nick-translated fragments were found to hybridize individually to the identical mouse mRNANGF of 1.35 kb in size (data not shown), which has been described previously (Scott et al., 1983). In all subsequent experiments shown here probes were used that were derived from the large PstI cleaved fragment (917 bp+C-tail). A 151-bp fragment complementary to the 3' non-coding region of the mRNANGF was not used here. Evaluation of optimal hybridization conditions using cDNANGF and RNANGF Two presumptive mRNANGF message sizes were found in heart atrium and vas deferens that migrated as a 1.35-kb band in the poly(A) + fraction and as a 4.7-kb band in the poly(A) - fraction, respectively (Figure 2). It was evident from Figure 2 that using the nick-translated cDNANGF probe only weak signals for the heart atrium could be achieved at the 1.35-kb band. Thus, there were two reasons for applying 3183
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skeletal muscle suggesting that NGF synthesis is predominantly determined by the corresponding levels of mRNANGF. Thus, unlike the tissue-specific post-translational processing found for pro-opiomelanocortin (Herbert et al., 1983), proteolytic cleavage of a pre-existing pro-NGF protein or control of initiation of translation seem not to be the predominant mechanisms of regulation of NGF synthesis under physiological conditions. In contrast to the good correlation between the relative levels of NGF protein and mRNA in heart ventricle and atrium, vas deferens and skeletal muscle, the ratio of NGF protein to mRNA is more than three orders of magnitude higher in both male and female submandibular glands. In this
subjected to thorough analysis. Interestingly, neither 18S rRNA nor yeast 28S rRNA interacted with the probes, indicating that perhaps the insertion of GC-rich oligonucleotides in the large rRNA of higher animals (Chan et al., 1983) is responsible for their 'pseudospecific' hybridization with the probe. Using probes with reversed polarity proved to be of invaluable help in distinguishing the specific hybridization at 1.35 kb from these pseudospecific (4.7 kb) signals. Subsequently it was found that raising the hybridization temperature from 60°C to 65°C resulted in removal of the 4.7-kb band in poly(A) - or total RNA blots, while high stringency washings after low stringency hybridization had previously been unsuccessful in reducing the unspecific hybridization. In addition, unspecific signals could be further suppressed by treatment of the blots with very low concentrations of RNase
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organ NGF is present in excessively high quantities (Table I) and is stored in large secretory vesicles (Schwab et al., 1976) of the tubular cells and is released into the saliva by a classical Ca2 +-dependent secretory mechanism (Hirata and Orth, 1980). In contrast, in organ cultures of rat irides (which like vas deferens, heart ventricle and atrium represent densely innervated peripheral organs) it has been demonstrated that NGF is released continuously into the medium also in the absence of exogenous calcium (Barth et al., 1984) suggesting a 'constitutive' mechanism of secretion (Tartakoff et al., 1978; Gumbiner and Kelly, 1982). These differences between the storage/release mechanism of the exocrine submandibular gland and the other organs provide a reasonable explanation for the differences in the ratio of NGF protein to mRNANGF. It is worth mentioning that in other species the submandibular gland does not show these excessively high NGF levels. In the rat, for instance, the NGF levels in this organ are within the framework of the density of innervation, i.e., between the level of the heart ventricle and atrium (Thoenen et al., 1983). The concept that NGF acts as a retrograde messenger (Schwab and Thoenen, 1983; Darling et al., 1983) implicates its synthesis in and release from peripheral target organs. Thus, the high levels of ganglionic NGF are expected to result predominantly from uptake by receptor-mediated endocytosis and subsequent retrograde transport rather than local synthesis. Previous observations provided direct evidence for retrograde axonal transport of endogenous NGF (Korsching and Thoenen, 1983b). This together with a rapid decrease of NGF levels in sympathetic ganglia (
