Brain Research, 85 (1975) 337-341

© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

337

R E T R O G R A D E AXONAL TRANSPORT OF NERVE G R O W T H FACTOR: SPECIFICITY AND BIOLOGICAL IMPORTANCE

K. STOECKEL AND H. THOENEN Department of Pharmacology, Biocenter of the University, Basel (Switzerland)

Nerve growth factor (NGF) is a protein which efficiently enhances both growth and differentiation of peripheral adrenergic neurons 15. The biochemical counterpart to the morphological manifestations of differentiation is the selective induction of tyrosine hydroxylase (TH) and dopamine-fl-hydroxylase (DBH)7, 2a. These two enzymes catalyze the rate-limiting steps in the synthesis of the adrenergic transmitter norepinephrine and are selectively located in adrenergic neurons ls,22,24. Since N G F is not exclusively synthesized in the submaxillary gland - - the richest source for the isolation 2 of this protein - - but also in adrenergically innervated effector organs s it has been suggested that N G F might act as atrophic factor, transferring information from the effector organ to the cell body of the innervating neuron 5. This hypothesis has been strengthened by the recent demonstration that labeled N G F is taken up by adrenergic nerve terminals and transported retrogradely at a rate of 2.5 mm/h to the perikaryon 6. The rate of this retrograde axonal transport and its susceptibility to colchicine show that not only the orthogradel,a, 20 but also the retrograde axonal transport 6 depends on an intact neurotubular system. Since a retrograde axonal transport has been shown to occur in both peripheral and central neurons9-11,13,17 for a number of proteins such as horseradish peroxidase and bovine albumin, which bear no perspicuous relationship to neuronal function, it could be assumed that retrograde axonai transport is a rather non-specific phenomenon and that macromolecules are sampled more or less indiscriminately by all the nerve terminals. Thus, uptake and retrograde transport would not contribute to the specificity of any effect which would entirely depend on the presence or absence of a target site for the axonally transported macromolecules. We, therefore, investigated whether a specificity for uptake and transport does exist, whether it depends on the molecular weight or the electrical charge of the molecule at physiological pH and whether there are differences between various species of neurons. Moreover, we investigated whether and to what extent the moiety of N G F reaching the cell bodies of adrenergic neurons by retrograde transport is responsible for the biological effect of N G F as far as characterized by the induction of TH in the perikaryon. In studies designed to establish the specificity of the retrograde axonal transport, N G F and a series of other proteins with various molecular weights (13,000-540,000)

338 TABLE I SPECIFICITY OF THE RETROGRADE

AXONAL TRANSPORT

IN A D R E N E R G I C

NEURONS

Comparison between the accumulation of radioactivity in superior cervical ganglia after unilateral intraocular injection of [~251]NGFand various other [125I]-labeledproteins. The ganglia were excised 14 h after injection. In each case the injected radioactivity was 1 /~Ci. Values are means ~ S.E.M. for groups of 7-10 adult male mice. Mol. wt.

lp

Difference in ganglionic radioactivity (counts~rain) (injected side -- noll-injected side)

fl-NGF Cytochrome C Insulin Horseradish peroxidase Ovalbumin Bovine albumin Ferritin

26,000 13,500 24,400 44,000 45,000 67,000 540,000

9.3 9.8 5.4 9.7 4.6 4.8 4.6

98l : 80* --3 i:: 14"* --3 _~= 9** ~ 6 " 11 * * --3 ± 6** ÷ 1 ~: 14"* ? 4 ~ 14" *

* Difference statistically significant (P < 0.0005). ** Difference not statistically significant (P > 0.05). and isoelectric points (4.6-9.7) were labeled with 125I. In each case 1 #Ci of identically labeled protein was injected into the right anterior eye chamber of adult male albino mice. The superior cervical ganglia of the injected and non-injected side were removed 14 h later and the radioactivity determined in a y-counter. The difference between the radioactivity accumulated in the ganglia of the injected and non-injected side reached its m a x i m u m between 12 and 16 h. The magnitude of this difference is a measure of the extent of the retrograde transport 6. As shown in Table I, of all the proteins investigated N G F was the only one which exhibited a statistically significant (P < 0.05) difference between the injected and non-injected side. This does not mean that the other proteins were not transported at all - - the amount transported was too small to be detected within the limits of the method - - but there was a striking preference for N G F . To obtain any information as to whether the radioactivity accumulated in the ganglia of the injected side is N G F the ganglia were homogenized in Tris buffer, p H 6.8 containing 5 ~ mercaptoethanol and 2.3 ~ SDS. Seventy per cent of the extracted radioactivity was accumulated at the position of N G F in a 12 ~ SDS gel 14. Although this finding does not prove that the N G F transported maintained its biological activity it shows at least that the majority of the radioactivity in the ganglia is associated with a molecule of the same molecular weight as N G F . After it had been shown that N G F exhibits a high selectivity for retrograde axonal transport in the adrenergic neurons it seemed to be of interest to study the effect of minor chemical modifications of the N G F molecule on the retrograde axonal transport in the adrenergic neuron. After partial oxidation of the tryptophan residues by N - b r o m o s u c c i n i m i d d l the retrograde transport of the modified N G F was markedly impaired. There was a good correlation between the reduction of the retrograde

339 transport and the reduction of the biological activity determined in the chicken dorsal root ganglia assay4. In order to obtain information as to whether the high specificity of the retrograde transport of NGF is confined to the adrenergic neuron or whether this preference depends on factors common to all neurons, we studied the retrograde axonal transport of NGF in the rat motor neuron. The experiments were performed under experimental conditions under which the retrograde transport of tetanus toxin had clearly been demonstrated12. Twenty #Ci of [12H]NGF were injected unilaterally into the musculus deltoideus and over the next 48 h the radioactivity accumulating in the right and left side of the spinal cord segments C6-Cs was determined. In contrast to tetanus toxin there was never a statistically significant (P > 0.05) difference between left and right side after injection of [I~H]NGF. Thus, the selectivity of retrograde transport of NGF does not depend on properties which are common to all neurons. Very recent experiments have shown that a highly selective retrograde transport of NGF also takes place in the sensory neuron of adult rats, although the biological response is confined to a very short period of embryonic life, at least as far as can be judged from morphological studies15. Therefore, in the sensory neuron the selectivity of uptake and transport seems to persist over the whole life span whereas the biological response is limited to a very short period. In order to investigate the biological importance of the moiety of NGF reaching the cell body by retrograde transport we injected 40 #g of NGF into the right eye and 100 #g into the right submaxillary gland. Forty-eight hours later we determined the TH activity in the superior cervical ganglia of the injected and non-injected side (Fig. 1). On the injected side the increase (expressed in per cent of controls) amounted to

Controls c

o

300

NGF

[~I

-injected side injected side

~non

oo.

-1-

100 •

I.-

Fig. 1. Effect of unilateral injection of N G F into the anterior eye chamber and submaxillary gland on tyrosine hydroxylase (TH) activity in the superior cervical ganglion (SCG). Adult mice (20-30 g) were injected unilaterally into the anterior eye chamber ( 2 / d ) and submaxillary gland (5/ai) with a 20 mg/ml solution of N G F . Forty-eight hours later the T H activity was determined 16,19 separately in the ganglia of the left and right side. The T H activity in controls was 0.0656 nm/h/ganglion (total) and 1.1777 rim/h/rag protein (specific) respectively. Results are expressed as per cent of controls. Values are means ± S.E.M. for groups of 5-8 animals. Black column, P < 0.01 as compared to both controls and non-injected side.

340 124 %, on the non-injected side to 66/o.°/The increase in TH activity on the non-injected side results from the moiety of N G F which inevitably escapes from the site of injection into the general circulation and reaches the ganglion of the contralateral side directly and/or by retrograde transport from the nerve terminals. The difference between the injected and non-injected side is even more impressive if one takes into account that only about 12 °k of the adrenergic neurons located in the superior cervical ganglion supply the anterior eye chamber and the submaxillary gland. Thus, if all the nerve terminals were (unilaterally) exposed to similarly high concentrations of N G F the magnitude of difference between sides would even be higher. Moreover, it has to be taken into account that the injection as such produces a general stress and in consequence a (bilateral) transsynaptic induction of TH of about 15-20/°~,. The subtraction of this bilateral increase, non-related to the effect of NGF, would contribute to an even greater increase in the relative difference between sides. Although our data do not exclude a direct (blood borne) action of N G F on the adrenergic cell body the moiety of N G F reaching the perikaryon by retrograde transport accounts for a considerable part of the biological effect. In conclusion, it has been demonstrated that N G F is transported retrogradely with a high selectivity in adrenergic and sensory, but not motor neurons, demonstrating that the selectivity does not depend on factors common to all neurons. Moreover, a considerable part of the biological effect of N G F on adrenergic neurons results from the moiety reaching the cell body by retrograde axonal transport. Thus, our observations are consistent with the hypothesis that N G F might act as atrophic factor transferring information from the effector organ to the cell bodies of the innervating neurons.

This work was supported by the Swiss National Foundation for Scientific Research (Grant No. 3.653.71).

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