Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor

A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.     

A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor

Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF-receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins.     

Evidence for Phosphatidylinositol 4-Kinase and Actin Involvement in the Regulation of 125I-β-Nerve Growth Factor Retrograde Axonal Transport

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled betaNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I-betaNGF retrograde transport. PAO (20 nM to 200 microM) also inhibited NGF-dependent survival of both sympathetic and sensory neuronal populations. F-actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-betaNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-betaNGF retrograde axonal transport in vivo.     

A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells

A nerve growth factor (NGF) receptor interactive monoclonal antibody (192-IgG) which enhances beta-NGF binding to PC12 cells has been produced. The hybridoma clone was obtained by fusing Sp2/0- Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside solubilized proteins from isolated PC12 cell plasma membranes. The antibody is an IgG, which does not bind beta-NGF. It binds to the same number of sites on PC12 cells at low temperature as does beta-NGF. The 192-IgG increases the apparent affinity of beta-NGF binding to fast receptors on PC12 cells at low temperature by a factor of 2.5- to 4-fold and enhances the photoactivatable cross-linking of beta-NGF to the same receptor while decreasing the cross-linking of beta-NGF to the slow NGF receptor. At 37 degrees C 192-IgG partially inhibits the regeneration of neurites from primed PC12 cells. The 192-IgG also reduces the rate of appearance of binding to slow NGF receptors and increases the proportion of beta-NGF bound to fast receptors at 37 degrees C. These results implicate the slow receptor as the mediator of the biological response. This antibody provides a tool for examining steps in the mechanism of action of beta-NGF after binding to the receptor.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.     

Retrograde axonal transport of specific macromolecules as a tool for characterizing nerve terminal membranes

The uptake of macromolecules by nerve terminals which is followed by retrograde axonal transport seems to occur by two different mechanisms, a specific and a nonspecific one. The nonspecific uptake depends on the presence of macromolecules (e.g., horseradish peroxidase) in the vicinity of the nerve terminals at very high concentrations and is enhanced by neuronal activity. In contrast, the specific uptake and subsequent retrograde axonal transport becomes apparent at much lower concentrations of the appropriate macromolecules, depends on the affinity of these ligands for specific binding sites on the surface of the neuronal membrane, and is independent of neuronal activity. The fact that lectins and some bacterial toxins bind to specific membrane glycoproteins or glycolipids allows conclusions to be drawn regarding qualitative and even quantitative aspects of the composition of the plasma membrane of the nerve terminals. ¹²⁵I-labelled nerve growth factor (NGF), tetanus toxin, cholera toxin, wheat germ agglutinin (WGA), ricin II, phytohemagglutinin (PHA), and concanavalin A (ConA) were injected into the anterior eye chamber of rats where they were taken up by adrenergic nerve terminals and transported retrogradely to the superior cervical ganglion. The saturation of the uptake-transport found for NGF, WGA, choleragenoid and an atoxic binding-fragment of tetanus toxin indicates that limited numbers of binding sites, which showed also different affinites, are present for each ligand on the membrane of the nerve terminals. Competition experiments showed that the binding sites for the ligands investigated are largely independent. Two different classes of binding sites (high affinity-low capacity and intermediate affinity-intermediate capacity) seem to be involved in the saturable retrograde axonal transport of NGF. In contrast, WGA seems to have only a single class of binding-uptake sites with high capacity and relatively low affinity. Strong evidence for positive cooperativity was obtained for the uptake and subsequent transport of the tetanus toxin fragment.     

High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

Nicotinic acetylcholine receptor of mammalian brain: naja toxin binding to subcellular fractions of rat brain

Abstract— Subcellular fractions of rat brain cortex bound 6.8 ± 0.4 fmol of ¹²⁵I-labeled α-naja toxin per milligram of protein. Toxin binding was saturable and specific e.g. nicotinic agonist and antagonist blockable. Regions in rat brain varied in saturable, specific toxin binding with hippocampus, olfactory area and corpus striatum displaying greatest toxin binding. We conclude that ¹²⁵I-α-naja toxin is a suitable probe for nicotinic cholinergic receptor in brain.     

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves

Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.     

Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding.

Nerve growth factor (NGF) binds to a low affinity cell surface receptor (p75NGFR) which contains four extracellular repeats, rich in cysteine residues and negatively charged. We have made mutations in the receptor cDNA by inserting linkers in specific domains of the receptor. Nearly all the mutations caused a change in the predicted charge, and resulted in either an insertion or deletion in the primary sequence. Stably transfected fibroblasts were assayed for NGF binding by affinity cross-linking with 125I-NGF. Appropriate expression of the mutated receptors was monitored by rosetting with monoclonal antibodies and by metabolic labeling followed by immunoprecipitation. Although the mutant receptors were recognized by monoclonal antibodies, insertions and deletions in the third and fourth cysteine-rich regions of the receptor had a detrimental effect upon NGF binding. Insertions made outside the cysteine-rich region or in the cytoplasmic domain did not inhibit the ability of 125I-NGF to bind to the receptor, as assessed by affinity cross-linking. A chimeric human-rat NGF receptor transfected into fibroblasts indicates that NGF binding and monoclonal antibody recognition sites are separated but contained within the four cysteine repeats.     

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.     

NGF-stimulated retrograde transport of trkA in the mammalian nervous system

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.     

The ontogeny of specific retrograde transport of nerve growth factor by motoneurons of the brainstem and spinal cord.

Radiolabeled Nerve Growth Factor (NGF) was injected into either the mandibular process of the first visceral arch or the limb bud of chick embryos at Days 3.5-14 or Days 4-13 of incubation, respectively. Control embryos received injections of labeled cytochrome-C or labeled NGF plus an excess of unlabeled NGF. The tissues were then processed for autoradiography. The 125I-NGF was retrogradely transported by motoneurons of the trigeminal (V) motor nucleus on Days 3.5-8 of incubation, but not at later stages. Similar transport was seen in motoneurons of the spinal cord lateral motor column from Days 4-10 of incubation, but not at later stages. Sensory neurons of the V ganglion and of the dorsal root ganglia transported NGF at all injection ages. In no instance was the 125I-cytochrome-C transported by sensory or motor neurons. The injection of an excess of cold NGF along with labeled NGF resulted in no evidence of retrograde transport of the labeled NGF indicating that the transport was saturable. The time of transport by these brainstem and spinal cord motoneurons corresponds closely to the points during development at which they have been found to exhibit specific NGF binding. The present results, then, provide further evidence for a possible biological role for NGF during early developmental stages of these motoneuron populations.     

Distribution and characteristics of nerve growth factor binding on cholinergic neurons of rat and monkey forebrain

In sections of rat forebrain, perikarya labeled radioautographically with¹²⁵I-NGF resembled cholinesterase-positive neurons in their distribution within striatum and basal forebrain. Neurons with NGF receptors were also visualized in radioautographs prepared from the basal forebrain of a cerebrus monkey. Present techniques fail to detect axons projecting from basal forebrain to hippocampus or cortex which have been shown to take up NGF selectively in retrograde transport studies. In studies with membrane-enriched preparations from rat, high-affinity binding of¹²⁵I-NGF (half maximal saturation in the 15–30 pM range) was detected in basal forebrain and striatum; lower levels of high-affinity binding were seen in hippocampus and neocortex. The binding and molecular properties of these receptors are similar to those described in other NGF-responsive tissues. These observations are further evidence supporting a biological role for NGF on some forebrain cholinergic neurons in adult rat.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).     

NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Target-derived NGF promotes the phenotypic maintenance of mature dorsal root ganglion (DRG) nociceptive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of endosomes containing NGF, activated TrkA, and signaling proteins of the Rap1/Erk1/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erk1/2, p-p38, and pAkt in these membranes. Conversely, NGF antibody injections decreased the retrograde transport of p-Erk1/2 and p-p38. Our results are evidence that signaling endosomes, with the characteristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.     

Nerve growth factor receptors in the peripubertal rat ovary.

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged recycling of internalized neurotrophins in the nerve terminal

BACKGROUND: Neurons require contact with their target tissue in order to survive and make correct connections. The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. However, the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body remains unclear. METHODS: Retrograde axonal transport was examined using the transport of I(125)-labeled neurotrophins from the eye to sympathetic and sensory ganglia. The phenomena was further studied by adding rhodamine-labeled nerve growth factor (NGF) to cultures of dissociated sympathetic ganglia and the movement of organelles followed with the aid of video microscopy. RESULTS: I(125)-labeled neurotrophins were transported from the eye to the sympathetic and sensory ganglia. A 100-fold excess of unlabeled neurotrophin, administered up to 4 h after the labeled material, completely prevented accumulation of labeled neurotrophin in the ganglia. The effect was specific for the labeled neurotrophin as administration of a high concentration of a different neurotrophin failed to inhibit the transport. In dissociated cultures, we found rapid binding of label, to surface membrane receptors, followed by an accumulation of labeled vesicles in the growth cone. Incubation of these cultures with unlabeled NGF led to a rapid loss of label in the growth cones. CONCLUSIONS: These results suggest that there is a pool of internalized neurotrophin, in vesicles in the nerve terminal, which is in rapid equilibrium with the external environment. It is from this pool that a small fraction of the neurotrophin-containing vesicles is targeted for retrograde transport. Potential models for this system are presented.     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.