Synaptic targeting of retrogradely transported trophic factors in motoneurons: comparison of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and cardiotrophin-1 with tetanus toxin

Abstract

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and cardiotrophin-1 (CT-1) are the most potent neurotrophic factors for motoneurons, but their fate after retrograde axonal transport is not known. Internalized trophic factors may be degraded, or they may be recycled and transferred to other neurons, similar to the known route of tetanus toxin. We tested whether neonatal rat hypoglossal motoneurons target retrogradely transported trophic factors to synaptic sites on their dendrites within the brainstem and subsequently transfer these trophins across the synaptic cleft to afferent synapses (transsynaptic transcytosis). Motoneurons retrogradely transport from the tongue radiolabeled GDNF, BDNF, and CT-1 as well as tetanus toxin. Quantitative autoradiographic electron microscopy showed that GDNF and BDNF were transported into motoneuron dendrites with labeling densities similar to those of tetanus toxin. Although tetanus toxin accumulated rapidly (within 8 h) at presynaptic sites, GDNF accumulated at synapses more slowly (within 15 h), and CT-1 never associated with synapses. Thus, some retrogradely transported neurotrophic factors are trafficked similarly but not identically to tetanus toxin. Both GDNF and BDNF accumulate at the external (limiting) membrane of multivesicular bodies within proximal dendrites. We conclude that tetanus toxin, GDNF, and BDNF are released from postsynaptic sites and are internalized by afferent presynaptic terminals, thus demonstrating transsynaptic transcytosis. CT-1, however, follows a strict degradation pathway after retrograde transport to the soma. Synaptic and transcytotic trafficking thus are restricted to particular neurotrophic factors such as GDNF and BDNF.

Figure 1.

A-F, Retrograde transport of radiolabeled GDNF (A), BDNF (B), CT-1 (C), and TTC (D) from the tongue of neonatal rat pups to the hypoglossal motor nucleus. Autoradiographs of paraffin sections exposed for 4-6 weeks show that all four macromolecules are retrogradely transported. E, Radiolabeled CT-1 accumulates predominantly in hypoglossal cell bodies (arrows). F, Radiolabeled GDNF distributes throughout the hypoglossal neuropil with grain accumulations over putative motoneuron dendrites (arrows). The area shown in F is indicated in the lower-magnification dark-field view in A. Scale bars: (in D) A-D, 200 μm; E, 100 μm; F, 20 μm.

Figure 2.

A-G, Specificity and involvement of receptors in transport of radiolabeled proteins from the tongue to the hypoglossal nucleus in neonatal rats as seen in representative sections viewed in dark-field illumination. In all cases between 10 and 20 ng, radiolabeled trophic factor was injected. A, Transport of radiolabeled GDNF predominantly to the ipsilateral hypoglossal nucleus (ipsi) when the injection is carefully restricted to one side of the tongue. Scale bar, 500 μm. B, Transport of radiolabeled GDNF only. C, Transport of radiolabeled GDNF with 50- to 100-fold excess cold GDNF. D, Transport of radiolabeled BDNF only. E, Transport of radiolabeled BDNF with 50- to 100-fold excess cold BDNF. F, Transport of radiolabeled CT-1 only. G, Transport of radiolabeled CT-1 with 50- to 100-fold excess cold CT-1. Scale bar: (in G) B-G, 200 μm.

Figure 3.

A-F, Representative examples of silver grain accumulation over organelles within hypoglossal motoneurons indicating the localization of radiolabeled proteins. Pictured organelles include GDNF in a light endosome (A, arrow), BDNF in a dense endosome (B, arrow), CT-1 in a lysosome (C, arrow), and GDNF in a heavily labeled MVB (D) in the endoplasmic reticulum (E) and the Golgi apparatus with Golgi (G)-associated vesicles (F, arrows). Scale bars, 500 nm.

Figure 4.

A-F, Representative examples of silver grain accumulation over MVBs and synapses within hypoglossal motoneuron dendrites indicative of localization of radiolabeled GDNF and BDNF. A, Boxed areas show radiolabeled GDNF associated with postsynaptic MVBs adjacent to afferent synapses. The boxed areas are shown at higher magnification in B and C. Note that in both cases, silver grains accumulate more over the external (limiting) membrane than the core of MVBs. The synaptic densities (SD) are indicated. D, Large proximal dendrite with three synaptic terminals (T). The center synapse (boxed) is labeled with GDNF silver grains in the presynaptic region, as shown at higher magnification in E. F, Silver grain indicating BDNF is located within a terminal (T). Scale bars: A, D, 500 nm; B, C, E, F, 200 nm.

Figure 5.

A, B, Retrogradely transported radiolabeled proteins accumulate in synapses on proximal dendrites of hypoglossal motoneurons. A, Diagram depicts the precise definitions used in this analysis for (nonsynaptic) plasma membrane, postsynaptic, presynaptic, and terminal areas. A distance of 150 nm on each side of the plasma membrane was considered within the plasma membrane area. B, LDs at 15 h of TTC and the trophic factors GDNF, BDNF, and CT-1 are shown for the four regions as indicated in A: PM (nonsynaptic), plasma membrane; post, postsynaptic; pre, presynaptic; term, terminal. The data are based on quantitative electron microscopic autoradiography and analysis of 110-279 silver grains for each treatment group as shown in Table 2. The dashed line indicates an LD of 1.0. An LD of ≤1.0 is considered nonspecific background. Note that the LD profiles for TTC, GDNF, and BDNF are similar but not identical and that CT-1 does not associate specifically with synapses (LDs of <1.0).

Figure 6.

A-D, Comparison of LDs and protein degradation after retrograde axonal transport to the neonatal hypoglossal nucleus. A, Comparison of LDs of TTC, GDNF, BDNF, and CT-1. Note that LDs of MVBs and lysosomes (Lys) in cell bodies are not correlated. The dotted line indicates background level. B, Quantification of MVB labeling in the vicinity (within 400 nm) of postsynaptic densities in dendrites of motoneurons treated with TTC, GDNF, BDNF, or CT-1. Note that large fractions of GDNF and BDNF containing MVBs are located close to synapses. C, LDs of lysosomes differ between GDNF and TTC (background level) and BDNF (modest LD) and CT-1 (high LD). D, The LDs in C correlate with the trends for the amount of degraded protein recovered after retrograde transport from the hypoglossal nucleus and processed for TCA precipitation. Error bars indicate SEM.

Figure 7.

A, B, Mechanisms of release from MVBs and quantification of the distribution of BDNF and GDNF within MVBs. A, Schematic drawing of two alternative mechanisms of ligand release from MVBs. Recent data have described fusion of the external membrane with the plasma membrane and release of internal MVB vesicles as exosomes (top panel). The classical mechanism postulates a vesicular intermediate (similar to the initial formation of internal vesicles by external membrane budding) and subsequent transfer of such vesicles to the plasma membrane and release (bottom panel). B, Quantification of the distribution of centers of silver grains representing GDNF and BDNF relative to the external (limiting) membrane of MVBs for dendritic and somal MVBs. Note that the majority of silver grains in dendritic MVBs distribute within 60 nm of the outer (limiting) membrane, whereas the peak of distribution for somal MVBs is at 120-180 nm inside the MVB. Differences between dendritic and somal MVBs are statistically significant (p < 0.01 and p < 0.05, Wilcoxon signed rank-sum test).