Rafal Butowt

Anterograde axonal transport, transcytosis, and recycling of neurotrophic factors

Traditional views of neurotrophic factor biology held that trophic factors are released from target cells, retrogradely transported along their axons, and rapidly degraded upon arrival in cell bodies. Increasing evidence indicates that several trophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), glial cell-line derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-I), and neurotrophin-3 (NT-3), can move anterogradely along axons. They can escape the degradative pathway upon internalization and are recycled for future uses. Internalized ligands can move through intermediary cells by transcytosis, presumably by endocytosis via endosomes to the Golgi system, by trafficking of the factor to dendrites or by sorting into anterograde axonal transport with subsequent release from axon terminals and uptake by second- or third-order target neurons. Such data suggest the existence of multiple “trophic currencies,” which may be used over several steps in neural networks to enable nurturing relationships between connected neurons or glial cells, not unlike currency exchanges between trading partners in the world economy. Functions of multistep transfer of trophic material through neural networks may include regulation of neuronal survival, differentiation of phenotypes and dendritic morphology, synapse plasticity, as well as excitatory neurotransmission. The molecular mechanisms of sorting, trafficking, and release of trophic factors from distinct neuronal compartments are important for an understanding of neurotrophism, but they present challenging tasks owing to the low levels of the endogeneous factors.

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.

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.

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells : Roles of neurotrophin receptors

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.

Connecting the dots: trafficking of neurotrophins, lectins and diverse pathogens by binding to the neurotrophin receptor p75NTR

The common receptor for neurotrophins, p75, has important roles in internalization and trafficking of neurotrophins along axons. Recent studies show that an astonishing array of proteins, including lectins, pathogens and neurotoxins, bind the p75 receptor, suggesting that they can hijack and utilize this receptor for trafficking between neuronal populations within the nervous system. Such pathogens include the neurologically important rabies viruses, prion proteins, β‐amyloid and possibly tetanus toxin. These proteins may hijack existing transport machineries designed to traffick neurotrophins, thus allowing the infiltration and distribution of pathogens and toxins among vulnerable neuronal populations with devastating effects, as seen in rabies, prion encephalopathies, Alzheimers disease and tetanic muscle spasm. The discovery of an entry and transport machinery that is potentially shared between pathogens and neurotrophins sheds light ono trafficking systems in the nervous system and may assist the design of novel therapeutic avenues that prevent or slow the progression of diverse chronic and acute neurological disorders.

GDNF increases the survival of developing oculomotor neurons through a target-derived mechanism

Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron survival factor. We show here that in the chick oculomotor system, endogenous GDNF is derived largely from extraocular muscle but less from glial cells and not from muscle spindles. Increased levels of GDNF exclusively in the target rescued 30% of oculomotor neurons that would normally die during developmental cell death, a rate of rescue similar to that with systemic GDNF application. Thus, GDNF supports motoneuron survival in a retrograde, target-derived fashion, as opposed to a local paracrine route or an indirect route via sensory afferents. Persephin, another member of the GDNF family, did not increase survival with target delivery, despite its retrograde transport from the target. Unlike GDNF, however, persephin increased neurite outgrowth from oculomotor nuclei in vitro. Thus, one GDNF family member acts as a muscle-derived retrograde survival factor, whereas another one has distinct functions on neurite outgrowth.

Presynaptic neurotrophin-3 increases the number of tectal synapses, vesicle density, and number of docked vesicles in chick embryos.

To determine whether presynaptically derived neurotrophins may contribute to synaptic plasticity, we examined whether neurotrophin‐3 (NT‐3) changed the number, size, vesicle content, or vesicle distribution of synapses within the retinorecipient layers of the chick optic tectum. In this system, endogenous NT‐3 derives presynaptically from retinal ganglion cell axons. Retinotectal synapses comprise the majority of synapses in superficial tectal layers, as demonstrated by destruction of retinotectal input by intraocular application of the drug monensin. To examine the effect of increased or decreased levels of NT‐3, either exogenous NT‐3 or monoclonal NT‐3 blocking antibodies were injected into the optic tectum of 19‐day‐old chick embryos, spiked with radiolabeled protein to verify the success of injections and estimate effective concentrations. After 48 hours, the ultrastructure of superficial tectal layers was analyzed and compared with samples from control tecta injected with cytochrome C. NT‐3 increased the number of synapses, synaptic vesicles/profile, synaptic vesicle densities, the number of docked vesicles, and the length of the synaptic profile. Deprivation of anterogradely transported endogenous NT‐3 with NT‐3 antibodies resulted in the opposite effect: decreased numbers of synapses, decreased vesicle densities, and decreased numbers of docked vesicles. Brain‐derived neurotrophic factor (BDNF) had a largely different effect than NT‐3. BDNF increased the density of vesicles and deprivation of endogenous TrkB ligands with TrkB fusion protein reduced the density of vesicles in the synapses, without effects on synapse number or docked vesicles. We conclude that anterogradely transported NT‐3 affects synapse strength in a way that differs from that of presumably postsynaptic‐derived BDNF. J. Comp. Neurol. 458:62–77, 2003.

Fates of neurotrophins after retrograde axonal transport: Phosphorylation of p75NTR is a sorting signal for delayed degradation

Neurotrophins can mediate survival or death of neurons. Opposing functions of neurotrophins are based on binding of these ligands to two distinct types of receptors: trk receptors and p75NTR. Previous work showed that target-derived NGF induces cell death, whereas BDNF and NT-3 enhance survival of neurons in the isthmo-optic nucleus of avian embryos. To determine the fate of retrogradely transported neurotrophins and test whether their sorting differs between neurotrophins mediating survival- or death-signaling pathways, we traced receptor-binding, sorting, and degradation kinetics of target-applied radiolabeled neurotrophins that bind in this system to trk receptors (BDNF, NT-3) or only to p75NTR (NGF). At the ultrastructural level, the p75NTR-bound NGF accumulates with a significant delay in multivesicular bodies and organelles of the degradation pathway on arrival in the cell body when compared with trk-bound BDNF or NT-3. This delayed lysosomal accumulation was restricted to target-derived NGF, but was not seen when NGF was supplied to the soma in vitro. The kinase inhibitors K252a and Gö6976 alter the kinetics of organelle accumulation: phosphorylation of p75NTR is a sorting signal for delayed sequestering of p75NTR-bound NGF in multivesicular bodies and delayed degradation in lysosomes when compared with trk-bound neurotrophins. Mutagenesis and mass spectrometry studies indicate that p75NTR is phosphorylated by conventional protein kinase C on serine 266. We conclude that, in addition to the known phosphorylation of trks, the phosphorylation of p75NTR can also significantly affect neuronal survival in vivo by changing the intracellular sorting and degradation kinetics of its ligands and thus signaling duration.

Purification of chick retinal ganglion cells for molecular analysis: combining retrograde labeling and immunopanning yields 100% purity.

Retinal ganglion cells (RGCs) from embryonic and posthatch chickens were 100% purified by a novel combination of three steps: (1) Retrograde labeling by injection of the fluorescent carbocyanine tracer DiI into the optic nerve, (2) immunopanning of dissociated retinal cells with Thy1 antibodies, and (3) micro-aspiration of labeled RGCs into glass capillaries. The retina was dissected and dissociated with trypsin 12-15 h after the injection of DiI. DiI-labeled cells were identified on immunopanned dishes by fluorescence and collected for molecular analysis within 3 h after dissociation. This technique allowed the collection of up to 500 RGCs per capillary tube and 1500 labeled RGCs per retina. Extraction of RNA and molecular analysis by RT-PCR from 600 RGCs shows that expression of rare genes, such as those of neurotrophic factors, can be detected. This is the first description of a rapid and reliable technique for a 100% purification of RGCs with sufficient yield for molecular analysis of rare gene expression. The protocol can be modified for the purification of other cell types. The advantages and limitations of the three-step purification method are compared with previous RGC purification protocols.

Conventional kinesin-I motors participate in the anterograde axonal transport of neurotrophins in the visual system

Retinal ganglion cells (RGCs) anterogradely transport neurotrophins to the midbrain tectum/superior colliculus with significant downstream effects. The molecular mechanism of this type of axonal transport of neurotrophins is not well characterized. We identified kinesin‐I proteins as a motor participating in the anterograde axonal movement of vesicular structures containing radiolabeled neurotrophins along the optic nerve. RT‐PCR analysis of purified murine RGCs showed that adult RGCs express all known members of the kinesin‐I family. After intraocular injection of 125I‐brain‐derived neurotrophic factor (BDNF) into the adult mouse or 125I‐neurotrophin‐3 (NT‐3) into the embryonic chicken eye, radioactivity was efficiently immunoprecipitated from the optic nerve lysates by anti‐kinesin heavy chain and anti‐kinesin light chain monoclonal antibodies (H2 and L1). Immunoreactivity for the BDNF receptor trkB is also present in the immunoprecipitates obtained by the anti‐kinesin‐I antibodies. The delivery of the H2 antibody in vivo into the mouse RGCs substantially reduced anterograde axonal transport of 125I‐BDNF. Anterograde transport of BDNF was not diminished in kinesin light chain 1 (KLC1) knockout mice. However, this may be due to redundancy in functions between two different isoforms of KLC present in the RGCs, as it was described previously for kinesin heavy chains (Kanai et al. [ 2000 ] J Neurosci 20:6374–6384). These data indicate that kinesin‐I is a protein motor that participates in the anterograde axonal transport of neurotrophins in the chicken and mouse visual pathways.

Anterograde axonal transport of the exogenous cellular isoform of prion protein in the chick visual system

The cellular isoform of endogenous, newly synthesized prion protein (PrPc) can be transported by axons in the anterograde direction. To determine whether a mechanism exists for secreted PrPc to be internalized and then axonally transported, we analyzed internalization and anterograde axonal transport of radiolabeled recombinant PrPc after its intraocular injection in chick embryos. Internalization and axonal transport of exogenous PrPc to the midbrain by retinal ganglion cells (RGCs) is efficient, saturable and likely receptor-mediated. Ultrastructural quantitative localization of radiolabeled PrPc within RGC soma showed significant labeling of vesicular/endosomal compartments and much less labeling present over the Golgi apparatus and lysosomes, which indicates slow degradation of exogenous PrPc in this system. These data show that a mechanism exists to internalize a secreted form of PrPc and then to axonally transport such PrPc in an anterograde direction. This may provide an additional, novel mechanism for prion protein to spread among neurons.