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Neurotrophin trafficking by anterograde transport
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Neurotrophin trafficking by anterograde transport C. Anthony Altar and Peter S. DiStefano The ever-unfolding biology of NGF is consistent with a target-derived retrograde mode of action in peripheral and central neurons. However, another member of the neurotrophin family, brain-derived neurotrophic factor (BDNF), is present within nerve terminals in certain regions of the brain and PNS that do not contain the corresponding mRNA. Recent studies have shown that the endogenous neurotrophins, BDNF and neurotrophin-3 (NT-3), are transported anterogradely by central and peripheral neurons. The supply of BDNF by afferents is consistent with their presynaptic synthesis, vesicular storage, release and postsynaptic actions. Anterograde axonal transport provides an ‘afferent supply’ of BDNF and NT-3 to neurons and target tissues, where they function as trophic factors and as neurotransmitters. Trends Neurosci. (1998) 21, 433–437
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HE TRADITIONAL ROLE OF NEUROTROPHINS as retrogradely transported, target-derived survival factors is well established in the developing PNS (Refs 1,2). Thus, NGF is produced by targets of NGF-dependent neurons in limiting quantities and promotes neuron survival through its retrograde transport and signaling to the cell body. Retrograde transport was first demonstrated with injections of 125I-labeled NGF into the terminal fields of sympathetic and sensory neurons in the PNS (Ref. 3) or cholinergic neurons in the CNS (Ref. 4). In these studies, [125I]-NGF accumulated in neuronal cell bodies in a saturable, receptor-mediated fashion. Crucial experiments from the laboratories of Thoenen and Johnson demonstrated the retrograde flow of endogenous NGF by the accumulation of immunoreactive NGF at a site distal, but not proximal, to a ligature or lesion of peripheral and central neurons5.
Indirect evidence for bidirectional neurotrophin trafficking The subsequent discovery of other members of the NGF-related family has revealed new candidates for Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0166 - 2236/98/$19.00
axonal trafficking by brain and peripheral neurons. Intracranial and intranerve injections of iodinated brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) result in their retrograde transport to adult PNS and CNS neuron cell bodies6–9. However, two sets of findings made it clear that these newer family members had roles in the CNS that extended beyond that of target-derived support (survival-promoting and phenotypic maintenance functions) mediated by retrograde transport. First, BDNF and NT-3, unlike NGF, were shown to produce rapid, neuromodulatory effects in a variety of adult neural systems. For example, infusions of BDNF into the cell bodies of adult brain nuclei can augment neuronal firing rates, neurotransmitter release, metabolism and the phenotypic expression of biosynthetic enzymes in cholinergic, dopaminergic, serotonergic, or peptidergic neurons (for reviews, see Refs 8, 10–12). A brief (minutes) exposure of amphibian motor neuron–muscle cocultures to NT-3 or BDNF can augment the electrical activity of innervated muscle cells13. Even more rapid effects of BDNF, in the order of seconds, PII: S0166-2236(98)01273-9
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C. Anthony Altar is at Global Neuroscience Research Otsuka America Pharmaceutical Inc., Rockville, MD 20850, USA and Peter S. DiStefano is at Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
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include increases in electrical activity and intracellular Ca2+ in cultured hippocampal and cortical neurons14,15 (see Refs 11,16 for reviews). Second, and perhaps most revealing, the use of highly specific and sensitive antibodies identified BDNF and NT-3 protein in nerve terminals. Importantly, such growth factor-laden terminals were found in regions of the brain and PNS that did not contain the corresponding mRNA. BDNF protein, but not its mRNA, was found in the neostriatum17,18, neocortex, mossy fibers of hippocampal granule neurons, medial habenula, central amygdala, lateral septum and spinal cord19–21. BDNF protein concentrations in these regions exceeded that of NGF by 100-fold. Conversely, cell bodies of neurons that project to these regions are rich in BDNF mRNA. In the PNS, BDNF was found in the central and peripheral processes of dorsal root ganglia (DRG) sensory neurons, whereas BDNF mRNA was found in the cell bodies of these neurons but not in their central targets22. It is important to note that these characteristics described for BDNF do not apply to NGF, and NGF does not appear to be anterogradely transported by CNS or PNS neurons. Together, these findings suggest that neurotrophins other than NGF, and potentially other growth factors, might be transported anterogradely and released in amounts that could affect their postsynaptic targets.
Anterograde transport of exogenous neurotrophins The presence of the NT-3-binding molecules TrkC, TrkB and p75 in the adult optic tectum, and their presence on developing tectal cells whose survival required an anterogradely transported signal23, prompted von Bartheld to inject 125I-labeled NT-3 into the chicken embryo eye and look for evidence of anterograde transport. Indeed, [125I]-NT-3 accumulated in the optic tectum, where it was associated with presynaptic vesicles. Even more tantalizing was the presence of 125I in the dendrites and somata of postsynaptic tectal neurons, whose survival was enhanced by the eye injections of [125I]-NT-3 (Ref. 23). These findings indicate that the anterogradely transported [125I]-NT-3 was released by retinotectal neurons and taken up by their targets. The anterograde transport of [125I]-NT-3 occurred independently of electrical activity. It was blocked by transport inhibitors and depended on a complex of TrkC (the high-affinity receptor for NT-3) and p75 (the lower-affinity neurotrophin receptor)23,24. A predominance, by 85% to 15%, of anterograde transport relative to retrograde transport was observed for [125I]-NT-3 and 125I-labeled BDNF, respectively, in the chick retinotectal pathway24. In the zebra finch, [125I]-BDNF or [125I]-NT-3 accumulated anterogradely in the archistriatum following their injection into a frontal cortex motor nucleus that provides the major input to the archistriatum25. Deafferentation of the cortical input to the archistriatum produced apoptotic death of archistriatal neurons. Such death was blocked by infusion of BDNF or NT-3 into the archistriatum. Thus, in the chick retinotectal pathway and zebra finch motor-cortical pathway, the anterograde transport of exogenous NT-3 or BDNF can promote the survival of postsynaptic neurons that normally die during development or following axotomy. 434
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Anterograde transport of endogenous neurotrophins in the PNS Recent studies have shown that BDNF is normally concentrated in the superficial layers of the dorsal spinal cord22,26. Dorsal rhizotomy, which disrupts the DRG inputs to the dorsal cord, abolished this labeling. Furthermore, Michael et al.26 have identified BDNF in dense-core vesicles within axon terminals located in the superficial layers of the dorsal spinal cord. Thus, BDNF appears to be packaged much like a neurotransmitter in central processes of DRG neurons. By using a double ligation of the sciatic nerve, several groups have revealed the accumulation of endogenous BDNF on proximal and distal sides of the nerve ligature22,26,27. In agreement with von Bartheld’s findings with iodinated neurotrophins24, a greater intensity of proximal BDNF immunostaining confirmed that the predominant form of transport is in the anterograde direction. That DRG neurons are the probable source of such transported BDNF was indicated by: (1) the robust expression of BDNF mRNA in DRG neurons, compared to motor neurons or Schwann cells, and (2) the accumulation of BDNF in the area of nerve proximal to the DRG, whether a ligature was made in the sciatic nerve or in the dorsal root26,27. The presence of BDNF protein in small-to-medium-sized DRG neurons and in terminals of the dorsal horn, where small unmyelinated neurons normally terminate, suggests a role for the anterograde transport of BDNF in mediating nociceptive transmission or pain modulation. The anterograde transport of BDNF is greatly altered by injury to DRG axons. Sciatic nerve transection or dorsal rhizotomy results in a dramatic increase of BDNF protein transport to the proximal side of a ligature. This is accompanied by increased BDNF mRNA and protein content in the DRG but not at other sites along the peripheral neuraxis27. Interestingly, the ability of NGF to increase BDNF mRNA and protein levels in the DRG (Ref. 26) shows that NGF might also regulate the anterograde transport of BDNF by DRG neurons. It is important to note that, in all these studies, there is no anterograde accumulation of NGF, and that the accumulation of NT-3 is much less than that of BDNF. While similar aspects of the anterograde transport of NT-4 are unknown, anterograde transport appears to be selective within the neurotrophin family. Figure 1 illustrates what is known of anterograde and retrograde transport of BDNF in the PNS.
Anterograde transport of endogenous neurotrophins in the CNS In a remarkably close analogy with the anterograde transport and function of radiolabeled neurotrophins in the developing zebra-finch archistriatum25, endogenous BDNF is also anterogradely transported from the cortex to the neostriatum in the adult rat. While devoid of BDNF mRNA, the adult rat neostriatum nonetheless contains BDNF protein at levels not far below those measured in the BDNF mRNA-rich hippocampus17. Because cortical and nigral neurons that innervate the neostriatum contain BDNF mRNA, we proposed that these cells anterogradely transport BDNF to the striatum18. To test this, axonal transport was inhibited with intracerebroventricular injections of colchicine. This greatly elevated BDNF protein staining in the cell bodies of cortical and nigral cell bodies, but depleted staining within the neostriatum
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(Fig. 2). Unilateral removal of the frontoparietal neocortex decreased BDNF staining in the dorsolateral striatum, where most frontoparietal cortical afferents terminate. Decortication depleted BDNF protein contents by two-thirds in the ipsilateral striatum and by about one-third in the contralateral striatum. These decreases are proportional to the density of striatal innervation by crossed and uncrossed cortical afferents, respectively. Unilateral lesions of the uncrossed nigrostriatal pathway decreased BDNF to a smaller degree, and only in the ipsilateral striatum. Selective ablation of intrinsic striatal neurons did not decrease neostriatal BDNF. Thus, BDNF in the neostriatum is derived largely by anterograde transport within cortical afferents, and to a lesser degree by nigral afferents. The fact that BDNF-gene deletion diminishes the number of parvalbumin-expressing striatal neurons in proportion to the loss in BDNF protein18 suggests a postsynaptic role for the anterogradely transported BDNF. Support for a broader CNS role of anterograde neurotrophin transport is provided by analyses in other brain regions. In the adult hippocampus, seizures induced by electroshock or kainic acid increase BDNF mRNA levels within dentate granule neurons and increase BDNF protein within mossy fibers21. The granule-cell mossy fibers synapse on the apical dendrites of CA3 pyramidal neurons. Because BDNF mRNA was not elevated in the pyramidal neurons by either form of seizure, and because kainate destroys the pyramidal neurons, it is likely that BDNF synthesized by granule neurons is anterogradely transported to hippocampal CA3 pyramidal neurons. The central amygdala and medial habenula contain BDNF-immunoreactive fibers, but not BDNF mRNA (Ref. 19). Lesions of the pontine parabrachial or septal nuclei, which contain high levels of BDNF mRNA, completely eliminated the BDNF-immunoreactive fibers in the amygdala and habenula, respectively. Thus, the BDNF in these regions is probably derived by anterograde transport from the pons to amygdala and from the septum to habenula. The presence of BDNF protein in axon terminals throughout the brain18–20 suggests that anterograde transport might be a common feature of neurotrophin trafficking in the CNS. Figure 3 summarizes anterograde and retrograde transport of BDNF in basal ganglia pathways.
Fig. 1. Schematic representation of the bidirectional axonal transport of neurotrophins in the PNS. Neurons in the dorsal root ganglion (DRG) expressing brain-derived neurotrophic factor (BDNF) mRNA and protein (blue symbols) are predominantly, but not exclusively, small diameter neurons whose central projections terminate in superficial layers of spinal cord gray matter. This is also a site of BDNF immunoreactivity (demarcated in blue) with no corresponding BDNF mRNA expression. Arrows illustrate the capacity for retrograde or anterograde transport of BDNF, either from endogenous or exogenous sources. Large arrows indicate a predominance in the direction of BDNF trafficking. In DRG, BDNF could also serve an autocrine/paracrine role for neighboring DRG neurons, especially after injury. Similarly, anterogradely trafficked BDNF might have trophic actions on targets of BDNF-expressing neurons or on neighboring terminals of non-BDNF-expressing neurons. The anterograde transport of BDNF in motor neurons is relatively weak, as indicated by an arrow and question mark. Similarly, exogenous BDNF can be retrogradely transported from spinal cord to DRG (P. DiStefano and Bregman, unpublished observations), but evidence does not support transport of endogenous neurotrophin in this direction.
dispose it for packaging into anterogradely transported synaptic vesicles. Cleavage to active forms could occur in distinct axonal compartments or upon release. The BDNF protein content of cortical synaptosomes is increased following a systemic treatment with kainic acid that greatly upregulates BDNF mRNA (Ref. 29). It will be of interest to determine whether pharmacological or other manipulations that augment BDNF mRNA also augment BDNF protein in the terminal regions. It will also be important to determine if BDNF is packaged, transported and released by vesicles in tandem with other growth factors [e.g. basic fibroblast growth factor (bFGF) and NT-3], transmitters (e.g. glutamate) or neuroprotective molecules (e.g. ascorbate) that are also found in striatal nerve terminals (Fig. 4).
A neurotransmitter-like role for anterogradely transported neurotrophins As summarized above, anterograde transport provides BDNF to presynaptic nerve terminals in a host of central and peripheral neurons. Thus, anterograde transport is in keeping with the proposed neurotransmitter-like role of neurotrophins11,12,28. In fact, BDNF fulfills all of the classical criteria for being considered a neurotransmitter, as follows.
Presynaptic synthesis and vesicular storage As reviewed above, an abundance of BDNF mRNA is found in the cell bodies of neurons in which BDNF protein is anterogradely transported. The BDNF immunoreactivity in rat brain is enriched in a synaptosomal fraction, and predominates over profiles that resemble dense-core vesicles29. Is the form of BDNF that is anterogradely transported different from that which is retrogradely transported? Neurotrophins are synthesized in pre-pro forms and are processed to mature forms. A longer, less mature form of BDNF could pre-
Fig. 2. Brain-derived neurotrophic factor (BDNF) immunostaining in the frontal cortex of a rat that had received an intraventricular injection of the microtubule-disrupting agent colchicine to block anterograde and retrograde axonal transport. The pial surface of the brain is at the top of the illustration. The BDNF staining is particularly intense in layers II and IV cortical pyramidal neurons. Similar increases in BDNF staining of pars compacta neurons of the substantia nigra18 represent an accumulation of BDNF that would otherwise have been anterogradely trafficked by cortical and nigral neurons to afferent targets such as the neostriatum.
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requires functional Na+ channels and intracellular, but not extracellular, Ca2+.
II
Postsynaptic Trk localization
Cortex V cc
Striatum
S. nigra pc Glutamate Parvalbumin
Consistent with a transmitter-like role for BDNF, full-length TrkB is found in postsynaptic membranes of neurons in the PNS and CNS. In the PNS, immunostaining reveals full-length TrkB on DRG neurons and in the dorsal root entry zone of the spinal cord32. Truncated forms of TrkB are also found in the spinal cord, but they also reside in various target regions in the periphery as well as on Schwann cells. In the CNS, full-length phosphorylated TrkB is present in the optic tectum of the chick23,24 and postsynaptic densities prepared from cortex and hippocampus33. Immunoreactivity for full-length TrkB is seen on the postsynaptic densities of dendritic shafts and spines and on the initial axon segment of hippocampal pyramidal and dentate granule neurons (Drake et al., unpublished observations). While the localization of full-length TrkB is well understood, that of the truncated form is just emerging (see below).
Postsynaptic actions
As discussed above, the rapid, postsynaptic effects of BDNF and NT-3 include their ability to elevate the frequency and amplitude of muscle cell depolarization Dopamine at the developing Xenopus neuromuscular junction13. GABA In the CNS, rapid effects of BDNF are best described for the long-term potentiation (LTP) of hippocampal Fig. 3. Cross sectional representation of brain-derived neurotrophic factor (BDNF) trafficking in corticostriatal, nigral and nigrostriatal synaptic responses. This role for BDNF has implineurons of the rat basal ganglia. There is no retrograde transport by cations for learning and memory, and the failure of layer II corticostriatal neurons or anterograde transport by striatal neur- cognitive abilities in dementia, including Alzheimer’s ons (note the absence of an arrow). Abbreviations: cc, corpus callosum; disease. Cultured hippocampal neurons show an elS. nigra pc, substantia nigra, pars compacta; S. nigra pr, substantia evated postsynaptic current amplitude within minutes nigra, pars reticulata; II, V, layers II and V of the frontal cortex. of BDNF exposure14. These effects are blocked with the kinase inhibitor K-252a, and potentiated by the phosphatase inhibitor okadaic acid. Also within minutes, Depolarization-evoked release The release of [125I]-NT-3 has been observed follow- BDNF or NT-3 can elevate for several hours the deing its anterograde transport by retinal ganglion cells polarization of postsynaptic Schaffer collateral–CA1 to chick tectum23. Endogenous BDNF in cortex30 and synapses in hippocampal slices15. The effects of these [125I]-BDNF preloaded into a synaptosomal fraction of changes include the potentiation of glutamatergic cortex and hippocampus31 can be released by depolar- transmission in the hippocampus14,34. Deletion of one izing stimuli (glutamate, potassium). [125I]-BDNF release or both alleles of the BDNF gene decreases basal synaptic transmission and weakens the LTP of postsynaptic responses Glia/ependyma in the CA1 region35,36. EAAT1, Also within several minutes, EAAT2 BDNF can enhance the phosphorylation of postsynaptic N-methyl-Daspartate (NMDA) receptor subunits Glu 1 (NR1)37 and 2 (NR2b)38 in hippoAA campus and neocortex (Fig. 4). The P LTP produced at synapses between BDNF P Schaffer collaterals and CA1 neurmRNA P Glu ons also elevates BDNF mRNA exP CREB-P NM1 pression in postsynaptic CA1 neurP NM2 P ons39, and in hippocampal dentate P BDNF Glu granule neurons in which LTP was TrkB kinase cAMP induced by perforant path stimuCa2+ Ca2+ Glu BDNF TrkB truncated lation40. Thus, hippocampal LTP mRNA VSCC might depend upon the ability of Schaffer collateral firing to increase presynaptic BDNF synthesis, anteroFig. 4. Brain-derived neurotrophic factor (BDNF) fulfills the classical criteria for being considered as a rapidly grade transport and release. The conacting synaptic modulator. These criteria are illustrated for a typical axodendritic connection between cortical glut- comitant release of glutamate would amatergic and striatal GABAergic interneurons. Abbreviations: AA, ascorbic acid; CREB-P, phosphorylated cyclic AMP increase BDNFergic and glutamatresponse-element-binding protein; EAAT, excitatory amino acid transporter; Glu, glutamate; NM1, NM2, NMDA recep- ergic signaling via a coordinated tor subunits 1 and 2; P, phosphate; TrkB, BDNF receptor; VSCC, voltage-sensitive Ca2+ channel. phosphorylation of TrkB and S. nigra pr
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Calbindin
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NMDA receptors on postsynaptic CA1 pyramidal neurons (Fig. 4).
Inactivation and recycling of BDNF The inactivation of BDNF can occur through one of at least three mechanisms. One form, mediated by retrograde transport, requires BDNF to bind full-length TrkB on the presynaptic nerve terminal. The internalization and retrograde transport of the TrkB–BDNF complex would represent a form of signaling to the cell body41 and inactivation of BDNF by its removal from the synapse. An alternative mode of termination might involve truncated TrkB receptors that reside on astrocytes and ependymal cells. BDNF can bind to and thereby be sequestered by these sites42,43. This contributes to the termination of the BDNF response by limiting BDNF diffusion within brain parenchyma and away from signal-transducing full-length Trks (Refs 8,42). The truncated TrkB on astrocytes can also internalize BDNF (Ref. 43). Without degradation, the internalized BDNF is released and can support neuron survival43. Thus, astrocytes might distribute BDNF to a much larger volume than was originally supplied by neuronal afferents. Such a broader distribution would be particularly relevant in injured brain, where astrocytes become hypertrophic and neuron survival becomes neurotrophin-dependent.
Other potential roles for anterograde transport Anterograde trafficking of BDNF appears to be a widespread phenomenon, the failure of which could compromise electrical, morphological and neurochemical plasticity in the adult and aged brain. Figures 1 and 2 are models for BDNF trafficking within representative peripheral and central pathways. The anterograde trafficking of endogenous BDNF and NT-3 concentrates them in nerve terminals, where they are stored in vesicles. Their release from terminals, and effects on postsynaptic target neurons (Fig. 4), contrast with the traditional role of NGF as a retrogradely transported, target-derived survival factor. Based on the properties of anterograde transport summarized here, there are multiple roles for the BDNF that is anterogradely trafficked by BDNF-expressing central and peripheral neurons: (1) a neurotransmitter-like role for rapid postsynaptic functions; (2) a source of BDNF to astrocytes, particularly in injured brain; (3) as a trophic agent for postsynaptic targets such as skin, muscle, or gut; and (4) as a signaling molecule between nerve terminals of homologous neurons (e.g. nigrostriatal dopamine neurons) or heterologous neurons (e.g. release from corticostriatal terminals and binding by nigrostriatal terminals), which can then internalize and retrogradely transport the protein. Such a scheme would represent an afferent-supplied adjunct to retrograde signaling that characterizes the neurotrophins. The anterograde transport of BDNF and NT-3 adds an alternate form of trafficking for this important class of molecules. It opens up new areas of inquiry of neurotrophin function in the biology and pathology of the nervous system. What role does anterograde transport play in the ontogeny of the nervous system and its many targets? Does neuronal maturation follow a developmental sequence of autocrine, paracrine, retrograde and anterograde modes of support? Are other endogenous growth factors, such as bFGF, transforming growth factor-b or leukemia inhibitory factor
and/or ciliary neurotrophic factor (LIF/CNTF) family members, provided by afferents, or is anterograde transport peculiar to BDNF and NT-3? Do other PNS and CNS systems, like the enteric nervous system or the cerebellum, receive an afferent supply of neurotrophins? For example, cortical neurons innervate and thus can potentially supply BDNF to a remarkably broad number of areas, including the neocortex itself, the striatum, limbic system, midbrain, brainstem and spinal cord. The BDNF requirement for depolarizationdependent survival of cultured cortical neurons44 is consistent with a supply of BDNF by corticocortical afferents, as well as by autocrine/paracrine modes of support8,44. What are the physiological and pathophysiological conditions that regulate neurotrophin expression and anterograde trafficking? The molecular motors for retrograde and anterograde transport (e.g. kinesins, kef-2, dynamins) are upregulated by nerve injury45. Do such changes influence the amounts and net balance of anterograde and retrograde flows of BDNF or NT-3? Upregulation of anterograde transport motors could be a compensatory strategy to overcome impairments of axonal transport. References 1 2 3 4 5 6 7 8 9 10
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Acknowledgements We thank Dr Ronald M. Lindsay for his insight and encouragement, and for his comments during the preparation of this article. The preparation of Fig. 2 by Dr James Conner is greatly appreciated.
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Neurotrophin trafficking by anterograde transport C. Anthony Altar and Peter S. DiStefano The ever-unfolding biology of NGF is consistent with a target-derived retrograde mode of action in peripheral and central neurons. However, another member of the neurotrophin family, brain-derived neurotrophic factor (BDNF), is present within nerve terminals in certain regions of the brain and PNS that do not contain the corresponding mRNA. Recent studies have shown that the endogenous neurotrophins, BDNF and neurotrophin-3 (NT-3), are transported anterogradely by central and peripheral neurons. The supply of BDNF by afferents is consistent with their presynaptic synthesis, vesicular storage, release and postsynaptic actions. Anterograde axonal transport provides an ‘afferent supply’ of BDNF and NT-3 to neurons and target tissues, where they function as trophic factors and as neurotransmitters. Trends Neurosci. (1998) 21, 433–437
T
HE TRADITIONAL ROLE OF NEUROTROPHINS as retrogradely transported, target-derived survival factors is well established in the developing PNS (Refs 1,2). Thus, NGF is produced by targets of NGF-dependent neurons in limiting quantities and promotes neuron survival through its retrograde transport and signaling to the cell body. Retrograde transport was first demonstrated with injections of 125I-labeled NGF into the terminal fields of sympathetic and sensory neurons in the PNS (Ref. 3) or cholinergic neurons in the CNS (Ref. 4). In these studies, [125I]-NGF accumulated in neuronal cell bodies in a saturable, receptor-mediated fashion. Crucial experiments from the laboratories of Thoenen and Johnson demonstrated the retrograde flow of endogenous NGF by the accumulation of immunoreactive NGF at a site distal, but not proximal, to a ligature or lesion of peripheral and central neurons5.
Indirect evidence for bidirectional neurotrophin trafficking The subsequent discovery of other members of the NGF-related family has revealed new candidates for Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0166 - 2236/98/$19.00
axonal trafficking by brain and peripheral neurons. Intracranial and intranerve injections of iodinated brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) result in their retrograde transport to adult PNS and CNS neuron cell bodies6–9. However, two sets of findings made it clear that these newer family members had roles in the CNS that extended beyond that of target-derived support (survival-promoting and phenotypic maintenance functions) mediated by retrograde transport. First, BDNF and NT-3, unlike NGF, were shown to produce rapid, neuromodulatory effects in a variety of adult neural systems. For example, infusions of BDNF into the cell bodies of adult brain nuclei can augment neuronal firing rates, neurotransmitter release, metabolism and the phenotypic expression of biosynthetic enzymes in cholinergic, dopaminergic, serotonergic, or peptidergic neurons (for reviews, see Refs 8, 10–12). A brief (minutes) exposure of amphibian motor neuron–muscle cocultures to NT-3 or BDNF can augment the electrical activity of innervated muscle cells13. Even more rapid effects of BDNF, in the order of seconds, PII: S0166-2236(98)01273-9
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C. Anthony Altar is at Global Neuroscience Research Otsuka America Pharmaceutical Inc., Rockville, MD 20850, USA and Peter S. DiStefano is at Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
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include increases in electrical activity and intracellular Ca2+ in cultured hippocampal and cortical neurons14,15 (see Refs 11,16 for reviews). Second, and perhaps most revealing, the use of highly specific and sensitive antibodies identified BDNF and NT-3 protein in nerve terminals. Importantly, such growth factor-laden terminals were found in regions of the brain and PNS that did not contain the corresponding mRNA. BDNF protein, but not its mRNA, was found in the neostriatum17,18, neocortex, mossy fibers of hippocampal granule neurons, medial habenula, central amygdala, lateral septum and spinal cord19–21. BDNF protein concentrations in these regions exceeded that of NGF by 100-fold. Conversely, cell bodies of neurons that project to these regions are rich in BDNF mRNA. In the PNS, BDNF was found in the central and peripheral processes of dorsal root ganglia (DRG) sensory neurons, whereas BDNF mRNA was found in the cell bodies of these neurons but not in their central targets22. It is important to note that these characteristics described for BDNF do not apply to NGF, and NGF does not appear to be anterogradely transported by CNS or PNS neurons. Together, these findings suggest that neurotrophins other than NGF, and potentially other growth factors, might be transported anterogradely and released in amounts that could affect their postsynaptic targets.
Anterograde transport of exogenous neurotrophins The presence of the NT-3-binding molecules TrkC, TrkB and p75 in the adult optic tectum, and their presence on developing tectal cells whose survival required an anterogradely transported signal23, prompted von Bartheld to inject 125I-labeled NT-3 into the chicken embryo eye and look for evidence of anterograde transport. Indeed, [125I]-NT-3 accumulated in the optic tectum, where it was associated with presynaptic vesicles. Even more tantalizing was the presence of 125I in the dendrites and somata of postsynaptic tectal neurons, whose survival was enhanced by the eye injections of [125I]-NT-3 (Ref. 23). These findings indicate that the anterogradely transported [125I]-NT-3 was released by retinotectal neurons and taken up by their targets. The anterograde transport of [125I]-NT-3 occurred independently of electrical activity. It was blocked by transport inhibitors and depended on a complex of TrkC (the high-affinity receptor for NT-3) and p75 (the lower-affinity neurotrophin receptor)23,24. A predominance, by 85% to 15%, of anterograde transport relative to retrograde transport was observed for [125I]-NT-3 and 125I-labeled BDNF, respectively, in the chick retinotectal pathway24. In the zebra finch, [125I]-BDNF or [125I]-NT-3 accumulated anterogradely in the archistriatum following their injection into a frontal cortex motor nucleus that provides the major input to the archistriatum25. Deafferentation of the cortical input to the archistriatum produced apoptotic death of archistriatal neurons. Such death was blocked by infusion of BDNF or NT-3 into the archistriatum. Thus, in the chick retinotectal pathway and zebra finch motor-cortical pathway, the anterograde transport of exogenous NT-3 or BDNF can promote the survival of postsynaptic neurons that normally die during development or following axotomy. 434
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Anterograde transport of endogenous neurotrophins in the PNS Recent studies have shown that BDNF is normally concentrated in the superficial layers of the dorsal spinal cord22,26. Dorsal rhizotomy, which disrupts the DRG inputs to the dorsal cord, abolished this labeling. Furthermore, Michael et al.26 have identified BDNF in dense-core vesicles within axon terminals located in the superficial layers of the dorsal spinal cord. Thus, BDNF appears to be packaged much like a neurotransmitter in central processes of DRG neurons. By using a double ligation of the sciatic nerve, several groups have revealed the accumulation of endogenous BDNF on proximal and distal sides of the nerve ligature22,26,27. In agreement with von Bartheld’s findings with iodinated neurotrophins24, a greater intensity of proximal BDNF immunostaining confirmed that the predominant form of transport is in the anterograde direction. That DRG neurons are the probable source of such transported BDNF was indicated by: (1) the robust expression of BDNF mRNA in DRG neurons, compared to motor neurons or Schwann cells, and (2) the accumulation of BDNF in the area of nerve proximal to the DRG, whether a ligature was made in the sciatic nerve or in the dorsal root26,27. The presence of BDNF protein in small-to-medium-sized DRG neurons and in terminals of the dorsal horn, where small unmyelinated neurons normally terminate, suggests a role for the anterograde transport of BDNF in mediating nociceptive transmission or pain modulation. The anterograde transport of BDNF is greatly altered by injury to DRG axons. Sciatic nerve transection or dorsal rhizotomy results in a dramatic increase of BDNF protein transport to the proximal side of a ligature. This is accompanied by increased BDNF mRNA and protein content in the DRG but not at other sites along the peripheral neuraxis27. Interestingly, the ability of NGF to increase BDNF mRNA and protein levels in the DRG (Ref. 26) shows that NGF might also regulate the anterograde transport of BDNF by DRG neurons. It is important to note that, in all these studies, there is no anterograde accumulation of NGF, and that the accumulation of NT-3 is much less than that of BDNF. While similar aspects of the anterograde transport of NT-4 are unknown, anterograde transport appears to be selective within the neurotrophin family. Figure 1 illustrates what is known of anterograde and retrograde transport of BDNF in the PNS.
Anterograde transport of endogenous neurotrophins in the CNS In a remarkably close analogy with the anterograde transport and function of radiolabeled neurotrophins in the developing zebra-finch archistriatum25, endogenous BDNF is also anterogradely transported from the cortex to the neostriatum in the adult rat. While devoid of BDNF mRNA, the adult rat neostriatum nonetheless contains BDNF protein at levels not far below those measured in the BDNF mRNA-rich hippocampus17. Because cortical and nigral neurons that innervate the neostriatum contain BDNF mRNA, we proposed that these cells anterogradely transport BDNF to the striatum18. To test this, axonal transport was inhibited with intracerebroventricular injections of colchicine. This greatly elevated BDNF protein staining in the cell bodies of cortical and nigral cell bodies, but depleted staining within the neostriatum
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(Fig. 2). Unilateral removal of the frontoparietal neocortex decreased BDNF staining in the dorsolateral striatum, where most frontoparietal cortical afferents terminate. Decortication depleted BDNF protein contents by two-thirds in the ipsilateral striatum and by about one-third in the contralateral striatum. These decreases are proportional to the density of striatal innervation by crossed and uncrossed cortical afferents, respectively. Unilateral lesions of the uncrossed nigrostriatal pathway decreased BDNF to a smaller degree, and only in the ipsilateral striatum. Selective ablation of intrinsic striatal neurons did not decrease neostriatal BDNF. Thus, BDNF in the neostriatum is derived largely by anterograde transport within cortical afferents, and to a lesser degree by nigral afferents. The fact that BDNF-gene deletion diminishes the number of parvalbumin-expressing striatal neurons in proportion to the loss in BDNF protein18 suggests a postsynaptic role for the anterogradely transported BDNF. Support for a broader CNS role of anterograde neurotrophin transport is provided by analyses in other brain regions. In the adult hippocampus, seizures induced by electroshock or kainic acid increase BDNF mRNA levels within dentate granule neurons and increase BDNF protein within mossy fibers21. The granule-cell mossy fibers synapse on the apical dendrites of CA3 pyramidal neurons. Because BDNF mRNA was not elevated in the pyramidal neurons by either form of seizure, and because kainate destroys the pyramidal neurons, it is likely that BDNF synthesized by granule neurons is anterogradely transported to hippocampal CA3 pyramidal neurons. The central amygdala and medial habenula contain BDNF-immunoreactive fibers, but not BDNF mRNA (Ref. 19). Lesions of the pontine parabrachial or septal nuclei, which contain high levels of BDNF mRNA, completely eliminated the BDNF-immunoreactive fibers in the amygdala and habenula, respectively. Thus, the BDNF in these regions is probably derived by anterograde transport from the pons to amygdala and from the septum to habenula. The presence of BDNF protein in axon terminals throughout the brain18–20 suggests that anterograde transport might be a common feature of neurotrophin trafficking in the CNS. Figure 3 summarizes anterograde and retrograde transport of BDNF in basal ganglia pathways.
Fig. 1. Schematic representation of the bidirectional axonal transport of neurotrophins in the PNS. Neurons in the dorsal root ganglion (DRG) expressing brain-derived neurotrophic factor (BDNF) mRNA and protein (blue symbols) are predominantly, but not exclusively, small diameter neurons whose central projections terminate in superficial layers of spinal cord gray matter. This is also a site of BDNF immunoreactivity (demarcated in blue) with no corresponding BDNF mRNA expression. Arrows illustrate the capacity for retrograde or anterograde transport of BDNF, either from endogenous or exogenous sources. Large arrows indicate a predominance in the direction of BDNF trafficking. In DRG, BDNF could also serve an autocrine/paracrine role for neighboring DRG neurons, especially after injury. Similarly, anterogradely trafficked BDNF might have trophic actions on targets of BDNF-expressing neurons or on neighboring terminals of non-BDNF-expressing neurons. The anterograde transport of BDNF in motor neurons is relatively weak, as indicated by an arrow and question mark. Similarly, exogenous BDNF can be retrogradely transported from spinal cord to DRG (P. DiStefano and Bregman, unpublished observations), but evidence does not support transport of endogenous neurotrophin in this direction.
dispose it for packaging into anterogradely transported synaptic vesicles. Cleavage to active forms could occur in distinct axonal compartments or upon release. The BDNF protein content of cortical synaptosomes is increased following a systemic treatment with kainic acid that greatly upregulates BDNF mRNA (Ref. 29). It will be of interest to determine whether pharmacological or other manipulations that augment BDNF mRNA also augment BDNF protein in the terminal regions. It will also be important to determine if BDNF is packaged, transported and released by vesicles in tandem with other growth factors [e.g. basic fibroblast growth factor (bFGF) and NT-3], transmitters (e.g. glutamate) or neuroprotective molecules (e.g. ascorbate) that are also found in striatal nerve terminals (Fig. 4).
A neurotransmitter-like role for anterogradely transported neurotrophins As summarized above, anterograde transport provides BDNF to presynaptic nerve terminals in a host of central and peripheral neurons. Thus, anterograde transport is in keeping with the proposed neurotransmitter-like role of neurotrophins11,12,28. In fact, BDNF fulfills all of the classical criteria for being considered a neurotransmitter, as follows.
Presynaptic synthesis and vesicular storage As reviewed above, an abundance of BDNF mRNA is found in the cell bodies of neurons in which BDNF protein is anterogradely transported. The BDNF immunoreactivity in rat brain is enriched in a synaptosomal fraction, and predominates over profiles that resemble dense-core vesicles29. Is the form of BDNF that is anterogradely transported different from that which is retrogradely transported? Neurotrophins are synthesized in pre-pro forms and are processed to mature forms. A longer, less mature form of BDNF could pre-
Fig. 2. Brain-derived neurotrophic factor (BDNF) immunostaining in the frontal cortex of a rat that had received an intraventricular injection of the microtubule-disrupting agent colchicine to block anterograde and retrograde axonal transport. The pial surface of the brain is at the top of the illustration. The BDNF staining is particularly intense in layers II and IV cortical pyramidal neurons. Similar increases in BDNF staining of pars compacta neurons of the substantia nigra18 represent an accumulation of BDNF that would otherwise have been anterogradely trafficked by cortical and nigral neurons to afferent targets such as the neostriatum.
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requires functional Na+ channels and intracellular, but not extracellular, Ca2+.
II
Postsynaptic Trk localization
Cortex V cc
Striatum
S. nigra pc Glutamate Parvalbumin
Consistent with a transmitter-like role for BDNF, full-length TrkB is found in postsynaptic membranes of neurons in the PNS and CNS. In the PNS, immunostaining reveals full-length TrkB on DRG neurons and in the dorsal root entry zone of the spinal cord32. Truncated forms of TrkB are also found in the spinal cord, but they also reside in various target regions in the periphery as well as on Schwann cells. In the CNS, full-length phosphorylated TrkB is present in the optic tectum of the chick23,24 and postsynaptic densities prepared from cortex and hippocampus33. Immunoreactivity for full-length TrkB is seen on the postsynaptic densities of dendritic shafts and spines and on the initial axon segment of hippocampal pyramidal and dentate granule neurons (Drake et al., unpublished observations). While the localization of full-length TrkB is well understood, that of the truncated form is just emerging (see below).
Postsynaptic actions
As discussed above, the rapid, postsynaptic effects of BDNF and NT-3 include their ability to elevate the frequency and amplitude of muscle cell depolarization Dopamine at the developing Xenopus neuromuscular junction13. GABA In the CNS, rapid effects of BDNF are best described for the long-term potentiation (LTP) of hippocampal Fig. 3. Cross sectional representation of brain-derived neurotrophic factor (BDNF) trafficking in corticostriatal, nigral and nigrostriatal synaptic responses. This role for BDNF has implineurons of the rat basal ganglia. There is no retrograde transport by cations for learning and memory, and the failure of layer II corticostriatal neurons or anterograde transport by striatal neur- cognitive abilities in dementia, including Alzheimer’s ons (note the absence of an arrow). Abbreviations: cc, corpus callosum; disease. Cultured hippocampal neurons show an elS. nigra pc, substantia nigra, pars compacta; S. nigra pr, substantia evated postsynaptic current amplitude within minutes nigra, pars reticulata; II, V, layers II and V of the frontal cortex. of BDNF exposure14. These effects are blocked with the kinase inhibitor K-252a, and potentiated by the phosphatase inhibitor okadaic acid. Also within minutes, Depolarization-evoked release The release of [125I]-NT-3 has been observed follow- BDNF or NT-3 can elevate for several hours the deing its anterograde transport by retinal ganglion cells polarization of postsynaptic Schaffer collateral–CA1 to chick tectum23. Endogenous BDNF in cortex30 and synapses in hippocampal slices15. The effects of these [125I]-BDNF preloaded into a synaptosomal fraction of changes include the potentiation of glutamatergic cortex and hippocampus31 can be released by depolar- transmission in the hippocampus14,34. Deletion of one izing stimuli (glutamate, potassium). [125I]-BDNF release or both alleles of the BDNF gene decreases basal synaptic transmission and weakens the LTP of postsynaptic responses Glia/ependyma in the CA1 region35,36. EAAT1, Also within several minutes, EAAT2 BDNF can enhance the phosphorylation of postsynaptic N-methyl-Daspartate (NMDA) receptor subunits Glu 1 (NR1)37 and 2 (NR2b)38 in hippoAA campus and neocortex (Fig. 4). The P LTP produced at synapses between BDNF P Schaffer collaterals and CA1 neurmRNA P Glu ons also elevates BDNF mRNA exP CREB-P NM1 pression in postsynaptic CA1 neurP NM2 P ons39, and in hippocampal dentate P BDNF Glu granule neurons in which LTP was TrkB kinase cAMP induced by perforant path stimuCa2+ Ca2+ Glu BDNF TrkB truncated lation40. Thus, hippocampal LTP mRNA VSCC might depend upon the ability of Schaffer collateral firing to increase presynaptic BDNF synthesis, anteroFig. 4. Brain-derived neurotrophic factor (BDNF) fulfills the classical criteria for being considered as a rapidly grade transport and release. The conacting synaptic modulator. These criteria are illustrated for a typical axodendritic connection between cortical glut- comitant release of glutamate would amatergic and striatal GABAergic interneurons. Abbreviations: AA, ascorbic acid; CREB-P, phosphorylated cyclic AMP increase BDNFergic and glutamatresponse-element-binding protein; EAAT, excitatory amino acid transporter; Glu, glutamate; NM1, NM2, NMDA recep- ergic signaling via a coordinated tor subunits 1 and 2; P, phosphate; TrkB, BDNF receptor; VSCC, voltage-sensitive Ca2+ channel. phosphorylation of TrkB and S. nigra pr
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NMDA receptors on postsynaptic CA1 pyramidal neurons (Fig. 4).
Inactivation and recycling of BDNF The inactivation of BDNF can occur through one of at least three mechanisms. One form, mediated by retrograde transport, requires BDNF to bind full-length TrkB on the presynaptic nerve terminal. The internalization and retrograde transport of the TrkB–BDNF complex would represent a form of signaling to the cell body41 and inactivation of BDNF by its removal from the synapse. An alternative mode of termination might involve truncated TrkB receptors that reside on astrocytes and ependymal cells. BDNF can bind to and thereby be sequestered by these sites42,43. This contributes to the termination of the BDNF response by limiting BDNF diffusion within brain parenchyma and away from signal-transducing full-length Trks (Refs 8,42). The truncated TrkB on astrocytes can also internalize BDNF (Ref. 43). Without degradation, the internalized BDNF is released and can support neuron survival43. Thus, astrocytes might distribute BDNF to a much larger volume than was originally supplied by neuronal afferents. Such a broader distribution would be particularly relevant in injured brain, where astrocytes become hypertrophic and neuron survival becomes neurotrophin-dependent.
Other potential roles for anterograde transport Anterograde trafficking of BDNF appears to be a widespread phenomenon, the failure of which could compromise electrical, morphological and neurochemical plasticity in the adult and aged brain. Figures 1 and 2 are models for BDNF trafficking within representative peripheral and central pathways. The anterograde trafficking of endogenous BDNF and NT-3 concentrates them in nerve terminals, where they are stored in vesicles. Their release from terminals, and effects on postsynaptic target neurons (Fig. 4), contrast with the traditional role of NGF as a retrogradely transported, target-derived survival factor. Based on the properties of anterograde transport summarized here, there are multiple roles for the BDNF that is anterogradely trafficked by BDNF-expressing central and peripheral neurons: (1) a neurotransmitter-like role for rapid postsynaptic functions; (2) a source of BDNF to astrocytes, particularly in injured brain; (3) as a trophic agent for postsynaptic targets such as skin, muscle, or gut; and (4) as a signaling molecule between nerve terminals of homologous neurons (e.g. nigrostriatal dopamine neurons) or heterologous neurons (e.g. release from corticostriatal terminals and binding by nigrostriatal terminals), which can then internalize and retrogradely transport the protein. Such a scheme would represent an afferent-supplied adjunct to retrograde signaling that characterizes the neurotrophins. The anterograde transport of BDNF and NT-3 adds an alternate form of trafficking for this important class of molecules. It opens up new areas of inquiry of neurotrophin function in the biology and pathology of the nervous system. What role does anterograde transport play in the ontogeny of the nervous system and its many targets? Does neuronal maturation follow a developmental sequence of autocrine, paracrine, retrograde and anterograde modes of support? Are other endogenous growth factors, such as bFGF, transforming growth factor-b or leukemia inhibitory factor
and/or ciliary neurotrophic factor (LIF/CNTF) family members, provided by afferents, or is anterograde transport peculiar to BDNF and NT-3? Do other PNS and CNS systems, like the enteric nervous system or the cerebellum, receive an afferent supply of neurotrophins? For example, cortical neurons innervate and thus can potentially supply BDNF to a remarkably broad number of areas, including the neocortex itself, the striatum, limbic system, midbrain, brainstem and spinal cord. The BDNF requirement for depolarizationdependent survival of cultured cortical neurons44 is consistent with a supply of BDNF by corticocortical afferents, as well as by autocrine/paracrine modes of support8,44. What are the physiological and pathophysiological conditions that regulate neurotrophin expression and anterograde trafficking? The molecular motors for retrograde and anterograde transport (e.g. kinesins, kef-2, dynamins) are upregulated by nerve injury45. Do such changes influence the amounts and net balance of anterograde and retrograde flows of BDNF or NT-3? Upregulation of anterograde transport motors could be a compensatory strategy to overcome impairments of axonal transport. References 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
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Acknowledgements We thank Dr Ronald M. Lindsay for his insight and encouragement, and for his comments during the preparation of this article. The preparation of Fig. 2 by Dr James Conner is greatly appreciated.
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