Classical and Novel Directions in Neurotrophin Transport andResearch: Anterograde Transport of Brain-DerivedNeurotrophic Factor by Sensory NeuronsJAMES R. TONRA*Millennium BioTherapeutics, Cambridge, Massachusetts

KEY WORDS pain; inflammation; axotomy; CGRP; BDNF; NGF; DRG

ABSTRACT After the discovery of nerve growth factor, a classic model of neurotrophin actionwas developed. In this model, nerve endings compete for limited quantities of neurotrophic factorsproduced in neuronal target tissues. Neurotrophins are bound with high-affinity receptors ex-pressed on the neuronal membrane and then endocytosed and retrogradely transported back to thecell body of responsive neurons. This classic model of target derived trophic support has beenutilized to explain a wide range of trophic actions including effects on neuronal survival, terminalbranching, and protein expression. However, a number of recent findings in the field of neurotrophinresearch cannot be explained using the classic model. In the peripheral nervous system (PNS),sensory neurons have been shown to contain mRNA for a member of the neurotrophin family,brain-derived neurotrophic factor (BDNF). Sensory neurons do not receive synaptic input soneurotrophin production by these cells does not fit into the classic target derived model. In contrastto target derived trophic support, BDNF produced by sensory neurons provides local autocrine andparacrine neurotrophic support in vitro. Furthermore, in vivo, sensory neurons transport BDNF inthe anterograde direction away from the cell body, and opposite to the retrograde direction utilizedin the classic model. Thus, out of necessity, a new direction for neurotrophin research has developedto study the production and anterograde transport of neurotrophins. The importance of this newmode of neurotrophin action in the PNS is indicated by results that implicate it in the response topain, inflammation, and nerve injury. Microsc. Res. Tech. 45:225–232, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTIONThe peripheral nervous system (PNS) is frequently

used as a model to study the role of neuronal targetsand the effects of axotomy because of the accesibility ofthe targets and axons of motor, sensory, and autonomicneurons. In the 1940s, through studies of motor andsensory neurons in the PNS, Levi-Montalcini and col-leagues demonstrated that limb bud excision in chickembryos results in a dramatic loss of sensory neuronsin the dorsal root ganglion (DRG) associated with anincrease in the number of degenerating neuronal pro-files (Hamburger, 1993; Levi-Montalcini, 1987). Theseresults lead to the proposal that neuronal survival isregulated by the tissues that neurons innervate. Usingmouse sarcoma grafts that become innervated by sen-sory and sympathetic neurons, but not motor neurons,after implantation into the body wall of chick embryos(Bueker, 1948; Levi-Montalcini and Hamburger, 1951),it became clear that diffusible factor(s) located incertain tissues are responsible for the trophic effects ofinnervated tissues. This conclusion was based on thefinding that the sarcoma induced a generalized hyper-trophy of the sympathetic nervous system when it wasin contact with the embryo directly or only through thecirculation (Levi-Montalcini and Hamburger, 1953).

The search for these diffusible factor(s) culminated inthe discovery of the protein nerve growth factor (NGF),which was purified from mouse submaxillary salivaryglands (Cohen, 1960). NGF was subsequently shown to

act as an endogenous survival factor in vivo by demon-strating the dramatic sympathetic neuronal loss andganglionic atrophy in newborn mice after sequestrationof endogenous NGF with an anti-sera raised againstthis antigen (anti-NGF) (Cohen, 1960; Levi-Montalciniand Angeletti, 1966). This effect is dramatic enoughthat atrophy of sympathetic ganglion can be used as aneasily measured positive control when treating animalswith anti-NGF (Fig. 1) (Urschel and Hulsebosch, 1990;Tonra and Mendell, 1998). Further research into themechanism of action for NGF resulted in the demonstra-tion of additional trophic actions and the developmentof a now classical model for NGF action. In this model,NGF is produced in neuronal target tissues where it isbound and endocytosed by the axonal processes andterminal branches of responsive neuronal populations,and then transported retrogradely to the cell soma.

TARGET DERIVED MODEL OFNEUROTROPHIN ACTION

NGF mRNA and protein are found in the targets ofsympathetic and sensory neurons, but at very low

Contract grant sponsor: Regeneron Pharmaceuticals, Inc.; Contract grantsponsor: NIH; Contract grant numbers: R01-NS32264, P01-NS14899, R01-NS16996.

*Correspondence to: James R. Tonra, Ph.D., Millennium BioTherapeutics, 620Memorial Drive, Cambridge, MA 02139.

Received 1 October 1998; accepted in revised form 1 December 1998

MICROSCOPY RESEARCH AND TECHNIQUE 45:225–232 (1999)

r 1999 WILEY-LISS, INC.

levels (Davies et al., 1987; Heumann et al., 1984a;Shelton and Reichardt, 1984; Wheeler and Bothwell,1992). By limiting neurotrophic support, innervatedtissues establish a competitive environment that sculptsthe neural innervation of the tissue. NGF can affectneural innervation through effects on the survival (seebelow) and terminal branching (Campenot, 1987; Dia-mond et al., 1992a,b; Kimpinski et al., 1997; Yasuda etal., 1990) of innervating neurons. Thus, NGF depriva-tion through treatment with anti-NGF in neonatal rats(Goedert et al., 1984; Levi-Montalcini and Angeletti,1966; Ritter et al., 1991; Yip et al., 1984) or autoimmu-nization to NGF in adult rats (Gorin and Johnson,1979, 1980), decreases neuronal survival and innerva-tion density (Hill et al., 1988). At normal levels of NGFproduction, trophic support is not maximal. This isillustrated by the ability of supranormal NGF to in-crease neuronal survival, innervation density, and ter-minal branching (Albers et al., 1994; Diamond et al.,1992a, b). Target tissues are, therefore, actively control-ling the extent of neuronal innervation through limitedproduction of neurotrophic factors, resulting in a posi-tive correlation between NGF mRNA levels and neuro-nal innervation density (Harper and Davies, 1990;Shelton and Reichardt, 1984). In addition to survivaland terminal branching, target derived neurotrophinscan regulate the protein expression (Miller et al., 1982;Verge et al., 1990) and somal size (Hendry 1977; Rich etal., 1984; Yasuda et al., 1990) of responsive neurons.

To gain access to the limited NGF supply, the firststep is the binding of target derived NGF with specificreceptors expressed on the membrane of responsiveneurons. Two transmembrane receptors for NGF havebeen described; p75 (Chao et al., 1986; Johnson, D. etal., 1986; Puma et al., 1983) and trkA (Kaplan et al.,1991; Loeb et al., 1991). The function of p75 is stillevolving (Bothwell, 1996; Chao et al., 1992; see below),but expression may increase the binding affinity ofNGF to trkA (Hempstead et al., 1991). Mice expressinga mutated form of p75 have a significant loss of sensoryinnervation of the foot pad although significant effects

on the NGF dependent sympathetic nervous system arenot observed (Lee et al., 1992). Sympathetic neuronsfrom mice lacking p75 respond normally to NGF in vitrowhile cutaneous sensory neurons have decreased sensi-tivity to NGF (Davies et al., 1993). Thus, p75 may beinvolved in increasing NGF sensitivity in certain neu-rons but it is not required for NGF signaling. Incontrast, animals lacking trkA exhibit a dramatic lossof both sensory and sympathetic neurons (Smeyne etal., 1994). In addition, in mutant rat pheochromocy-toma cells (PC12 cells) that have low trkA expressionand lack NGF responsiveness, transfection with trkAreturns the ability to bind NGF with high affinity andrespond to NGF by increasing neurite outgrowth, cellsize, and neuronal survival in serum-free medium(Loeb et al., 1991). Thus, binding to trkA is thought tobe critical for NGF action. Subsequent to binding, NGFis endocytosed.

NGF and activated trkA, indicated by NGF inducedphosphorylation, are endocytosed in NGF responsivePC-12 cells (Grimes et al., 1996). Since NGF injectedintracellularly does not mimick the effects of extracellu-lar NGF (Heumann et al., 1984b), the endocytosed andmembrane bound activated trk receptor may mediatethe effects of NGF treatment on cell survival andmorphology (Grimes et al., 1996). Some actions of NGFare rapid, occurring shortly after binding. In cultures ofsympathetic neurons from the superior cervical gan-glion (SCG) that have been deprived of trophic supportfor a period of time, NGF treatment causes morphologi-cal changes in growth cones just 30 seconds to 2minutes after addition (Seeley and Greene, 1983; Con-nolly et al., 1985). Furthermore, when SCG neurons aregrown so that individual neurons have processes ineach of three compartmentalized cultures, neurite ex-tension is only stimulated by NGF in the compartmentthat was exposed to the higher NGF concentration(Campenot, 1987), indicating local rather than distantaction. However, some actions of NGF are distant to thesite of binding. Results suggest that the signal for thisaction is carried by the retrograde transport of NGF,and possibly activated trkA, towards the neuronal cellbody.

Retrograde transport after endocytosis of NGF hasbeen demonstrated for both postganglionic sympatheticneurons (Hendry et al., 1974; Johnson et al., 1978) andDRG sensory neurons (Stoeckel et al., 1974) by inject-ing 125I labeled NGF into the anterior eye chamber orsubcutaneously into the forepaw, respectively. Intracel-lular accumulation of 125I-NGF was demonstrated ipsi-lateral, but not contralateral to the injection site andwas abolished by cutting the nerves from the ganglionto the target. After treatment with an intraocularlong-acting NGF preparation, only those postganglionicsympathetic neurons that retrogradely transported io-dinated NGF had increased somal size (Hendry, 1977),demonstrating that cells that retrogradely transportedNGF had a physiological response to NGF. A specificrole for retrograde transport of NGF, possibly as areceptor linked complex, is supported by the findingthat axotomy or colchicine treatment both inhibit theresponse of sympathetic neurons to subcutaneouslyinjected NGF, ipsilateral to the treatment (Paravicini etal., 1975). Thus, systemic NGF is less potent if retro-grade transport is blocked, even when direct access to

Fig. 1. Superior cervical ganglia (SCG) from adult rats (5 weeks ofage) treated from postnatal day 2–14 with normal rabbit serum (twoganglia on left) or rabbit serum raised against nerve growth factor(anti-NGF; two ganglia on right). SCGs were dissected and stained for2 minutes in cresyl violet. Anti-NGF treated SCGs are atrophied dueto neuronal death after sequestration of endogenous NGF by thetreatment. For details of the study see Tonra and Mendell (1998). X6.

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the cell body is equivalent, indicating that some of theeffects of neurotrophins are caused by retrogradelytransported compounds. NGF also stimulates the retro-grade transport of activated trkA (Ehlers et al., 1995),suggesting that the internalized NGF-trkA complex(Grimes et al., 1996) is a retrograde signal of targetderived trophic support.

Thus, NGF acts as a target derived trophic factorthrough binding, internalization, and retrograde trans-port. Targets of neurons in the PNS produce NGF inlimited amounts to determine cellular characteristicssuch as protein expression, somal size, terminal branch-ing, and survival (see above). These trophic effects onneurons resulted in the classification of NGF as aneurotrophin (Korsching, 1993). It was clear that addi-tional neurotrophins existed because embryonic sen-sory neurons could be supported by a factor in mediumand brain extracts that could be immunologically differ-entiated from NGF. These experiments lead to thepurification of brain derived neurotrophic factor (Bardeet al., 1982), the second member of a growing family ofproteins related to NGF (Rodriguez-Tebar et al., 1991).

BDNF AS A TARGET DERIVEDNEUROTROPHIN

The target derived model of neurotrophic support canalso be used to model the actions of BDNF on respon-sive neurons. BDNF is produced in peripheral targettissues of PNS neurons (Koliatsos et al., 1993; Schecter-son and Bothwell, 1992), and binds to both the pan-neurotrophin receptor, p75, and a specific tyrosinekinase receptor (trkB). BDNF selectivity for trkB (Ip etal., 1993; Squinto et al., 1991) may be imparted by thecoexpression of p75 in neuronal cells because trkA andtrkB are not as selective for a given neurotrophin ifexpressed alone in non-neuronal cells lacking p75(Meakin and Shooter, 1992). Subsequent to binding,BDNF is endocytosed and retrogradely transported tothe neuronal cell body (DiStefano et al., 1992; Koliatsoset al., 1993), possibly as a complex with activated trkB(Bhattacharyya et al., 1997). Justifying its classifica-tion as a neurotrophin, BDNF treatment has beenshown to reduce naturally occurring degeneration ofsensory neurons (Hofer and Barde, 1988) and increasethe survival of sensory and motor neurons after axotomy(Eriksson et al., 1994; Koliatsos et al., 1993; Vejsada etal., 1995;Yan et al., 1992). In addition, similar to NGF(Albers et al., 1994), BDNF enhances axonal regenera-tion of adult sensory neurons in vitro (Lindsay, 1988),and overexpression of BDNF in the target field ofpreganglionic sympathetic neurons expressing trkBincreases innervation density (Causing et al., 1997).Thus, BDNF is similar to NGF with regard to bothstructure and mode of action.

BDNF PRODUCTION BY NEURONSWith the target derived model widely accepted as the

mechanism of action for neurotrophins, it was surpris-ing to learn that sensory neurons in the DRG expressBDNF (Ernfors et al., 1990, 1993; Schecterson andBothwell, 1992; Zhang et al., 1994). Neurotrophinproduction by neurons is not novel and can fit into theclassic target derived model. For example, NGF isproduced by hippocampal neurons and is thought toserve as trophic support for innervating basal forebrain

cholinergic neurons (Ayer-LeLievre et al., 1988; Hefti,1986; Williams et al., 1986). However, DRG sensoryneurons do not receive synaptic contacts so BDNFproduced by these cells is not thought to serve as targetderived trophic support for an innervating neuronalpopulation. Since some sensory neurons are known toexpress trkB (McMahon et al., 1994; Schecterson andBothwell, 1992;) it was suggested that by producingBDNF sensory neurons were providing autocrine orparacrine trophic support to themselves or neighboringcells, respectively (Ernfors et al., 1990; Korsching,1993).

This possibility was supported in vitro utilizing adultrat DRG neurons that express BDNF (Acheson et al.,1995). Survival of adult sensory neurons is muchgreater than neonatal neurons after denervation in-duced target deprivation (Johnson, EM Jr. et al., 1986)or when cultured in vitro without exogenous growthfactors (Lindsay, 1988). When BDNF synthesis bysensory neurons is inhibited by incubation with anti-sense oligonucleotides to BDNF, neuronal survival isreduced by 35% showing that sensory neurons canprovide neurotrophic support for one another. Para-crine support of neighboring neurons can be consideredsimilar to the classical model where target cells supportneurons through nearby terminal branches. However,it was also shown that BDNF from sensory neuronsprovides autocrine neurotrophic support in vitro (Ache-son et al., 1995), which deviates dramatically from theclassic model. It has not been determined whetherBDNF is released and then bound by the same neuronor whether it is enclosed in organelles containing trkB,resulting in an intracellular BDNF-activated trkB com-plex. Nevertheless, this mechanism of action for neuro-trophins differs from the classic model in the pathwayutilized by BDNF to support the survival of a popula-tion of neurons.

ANTEROGRADE TRANSPORT OF BDNFThe production of BDNF by sensory neurons also

leads to the suggestion that sensory neurons mighttransport BDNF anterogradely towards the targets oftheir axonal processes (Ernfors et al., 1990) and in theopposite direction to retrograde transport used fortarget derived neurotrophic support. Anterograde trans-port of endogenous BDNF by sensory neurons wasverified in adult rats, and it was shown that BDNF istransported by sensory neurons towards both periph-eral and central targets (Zhou and Rush, 1996). BDNFimmunostaining in the central target of sensory neu-rons, the spinal cord, was observed in the superficialdorsal horn and was dramatically reduced by priorcrushing of the dorsal roots that contain the centralprojections of DRG neurons. Thus, anterogradely trans-ported BDNF was not simply released along the nerve,it was transported to the target (Zhou and Rush, 1996).BDNF immunostaining has also been localized to nervefibers and axons in the dermis and epidermis of skin(Michael et al., 1997). This suggests that peripherallytransported BDNF reaches the targets of sensoryneurons. Thus, in the peripheral nervous system BDNFis utilized as both a classical target derived neuro-trophin (see above), and an anterograde source ofneurotrophin to neuronal targets for as yet undeter-mined functions. Support for the anterograde transport

227BDNF ANTEROGRADE TRANSPORT BY SENSORY NEURONS

of BDNF and NT-3, but not NGF, in the central nervoussystem is also accumulating (see Altar and DiStefano,1998 and Conner et al., 1998 for review) so thismechanism of neurotrophin action may be common to anumber of neuronal systems.

Anterograde BDNF transport by sensory neurons isoccurring in the normal, untreated state since BDNFaccumulates at a ligation of the dorsal root or sciaticnerve on the same side as the cell body in otherwiseuntreated rats (Tonra et al., 1998; Zhou and Rush,1996). In addition, BDNF immunoreactivity in thespinal cord is diminished after cutting off the flow fromthe DRG by crushing the dorsal roots (Zhou and Rush,1996). However, the function of anterogradely trans-ported BDNF in the spinal cord and peripheral sensoryneuron targets is presently unknown. One means togain insight into function is to determine the type ofsensory stimuli recognized by the sensory neurons thatproduce and anterogradely transport BDNF. Sensoryneurons are often classified by cellular characteristicssuch as morphology and protein expression (Lawson,1992). For BDNF expressing neurons, data is availableon the protein content of the neurons, and it is throughthis colocalization data that inferences can be madeabout the function of some of the neurons that produceand anterogradely transport BDNF.

In the untreated condition, most BDNF expressingrat DRG neurons are trkA positive (Kashiba et al.,1997; Michael et al., 1997). In addition, both BDNF(Michael et al., 1997) and trkA (Averill et al., 1995)immunoreactivity in the spinal cord are heaviest inlaminae I and II of the dorsal horn, and both aredramatically reduced by dorsal rhizotomy (Averill et al.,1995; Zhou and Rush, 1996). TrkA expressing DRGsensory neurons are thought to be involved in sensingand transmitting painful stimuli. Loss of trkA positive,NGF responsive DRG neurons through anti-NGF treat-ment (Carroll et al., 1992; Gorin and Johnson, 1979) orknockout of trkA results in a blunted or absent responseto noxious mechanical or thermal stimuli (Smeyne etal., 1994; Urschel et al., 1991). Since BDNF is alsopresent in these cells, anterogradely transported BDNFmay be involved in the sensation or transmission ofpain (Altar and DiStefano, 1998).

In sensory neurons, BDNF also colocalizes withCGRP, another anterogradely transported protein. Thegreat majority of sensory neurons that bind NGF withhigh affinity (Verge et al., 1989) and express trkA(Averill et al., 1995; Verge et al., 1992) also expresscalcitonin gene-related peptide (CGRP), supporting thecolocalization of BDNF, trkA, and CGRP in some sen-sory neurons (see above). Similar to BDNF, CGRP hasbeen implicated in pain sensation and the peripheralresponse to painful stimuli because of its colocalizationwith trkA. However, in addition to this colocalizationdata, CGRP excites dorsal horn neurons involved in thetransmission of nociceptive stimuli (Biella et al., 1991;Miletic and Tan, 1988; Ryu et al., 1988). Furthermore,the anterograde transport of CGRP to the peripheraltargets such as skin has been implicated in the vasodi-lation caused by stimulation of pain sensing unmyelin-ated and thinly myelinated axons (Brain et al., 1985;Janig and Lisney, 1989; Delay-Goyet et al., 1992).

Assigning a function to anterogradely transportedBDNF in the response to painful stimuli is at a more

preliminary stage than CGRP. One possibility is thatBDNF provides trophic support to trkB expressing cellsin the targets. Motoneurons and sensory neurons areknown to express trkB (Koliatsos et al., 1993; McMahonet al., 1994; Schecterson and Bothwell, 1992) so sensoryneurons could be supporting these cells to some extentat the level of the nerve or axon terminal field. BDNF,like CGRP, may also play a role in synaptic transmis-sion of sensory stimuli in the spinal cord. In thehippocampus, BDNF has been shown to increase synap-tic responses to afferent stimulation (Figurov et al.,1996; Kang and Schuman, 1995; Song et al., 1998).Similar effects are also reported in the visual cortexwhere BDNF increased excitatory postsynaptic cur-rents (Carmignoto et al., 1997) and facilitated theinduction of long-term potentiation after low-frequencytetanus (Huber et al., 1998). BDNF is localized to densecore vesicles in the dorsal horn of the spinal cord(Michael et al., 1997) so it could also be released uponsynaptic activation (Griesbeck et al., 1999) to play arole in transmission of sensory stimuli. Interestingly,BDNF affects neuromuscular transmission during de-velopment (Lohof et al., 1993; Wang et al., 1995), so ifBDNF is anterogradely transported to muscle, it couldplay a role in synaptic transmission in the periphery aswell.

ROLE IN INFLAMMATIONAdditional roles for anterogradely transported BDNF

are suggested by reports of treatments that regulateBDNF production by sensory neurons. In particular,inflammation induced by subcutaneous injection ofcomplete Freund’s adjuvant into the hindpaw causes anupregulation of BDNF immunoreactivity in DRG sen-sory neurons (Cho et al., 1997). Increased BDNF insensory neurons after inflammation is associated withan increase in BDNF immunoreactivity in the spinalcord dorsal horn, indicating that the increased BDNFproduced in the DRG in response to inflammation isanterogradely transported to sensory neuron targets.BDNF is being regulated by inflammation in parallelwith the regulation of CGRP and Substance P produc-tion and anterograde transport by sensory neurons(Donnerer et al., 1992; Leslie et al., 1995).

The changes in BDNF (Cho et al., 1997), CGRP andSubstance P (Donnerer et al., 1992; Leslie et al., 1995)after inflammation can all be prevented by sequestra-tion of the excess NGF produced in inflamed tissue(Donnerer et al., 1992) with anti-NGF. Thus, NGF isthought to regulate the expression and transport ofthese proteins by sensory neurons. In support of this,NGF treatment increases BDNF in trkA positive DRGneurons and their projections into the spinal corddorsal horn (Apfel et al., 1996; Michael et al., 1997).NGF also increases BDNF accumulation on the side of asciatic nerve ligature closest to the neuronal cell body(Michael et al., 1997), supporting an upregulation ofBDNF anterograde transport into the periphery. Simi-lar to the effects on BDNF, NGF treatment increasesCGRP and Substance P in the sciatic nerve (Donnereret. al., 1992), and Substance P in the DRG (Leslie et al.,1995).

The regulation of the production and transport ofCGRP and Substance P after treatment with completeFreund’s adjuvant supports their involvement in the

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response to inflammation. Furthermore, the vasodilata-tion and plasma extravasation observed in inflamedtissue has been related to the release of CGRP andSubstance P by sensory neurons (Delay-Goyet et al.,1992; Green et al., 1992; Kenins et al., 1984; Louis etal., 1989). Inflammation also results in reduced painthresholds and increased excitability of dorsal hornneurons receiving synaptic input from sensory neurons(Ma and Woolf, 1996; Menetrey and Besson, 1982;Randich et al., 1997). These changes may be partiallyrelated to an increased release of CGRP and SubstanceP in the spinal cord resulting in excitatory effects on thesynaptic transmission of sensory information in thedorsal horn (Biella et al., 1991; Miletic and Tan, 1988;Ryu et al., 1988). BDNF could also be involved in theresponse to inflammation because BDNF productionand anterograde transport are regulated by inflamma-tion in the same manner as CGRP and Substance P. Inaddition, BDNF and CGRP are colocalized in DRGneurons and peripheral axonal processes indicatingthat they are produced and transported by many of thesame sensory neurons (Michael et al., 1997). The roleplayed by BDNF in inflammation remains unclear buteffects on synaptic transmission are possible (see above).Additionally, BDNF has been shown to reduce tissuedamage in metabolically stressed muscle (Lian et al.,1998) so increased anterograde transport of BDNF mayserve as trophic support for damaged tissue.

ROLE IN RESPONSE TO DENERVATIONStudies on the regulation of anterograde transport of

BDNF have also suggested a role in the response toaxonal injury. Axotomy of the peripheral process ofsensory neurons through crush injury of the sciaticnerve increases BDNF mRNA and protein in ipsilateralDRG (Ernfors et al., 1993; Sebert and Shooter, 1993;Tonra et al., 1998). Increased BDNF expression bysensory neurons after axotomy is observed in associa-tion with an upregulation of anterograde transport by300% (Tonra et al., 1998). The response to axotomy is

not dependent on whether the central or peripheralsensory neuron process is damaged because rhizotomyalso causes an upregulation of BDNF mRNA in DRGneurons (Fig. 2) and increased anterograde transportinto the peripheral processes. However, there are differ-ences in the regulation of anterograde transport follow-ing either central or peripheral injury. BDNF antero-grade transport is elevated for at least 3 days afterrhizotomy, whereas transport is elevated at one dayafter a crush injury of the sciatic nerve but returns tonormal levels by 2 days post-injury. This suggests atransient role for increased anterograde transport ofBDNF at the site of peripheral axonal damage whenreinnervation of the distal stump is permitted (crushinjury).

In contrast to the parallel control of BDNF, CGRP,and Substance P during inflammation (see above),CGRP and Substance P production and anterogradetransport by DRG neurons were not affected by sciaticnerve crush (Tonra et al., 1998). NGF is upregulated inthe nerve at the site of nerve injury (Heumann et al.,1987a,b) so the upregulation of BDNF in the DRG aftercrush could be a response to NGF (Apfel et al., 1996;Michael et al., 1997). However, an increase in CGRPand Substance P expression would be expected if NGFtrophic support were increased (Donnerer et al., 1992;Leslie et al., 1995). This suggests that a differentstimulus exists for upregulation of BDNF in sensoryneurons in response to nerve injury. Preliminary find-ings that BDNF mRNA is increased in trkA, as well astrkB and trkC positive sensory neurons (Averill et al.,1997), support this possibility because NGF would notbe expected to affect trkB and trkC positive neurons (Ipet al., 1993).

In injured nerve, anterogradely transported BDNFmay function more as a classical neurotrophin, provid-ing trophic support to axotomized neurons that inner-vate the site of injury. BDNF treatment can havedramatic effects on axotomized sensory and motorneurons. In neonates, BDNF prevents or delays sensory

Fig. 2. In situ hybridization for BDNF mRNA in the fifth lumbardorsal root ganglion (DRG) ipsilateral or contralateral to a rhizotomyperformed one day earlier. Ganglia are from the same rat and areprocessed on the same slide. BDNF mRNA is dramatically increased inDRG sensory neurons whose axonal projections to the spinal cord werecut (ipsilateral), compared to neurons whose projections are left intact

(contralateral). Increased BDNF message was found to occur at thesame time that BDNF anterograde transport into the peripheralprojections of sensory neurons was increased, indicating a coordinatedupregulation in response to nerve injury. For a detailed description ofthe study see Tonra et al. (1998). X75.

229BDNF ANTEROGRADE TRANSPORT BY SENSORY NEURONS

and motor neuron death after sciatic nerve section(Eriksson et al., 1994; Yan et al., 1992). In adults,BDNF has been shown to attenuate the sciatic nervelesion-induced decrease of choline acetyltransferaseimmunoreactivity in motor neurons (Friedman et al.,1995). A trophic role for BDNF at the site of nerveinjury is also suggested by the dramatic increase insensory and motor neuron transport of 125I labeledBDNF from the site of nerve damage compared totransport from intact nerve (Curtis et al., 1998; Di-Stefano and Curtis, 1994). In addition to neurotrophiceffects, BDNF could affect Schwaan cell responses tonerve injury, acting through p75, which is expressed inincreasing amounts by these cells at the site of nerveinjury (Funakoshi et al., 1993). Effects on Schwaancells through p75 could include promoting cell deathand guiding cell migration (Anton et al., 1994; Casaccia-Bonnefil et al., 1996; Rabiazadeh et al., 1993).

CONCLUSIONFor the majority of published neurotrophin research,

the classic model of target derived neurotrophic supportserves as a successful framework to understand themechanism of action for neurotrophins. However, a newdirection in neurotrophin research, towards the studyof the anterograde transport of BDNF, has developedout of the inability to explain certain experimentalresults using the classic model. The peripheral nervoussystem is providing an accessible environment to studythe production and anterograde transport of neurotroph-ins by neurons, similar to its role in the development ofthe classic model. This was made possible by the findingthat sensory neurons are among the neuronal popula-tions recognized to be utilizing this novel mechanism ofaction. Although many details remain to be discovered,the production and anterograde transport of BDNF bysensory neurons has already been implicated in theresponse to pain, inflammation, and nerve injury. Thusthe understanding of anterograde transport of BDNFhas potential clinical importance and further researchwill uncover the specific role BDNF plays in thesepathological states.

ACKNOWLEDGMENTSI am grateful to Drs. Peter DiStefano and Lorne

Mendell for their significant contributions to my re-search.

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