brain-derived neurotrophic factor induces excitotoxic sensitivity in cultured embryonic rat spinal...

Brain-Derived Neurotrophic Factor Induces ExcitotoxicSensitivity in Cultured Embryonic Rat Spinal Motor Neurons

Through Activation of the Phosphatidylinositol3-Kinase Pathway

*‡Hugh J. L. Fryer, *Daniel H. Wolf, †Ronald J. Knox, *‡Stephen M. Strittmatter,§Diane Pennica, §Rhona M. O’Leary, *David S. Russell, and *†Robert G. Kalb

Departments of*Neurology and†Pharmacology and‡Section of Neurobiology, Yale University School of Medicine, New Haven,Connecticut, and§Molecular Oncology Department, Genentech, Inc., South San Francisco, California, U.S.A.

Abstract: Neurotrophic factors (NTFs) can protectagainst or sensitize neurons to excitotoxicity. We studiedthe role played by various NTFs in the excitotoxic deathof purified embryonic rat motor neurons. Motor neuronscultured in brain-derived neurotrophic factor, but notneurotrophin 3, glial-derived neurotrophic factor, or car-diotrophin 1, were sensitive to excitotoxic insult. BDNFalso induces excitotoxic sensitivity (ES) in motor neuronswhen BDNF is combined with these other NTFs. Theeffect of BDNF depends on de novo protein and mRNAsynthesis. Reagents that either activate or inhibit the75-kDa NTF receptor p75NTR do not affect BDNF-in-duced ES. The low EC50 for BDNF-induced survival andES suggests that TrkB mediates both of these biologicalactivities. BDNF does not alter glutamate-evoked rises ofintracellular Ca21, suggesting BDNF acts downstream.Both wortmannin and LY294002, which specifically blockthe phosphatidylinositol 3-kinase (PI3K) intracellular sig-naling pathway in motor neurons, inhibit BDNF-inducedES. We confirm this finding using a herpes simplex virus(HSV) that expresses the dominant negative p85 subunitof PI3K. Infecting motor neurons with this HSV, but not acontrol HSV, blocks activation of the PI3K pathway andBDNF-induced ES. Through the activation of TrkB andthe PI3K signaling pathway, BDNF renders developingmotor neurons susceptible to glutamate receptor-medi-ated cell death. Key Words: Ionotropic glutamate recep-tors—Motor neurons—Excitotoxicity—Phosphatidylino-sitol 3-kinase—TrkB—Low-affinity neurotrophin receptor.J. Neurochem. 74, 582–595 (2000).

The death of neurons throughout the CNS can beinduced by the prolonged activation of ionotropic gluta-mate receptors (IGRs). This phenomenon, known as ex-citotoxicity, has been implicated in the death of neuronsin vivo in a variety of pathological conditions includingoxygen–glucose deprivation, seizures, trauma, and neu-rodegenerative disease (Choi, 1988, 1990; Lees, 1993;Lipton and Rosenberg, 1994; Shaw, 1994). During de-

velopment, excitotoxicity may contribute to the death ofneurons during naturally occurring neuronal death peri-ods (Caldero´ et al., 1997; Solum et al., 1997). In vitromodels of excitotoxicity have shown that the excitotoxicdeath of neurons is caused in part by sustained patho-logical rises in intracellular Ca21 concentration ([Ca21]i)(Randall and Thayer, 1992; Frandsen and Schousboe,1993; Hartley et al., 1993; Harman and Maxwell, 1995;Hyrc et al., 1997).

Neurotrophic factors (NTFs) are required for the sur-vival of developing and adult CNS neurons (Oppenheim,1989; Davies, 1994). Knowledge of the biological ac-tions of NTFs has led to the hypothesis that this class ofgrowth factors might aid in the prevention of neuronaldeath induced by a variety of noxious insults (Hefti,1994; Lindsay, 1994). In fact, a variety of NTFs abrogateIGR-induced death of CNS neurons in vitro and in vivo(Lindholm, 1994; Tatter et al., 1995). Data from in vitrosystems suggest that NTFs may protect neurons by (1)reducing the extent of the sustained rise of [Ca21]i in-duced by chronic IGR activation, (2) reducing cell sur-face-expressed IGRs, (3) inducing proteins that bufferrises of [Ca21]i, or (4) reducing the accumulation ofintracellular superoxides caused by the sustained rise of

Received August 3, 1999; revised manuscript received September23, 1999; accepted September 28, 1999.

Address correspondence and reprint requests to Dr. R. G. Kalb atDepartment of Neurology, Yale University School of Medicine, P.O.Box 208018, 333 Cedar St., New Haven, CT 06520-8018, U.S.A.E-mail: [email protected]

Abbreviations used:BDNF, brain-derived neurotrophic factor;[Ca21]i, intracellular calcium concentration; CT-1, cardiotrophin 1;ERK, extracellular signal-regulated kinase; ES, excitotoxic sensitivity;GDNF, glial-derived neurotrophic factor; HSV, herpes simplex virus;IGR, ionotropic glutamate receptor; MAP, mitogen-activated protein;NGF, nerve growth factor; NT, neurotrophin; NTF, neurotrophic fac-tor; PI3K, phosphatidylinositol 3-kinase.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

[Ca21]i (Mattson et al., 1993, 1995; Brandoli et al., 1998;Klocker et al., 1998).

NTFs have also been shown, however, to make neu-rons more vulnerable to excitotoxicity. In an in vitrohypoxic/ischemic model, in which the death of corticalneurons is caused by excitotoxicity, incubation of thecells with brain-derived neurotrophic factor (BDNF) andneurotrophin (NT) 3 and 4/5 exacerbates neuronal death(Koh et al., 1995). NTFs potentiate excitotoxicity inculture systems of other neurons as well (Prehn, 1996;Morrison and Mason, 1998). As mature cultures of neu-rons undergo a higher percentage of excitotoxicity-in-duced death than developing neurons, it has been hy-pothesized that NTFs may make neurons more vulnera-ble to excitotoxicity by accelerating their maturation(Samdani et al., 1997). NTFs have also been shown tomake neurons more vulnerable to excitotoxicity in vivo;BDNF, for example, injected into the hippocampus ex-acerbates kainate toxicity (Rudge et al., 1998).

As a consequence of their responsiveness to a numberof NTFs, cultured embryonic motor neurons have beenan ideal system with which to study NTF actions (Camuand Henderson, 1992; Henderson et al., 1997). In addi-tion, we have previously shown that prolonged activationof IGRs causes the death of a subset of motor neuronspurified from embryonic rats and that this effect is de-pendent on Ca21 influx (Fryer et al., 1999). In this study,the motor neurons were cultured in a mixture of BDNF,NT3, and NT4/5. Considering the contradictory rolesplayed by NTFs in other systems, we wanted to investi-gate the role that various individual NTFs play in theexcitotoxic death of embryonic spinal motor neurons. Inaddition, we wanted to determine which NTF receptorstransduce this signal and the intracellular signaling sys-tems that mediate this response.

MATERIALS AND METHODS

MaterialsTimed pregnant Sprague–Dawley rats were obtained from

Charles River (Kingstown, NY, U.S.A.). Leibowitz L15 me-dium, glutamine, penicillin/streptomycin, Ca21/Mg21-freephosphate-buffered saline, horse serum, mouse laminin, andsodium bicarbonate were purchased from GibcoBRL (GrandIsland, NY, U.S.A.). All other culture reagents, glutamate,glycine, and wortmannin were purchased from Sigma (St.Louis, MO, U.S.A.). LY294002 was purchased from Calbio-chem (La Jolla, CA, U.S.A.). Dr. Eugene Johnson (WashingtonUniversity, St. Louis, MO, U.S.A.) kindly provided the hybrid-oma cell line 192 [anti-rat 75-kDa NTF receptor (p75NTR)monoclonal antibody]. Recombinant NTFs were obtained fromthe following sources: Human BDNF was obtained fromCephalon (West Chester, PA, U.S.A.), Genentech (S. San Fran-cisco, CA, U.S.A.), Regeneron (Tarrytown, NY, U.S.A.), andAlomone Labs (Jerusalem, Israel); recombinant NT3 from Re-generon and Alomone Labs; cardiotrophin 1 (CT-1) from Ge-nentech; and glial-derived neurotrophic factor (GDNF) fromAmgen (Thousand Oaks, CA, U.S.A.). For the majority of theexperiments in this study, we used BDNF obtained from Re-generon. Two different antibodies to TrkB, which gave identi-cal results, were used (Santa Cruz Biotechnology, Santa Cruz,

CA, and Transduction Laboratories, Lexington, KY, U.S.A.).Other antibodies for immunocytochemistry and biochemistrywere obtained from the following sources: p85 (Upstate Bio-technology, Lake Placid, NY, U.S.A.);b-galactosidase (59-39,Inc., Boulder, CO, U.S.A.); phospho-Trk490 and phospho-Akt(New England BioLabs, Beverly, MA, U.S.A.); and phospho-ERK (Promega, Madison, WI, U.S.A.).

Motor neuron purification, drug treatments, andquantification of cell survival

Motor neurons from embryonic rat embryos were purified aspreviously described (Fryer et al., 1999). In brief, dissociatedventral spinal cords from the embryos of 15–16-day pregnantrats were first enriched for motor neurons on a cushion ofmetrizamide. These cells were further purified by panning onplates coated with a monoclonal antibody (antibody 192) thatrecognizes the low-affinity neurotrophin receptor p75NTR,which is expressed only on motor neurons of the embryonicventral horn (Yan and Johnson, 1988). Rather than eluting theimmunopanned motor neurons with monoclonal antibody 192,as we had previously reported, the cells for these experimentswere washed from the immunopanning plates with L15 me-dium, which effectively but gently removed the cells from theplate. Purified motor neurons were diluted with L15 mediumsupplemented with 0.63 mg/ml sodium bicarbonate, 100 IU/mlpenicillin, 100mg/ml streptomycin, 2% (vol/vol) horse serum,20 mM glucose, 5mg/ml insulin, 0.1 mM putrescine, 20 nMprogesterone, 0.1 mg/ml conalbumin, and 30 nM sodium se-lenite and were seeded at low density (4,500 cells or 4.5cells/mm2) onto 33-mm plates (Nunc, Boston, MA, U.S.A.)that had been coated sequentially with poly-D-ornithine andmouse laminin.

Excitotoxic sensitivity (ES) assays were performed aftermotor neurons were cultured overnight at 37°C and 5% CO2.Stock solutions of glutamate and glycine were prepared inLocke’s buffer (see below). For ES assays, the culture mediumwas replaced with Locke’s buffer (in mM: 134 NaCl, 25 KCl,2.3 CaCl2, 5 dextrose, 4 NaHCO3, and 5 HEPES, pH 7.2)containing vehicle (controls) or 200mM glutamate1 20 mMglycine, and the cultures were incubated at 37°C in 5% CO2.After 1 h, the Locke’s buffer was removed, the plates werewashed three times with fresh Locke’s buffer, and the originalor fresh medium was added to the plates.

Cell survival was quantified as follows. The initial numberof cells per plate was determined by counting cells from at leastthree nonadjacent 33 3-mm grids using a Nikon TMS invertedphase-contrast microscope at a magnification of 1003 fromthree to five plates 2–3 h after plating. Following varioustreatments (see text), cell survival was once again quantified.The percentage of initially plated cells is the survival per platedivided by the number of initially plated cells times 100. Eachdata point is the mean6 SEM of two to four independentexperiments. Significance was determined using ANOVA withScheffe’s test.

Virus studiesReplication-deficient herpes simplex viruses (HSVs) were

used to introduce genes into cultured motor neurons. Methodsfor the generation and titering of viral stocks have been de-scribed (Neve et al., 1997). The titers of the viruses employedin this study were routinely in the range of 3–53 107 plaque-forming units/ml.

A dominant negative form of p85 (Hara et al., 1994; Kotaniet al., 1994), the regulatory subunit of phosphatidylinositol3-kinase (PI3K), designated DNp85 (provided by Dr. M. Ka-

J. Neurochem., Vol. 74, No. 2, 2000

583BDNF-INDUCED EXCITOTOXIC SENSITIVITY

suga, Kobe University School of Medicine, Kobe, Japan), wascloned into the amplicon vector pRCUC and used to generaterecombinant virus. HSV engineered to express LacZ served asa control.

For use in the ES assays, motor neurons were plated inmedium containing 10 ng/ml CT-1 and cultured for 6–8 h. Onemicroliter of viral stock was added to cell cultured in 33-mmplates. When higher numbers of cells were plated for biochem-ical studies (see below), 5ml of viral stock was added. Weempirically determined that this concentration of either HSVconstruct gave the highest level of infection (determined im-munocytochemically) without evident toxicity.

ImmunocytochemistryFor immunocytological studies, 5,000 cells in 50ml of

medium were plated onto the center of 14-mm round glasscoverslips (Assistent, Germany). After a wait of 6 h for cells toattach, more medium was added to the well or dish to com-pletely cover the coverslip. Twenty-four to 48 h after the initialplating, cells were fixed with 4% (wt/vol) paraformaldehydeand 0.1% (vol/vol) glutaraldehyde in 0.1M phosphate buffer(pH 7.4) for 10–20 min at room temperature. Following severalwashes in phosphate buffer, cells were blocked and permeabil-ized in antibody dilution buffer [Dulbecco’s modified Eagle’smedium containing 5% (vol/vol) fetal calf serum and 0.2%(wt/vol) sodium azide] with 0.2% (vol/vol) Triton X-100 for10–20 min at room temperature. Coverglasses were incubatedat 4°C for 1–3 days with antibodies diluted in antibody dilutionbuffer. Primary antibody was visualized using species-specificfluorescein isothiocyanate- or horseradish peroxidase-conju-gated secondary antibodies (Jackson ImmunoResearch Labora-tories, West Grove, PA, U.S.A.). Stained cells were mountedon glass slides in Vectashield (Vector Labs, Burlingame, CA,U.S.A.) and photographed on a Zeiss Axioscope.

Single-cell [Ca21]i imagingSingle-cell fluorescence ratio [Ca21]i imaging from purified

embryonic motor neurons was performed as previously de-scribed (Fryer et al., 1999). Neurons that were cultured ontoglass coverslips were loaded with the acetoxymethyl esterCa21 indicator dye fura PE3 (TefLabs, Austin, TX, U.S.A.).Fura PE3 fluorescence was measured using a Nikon Diaphotequipped with a 403 Nikon Plan Fluor objective (NA5 1.3)and recorded with a Hammamatsu C2400 iCCD camera. Exci-tation (345 and 380 nm) illumination of fura PE3 was per-formed using a 75-W xenon arc lamp and a PC-controlledmonochromator coupled to the microscope by fiberoptic cable(Photon Technology Int., South Brunswick, NJ, U.S.A.).[Ca21]i concentrations were calculated from ratioed images asdescribed previously (Fryer et al., 1999).

BiochemistryApproximately 50,000 purified motor neurons were cultured

overnight in the wells of a 96-well plate in medium containing10 ng/ml CT-1. One hour prior to treatment with drugs and/orNTs (see text), the culture medium was replaced with L15medium containing 0.1% bovine serum albumin, 1% NaHCO3,and 10 ng/ml CT-1. Cells were treated with NTs for 5 min,washed once in phosphate-buffered saline containing 1 mMsodium orthovanadate and 10 mM sodium fluoride, and har-vested with RIPA buffer (Tris-buffered saline containing 1%Triton X100, 0.5% sodium deoxycholate, 1 mM sodium or-thovanadate, 10 mM sodium fluoride, 5 mM EDTA, 1 mMphenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, 5 mM«-aminocaproic acid, 5mg/ml leupeptin, and 5mg/ml pepstatin

A). Cell lysates from individual wells containing an identicalnumber of plated cells were centrifuged for 10 min at 10,000gto pellet DNA. Extracts were separated on 7.5% sodium dode-cyl sulfate–polyacrylamide gels and transferred to nitrocellu-lose. Sample loading was controlled by staining the membranesfor total protein with Ponceau S, probing with anti-ERK (notphosphospecific) and anti-Akt (not phosphospecific). None ofthese showed any significant differences. Membranes wereblocked with 5% nonfat dry milk diluted in Tris-buffered salineand incubated sequentially with various primary antibodies andhorseradish peroxidase-conjugated secondary antibodies. Im-munopositive bands were visualized by exposing blots, whichhad been reacted with chemiluminescent chromogen (ECL orECL Plus; Amersham, Arlington Heights, IL, U.S.A.), to au-toradiographic film.

RESULTS

BDNF induces ES of motor neuronsIn the following set of experiments, we wanted to

determine the effect of various NTFs on IGR-inducedexcitotoxicity. It has been shown that motor neuronsexpress receptors for, and are supported by, a number ofNTFs including BDNF, NT3, GDNF, and CT-1 (Hen-derson et al., 1993; Pennica et al., 1996; Treanor et al.,1996; Trupp et al., 1996; Klein et al., 1997). When usedat concentrations reported by others, we confirmed thatthese NTFs support the survival of purified motor neu-rons for several days in culture (Fig. 1A, lightly shadedcolumns). Each of these NTFs, used at saturating con-centrations (1, 1, 10, and 10 ng/ml, respectively), wasequally effective, supporting the survival of;90% of theinitially plated motor neurons during a 2-day cultureperiod. When all of these NTFs were combined, survivalduring the 2-day culture was similar to that when cellswere cultured in the other NTFs alone, indicating that theeffect of these factors was not additive during this timeinterval. We have also found ED50 values for survival foreach of these NTFs to be similar to those reported byother investigators (see Fig. 4; not shown). In compari-son, 40% of the plated motor neurons die in the absenceof NTFs.

To determine the effect that these NTFs have onIGR-induced toxicity, the following experiment was per-formed. Neurons were plated and cultured for 18–24 h inone of the NTFs at a concentration we found to bemaximal for motor neuron survival (see above). Themedium was then removed and replaced with Locke’sbuffer (see Materials and Methods) that was supple-mented with 200mM glutamate1 20 mM glycine orvehicle, concentrations that we have previously reportedto cause maximal motor neuron death (Fryer et al.,1999). After 1 h, the Locke’s buffer was removed, thecells were washed three times in fresh Locke’s bufferwithout agonists, and the original medium was replaced.After culturing the cells for an additional 18–24 h, motorneuron survival was determined (see Materials andMethods). This assay is used throughout this study andwill be referred to as the ES assay, with the term ESdefined as the ability of a NTF to make neurons vulner-able to glutamate receptor-mediated toxicity.


584 H. J. L. FRYER ET AL.

When cultured in BDNF under our assay conditions, a1-h exposure to glutamate causes the loss of;40% ofmotor neurons. There was no cell death of motor neuronscultured in NT3, GDNF, or CT-1 when subjected to theidentical toxicity assay. In the absence of NTFs, gluta-mate does not kill motor neurons. The effect of BDNFwas seen using recombinant protein purchased from fourdifferent vendors (see Materials and Methods). This ob-servation and the finding that all preparations of BDNFpromoted basal survival of motor neurons (when sup-plied in the picogram to nanogram per milliliter range)argue for a specific effect of BDNF on motor neuron ES.

These data could be interpreted in one of two ways:Either BDNF is the one NTF of those tested that fails tobe neuroprotective, or the other NTFs fail to induce ES inmotor neurons; that is, BDNF may cause a phenotypicchange in motor neurons that makes them vulnerable toexcitotoxic injury that the other NTFs fail to induce. To

distinguish between these two possibilities, motor neu-rons were cultured in medium containing either NT3,GDNF, or CT-1 with or without BDNF and then assayedfor ES. If BDNF is not neuroprotective, motor neuronscultured in BDNF with any other NTF should survive theexcitotoxic challenge. On the other hand, if BDNF in-duces ES, motor neurons cultured in either NT3, GDNF,or CT-1 might be made sensitive to IGR-mediated tox-icity by including BDNF in the culture medium. The datain Fig. 1B provide evidence for the second possibility.As was found for motor neurons cultured in BDNFalone,;40% of those cells cultured with BDNF and anyof the other NTFs were sensitive to a 1-h exposure toglutamate. Glutamate was not toxic to motor neurons ifBDNF was added to the medium after glutamate chal-lenge (see also below).

Our data indicate that embryonic motor neurons arenot inherently sensitive to prolonged activation of IGRsbut can be made so when cultured in BDNF. Addition-ally, of the other NTFs we have tested, none is neuro-protective, even when the cells are cultured in a combi-nation of all of the NTFs (Fig. 1B, combined). Our datasuggest that BDNF induces a phenotypic change in mo-tor neurons that renders them sensitive to excitotoxicity.

BDNF does not alter glutamate-evoked Ca21 influxNTFs can regulate the physiology of neurons in a

variety of different ways, including induction of expres-sion of voltage-gated and ligand-gated ion channels(D’Arcangelo et al., 1993; Fanger et al., 1995; Levineet al., 1995b; Bai and Kusiak, 1997). Furthermore, stud-ies on the effects of BDNF on long-term potentiationdemonstrate that BDNF can influence the responsivenessof the postsynaptic cell to glutamate (Levine et al.,1995a). BDNF induction of ES might be mediated byincreasing motor neuron physiological responsiveness toglutamate receptor activation (Levine et al., 1998). Toexamine the responsiveness of motor neurons to IGRstimulation, we monitored the changes of [Ca21]i levelsevoked by glutamate of cells that had been cultured inmedium containing various NTFs. Because many neu-rons can be monitored simultaneously, measurement offree [Ca21]i levels was an effective means of gaugingIGR responsiveness in motor neurons. We previouslyshowed that prolonged incubation of motor neurons withagonists of IGRs induces a sustained elevated level offree [Ca21]i and that rises in [Ca21]i are required forglutamate-induced death of motor neurons (Fryer et al.,1999).

To compare IGR responsiveness in cells cultured un-der conditions that induce ES with those that do not,motor neurons were plated in medium containing CT-1alone, which does not promote ES, or in CT-11 BDNF,which does. Motor neurons were cultured at high densityso that the free [Ca21]i level of 20–30 neurons could bemonitored simultaneously. Even at these higher densi-ties, BDNF is required for ES (data not shown). Afterovernight culture, neurons were loaded with the Ca21-

FIG. 1. Embryonic rat motor neurons cultured in BDNF, with orwithout other NTFs, can die upon glutamate receptor activation.A: Following overnight culture in BDNF, NT3, GDNF, or CT-1,embryonic rat motor neurons were treated with either 200 mMglutamate 1 20 mM glycine (lightly shaded columns) or vehicle(white columns) for 1 h. Afterward, medium was replaced, thecells were cultured overnight, and cell survival was quantified.Cells cultured in BDNF, but not in the other NTFs, were sensitiveto glutamate toxicity. Survival is reported as percentage of cellsremaining of those initially plated. B: Motor neurons were cul-tured overnight in either NT3, GDNF, or CT-1 (or these threecombined) in the presence (darkly shaded columns) or absence(black columns) of BDNF. The following day, all of the cells weretreated with glutamate/glycine, and motor neuron survival wasdetermined the next day. In the presence of BDNF, motor neu-rons were sensitive to excitotoxic insult. **p ! 0.001, ANOVAwith Scheffe’s test.



sensitive dye fura PE3, and glutamate-evoked rises in[Ca21]i were examined.

Figure 2 shows the free [Ca21]i level in neurons stim-ulated with glutamate. The baseline [Ca21]i level in cellscultured in any of the NTFs is;100 nM. Followingaddition of glutamate to the bathing medium, a transientspike of [Ca21]i is seen, followed by a sustained eleva-tion that lasts for the duration of exposure to glutamate.These population [Ca21]i dynamics are similar to thosewe have reported previously (Fryer et al., 1999). Themean level of the sustained rise in [Ca21]i did not differin cells cultured in CT-1 alone (four separate experi-ments, 97 total cells imaged) or CT-11 BDNF (sixseparate experiments, 128 total cells imaged). In com-parison with glutamate, cells stimulated with a concen-tration of extracellular K1 that was found to be maxi-mally depolarizing (25 mM) have much lower meansustained [Ca21]i.

The analysis of the mean population dynamics couldobscure the possibility that BDNF could increase theresponsiveness of a subpopulation of cells to glutamate.We thus wanted to compare the distribution of plateau[Ca21]i levels evoked by glutamate in cells cultured inCT-1 alone with those cultured in CT-11 BDNF. Themean plateaus of responsiveness of cells cultured inCT-1 alone or CT-11 BDNF are not significantly dif-ferent (4986 30 vs. 4376 26 nM; p . 0.4). Similarly,the percentage of cells that have lower or higher evokedlevels of [Ca21]i is also not significantly different be-tween the cells cultured under different conditions. Thesedata suggest that the mechanism whereby BDNF inducesES in motor neurons is not linked to increased electro-physiological responsiveness or exaggerated rises in[Ca21]i in susceptible cells. These findings reinforce theidea that glutamate-evoked rises in [Ca21]i are necessarybut not sufficient to induce excitotoxic death of motorneurons in our culture system.

TrkB, not p75NTR, is likely to mediate BDNF-induced ES

p75NTR and TrkB are two receptors that transduceBDNF signaling intracellularly (Barbacid, 1994; Chao,1994; Chao and Hempstead, 1995). As both receptorshave intracellular signaling domains, we sought to deter-mine if one or both are responsible for transducingBDNF-induced ES. The p75NTR component of the recep-tor is of particular interest because this tumor necrosisfactor-a receptor family member has an intracellulardeath domain that, upon ligand binding, can induce ap-optotic cell death (Rabizadeh et al., 1993; Casaccia-Bonnefil et al., 1996; Frade et al., 1996; Carter andLewin, 1997). For our first approach, we wanted todetermine if selective activation of p75NTR induces ES.To test for this, motor neurons were cultured in mediumcontaining nerve growth factor (NGF), which selectivelyactivates p75NTR but does not support basal motor neu-ron survival, and CT-1, which supports basal survival butneither abrogates nor promotes ES (Fig. 1). Under theseconditions, motor neurons were not sensitive to excito-toxicity (Fig. 3A).

Whereas both BDNF and NGF bind p75NTR withapproximately the same affinity, it is possible these li-

FIG. 3. p75NTR is unlikely to participate in induction of ES byBDNF. A: Motor neurons were cultured in CT-1, CT-1 1 NGF, orCT-1 1 REX antibody, which is a p75NTR function-blockingantibody, overnight, were treated with glutamate/glycine (opencolumns) or vehicle (filled columns), and cell survival was deter-mined the next day. None of these culture conditions induced ESin embryonic rat motor neurons. B: Cells were cultured in BDNF,BDNF 1 NGF, or BDNF 1 REX antibody overnight, were treatedwith glutamate/glycine (open columns) or vehicle (filled col-umns), and cell survival was determined the next day. Neitheractivating p75NTR with NGF nor blocking it with anti-REX anti-body inhibited the ability of BDNF to induce ES. **p ! 0.001.

FIG. 2. BDNF does not alter glutamate-evoked rises of [Ca21]i.Motor neurons were cultured in CT-1 alone or in CT-1 1 BDNFfor 2 days, after which evoked [Ca21]i was measured (see Ma-terials and Methods). After baseline [Ca21]i was established,10-fold stock of K1 or glutamate was added to the incubationbuffer. Evoked changes in [Ca21]i by 200 mM glutamate weresimilar for cells cultured in either CT-1 alone (squares) or in CT-11 BDNF (filled circles). In comparison, 25 mM K1 evoked lowerchanges in [Ca21]i (open circles).



gands activate p75NTR differently, resulting in distinctbiological effects. If this were the case, exposure ofmotor neurons to BDNF and high concentrations of NGFwould competitively block BDNF activation of p75NTR

but should not affect BDNF signaling through TrkB.Thus, in our next experiments, motor neurons were cul-tured in medium containing 1 ng/ml BDNF with NGF ata 100-fold higher concentration (100 ng/ml). We foundthat in this combination of NTFs, 40% of motor neuronswere sensitive to a 1-h exposure to glutamate (Fig. 3B),indicating that NGF neither accentuates nor inhibitsIGR-induced death of motor neurons. Recently, it hasbeen reported that activation of p75NTR on mouse motorneurons with NGF decreases the survival of mouse mo-tor neurons cultured in BDNF (Wiese et al., 1999).Contrary to this report, we find that the basal survival ofrat motor neurons cultured in BDNF and high concen-trations of NGF was not affected (Fig. 3B).

Although activation of p75NTR alone is insufficient toinduce ES, it is possible that p75NTR activation is re-quired. Thus, to determine if p75NTR activation is re-quired for BDNF-induced ES, we used the anti-REXantibody to block motor neuron p75NTR. The anti-REXantibody is a polyclonal antibody that blocks NGF bind-ing to p75NTR and has been shown to inhibit the biolog-ical activity of p75NTR (Weskamp and Reichardt, 1991;Casaccia-Bonnefil et al., 1996; Kohn et al., 1999). Be-cause the anti-REX antibody stains cultured motor neu-rons, whereas control rabbit antiserum does not (notshown), it is clear that this antibody recognizes p75NTR

on embryonic rat motor neurons. Motor neurons wereplated and cultured in medium containing BDNF and theanti-REX antibody, and the ES assay was performed.The anti-REX antibody was used at a concentration thatwas previously found to block the biological activationof p75NTR (Weskamp and Reichardt, 1991; Casaccia-Bonnefil et al., 1996; Kohn et al., 1999). In Fig. 3B, itcan be seen that the anti-REX antibody affects neitherthe basal survival of embryonic motor neurons norBDNF-induced ES. Thus, the data from these three ap-proaches suggest that p75NTR is not required for BDNF-induced ES in cultured embryonic rat motor neurons.

Considering that p75NTR is probably not required forBDNF-induced ES signaling, we next wanted to test ifTrkB, the most likely alternative, is necessary. Data frommany different sources indicate that TrkB is the majorreceptor used by BDNF for neuronal survival and differ-entiation (Barbacid, 1994). Greater than 90% of themotor neurons cultured for 24 h express TrkB, as deter-mined immunocytochemically using anti-TrkB antibod-ies (Fig. 4A). This TrkB, however, may not be expressedon the cell surface or may not be capable of beingactivated. Ligand binding to Trk receptors induces auto-phosphorylation, which is required for Trk signal trans-duction (Kaplan and Stephens, 1994; Obermeier et al.,1994; Baxter et al., 1995; Dikic et al., 1995). Thus, todetermine if TrkB can be activated by BDNF, we used anantibody that recognizes phospho-epitopes on activatedTrk (-A, -B, and -C) receptors in biochemical assays. In

the wells of a 96-well plate, 50,000 motor neurons wereplated and cultured in medium containing CT-1, whichsupports motor neuron survival but does not activateTrkB. The cells were stimulated for 5 min with BDNF,washed with phosphate-buffered saline, and extractedwith detergent-containing buffers (see Materials andMethods). When subjected to immunoblot analysis withan antibody that recognizes phosphorylated epitopes on

FIG. 4. Cultured embryonic rat motor neurons express TrkB thatcan be activated by BDNF. A: Motor neurons cultured for 1 daywere fixed and stained for TrkB. Staining is seen on .90% of thecells in the culture. B: Extracts of cells, which were cultured for1 day and incubated with BDNF (lane 2), NT3 (lane 3), or vehicle(lane 1) for 5 min, were western blotted with an antibody toactivated Trk. BDNF and NT3, but not vehicle, induced thephosphorylation of a 145-kDa band recognized by the antibodyto activated Trk. C: Motor neurons were cultured in variousconcentrations of BDNF or nothing, and survival was assessed 2days later. The EC50 of BDNF-dependent motor neuron survivalwas ;100 pg/ml. D: Motor neurons were cultured in CT-1 andvarious concentrations of BDNF overnight, exposed to gluta-mate/glycine for 1 h, washed, the medium was replaced, and cellsurvival was assessed the following day. CT-1 was included inthese experiments to support maximal cell survival regardless ofthe concentration of BDNF added. Motor neuron ES was depen-dent on the dose of BDNF used, with an EC50 of ;100 pg/ml.The low EC50 values for survival and ES suggest the involvementof TrkB in both of these biological activities.



activated Trk receptors, a large amount of activated Trkis detected from cultures in which BDNF was incubatedfor 5 min (Fig. 4B, lane 2). In cultures of cells that werenot stimulated with BDNF, very little activated Trk isdetected (Fig. 4B, lane 1). NT3 also stimulates Trkreceptor phosphorylation (Fig. 4B, lane 3). Consideringthe differences in affinity of NT3 to TrkB and TrkC(Lamballe et al., 1991; Soppet et al., 1991; Escandonet al., 1994), it seems likely that the majority of phospho-Trk induced by NT3 in Fig. 4 is due to TrkC. The motorneuron survival-promoting activity of NT3 occurs withan EC50 of ;200 pg/ml (Henderson et al., 1993),strongly suggesting that biologically relevant actions ofNT3 operate through activation of TrkC. Equal loadingof each lane in Fig. 4B was confirmed using an antibodythat recognizes all forms of phosphorylated and non-phosphorylated Trk receptors. These results suggest thatwhereas Trk activation isnecessaryfor ES induction, itis not sufficient.

To determine if the same receptor is responsible forboth survival and ES, we compared the EC50 of BDNFfor these biological activities. To assess survival support,motor neurons were plated in medium containing dilu-tions of BDNF and survival was assessed after 2 days ofculture. Survival support by BDNF was approximatelysigmoidal, with an EC50 of ;100 pg/ml (3.853 10212

M; Fig. 4C). A similar assay was performed to determinethe EC50 for BDNF-induced ES. Motor neurons werecultured overnight in CT-1 and dilutions of BDNF, afterwhich the ES assay was performed as described above.Inclusion of CT-1 was required for these studies topromote the basal survival of motor neurons. The induc-tion of ES in motor neurons by BDNF was also found tobe sigmoidal (Fig. 4D). In the absence of BDNF, motorneurons were not sensitive to glutamate. At 1 ng/ml, theresponse was maximal. The EC50 for induction of ES byBDNF was;100 pg/ml (3.853 10212 M). The dose–response curves for survival support and ES inductionwere found to be similar, albeit complementary, suggest-ing that the same receptor is responsible for both of thesebiological activities. The EC50 for these biological activ-ities are consistent with the EC50 for the activation ofTrkB by BDNF in other systems (Soppet et al., 1991; Ipet al., 1993), suggesting that this receptor is involved inboth survival support and induction of ES.

BDNF induction of ES requires mRNA and proteinsynthesis

We next wanted to determine if BDNF induction ofES is dependent on new gene expression. Because thecompounds that block protein and mRNA synthesis aretoxic to motor neurons when incubated for prolongedperiods (not shown), the minimum time required forBDNF-induced ES was first determined. BDNF wasadded to the medium at different times prior to the ESassay. CT-1 was included in the medium to ensure max-imal neuron survival during the time that motor neuronswere not incubated with BDNF. Motor neurons culturedfor $3 h in medium containing BDNF became sensitive

to glutamate toxicity, whereas cells cultured for only 1 hdid not (Fig. 5A).

The effects of protein and mRNA synthesis inhibitorswere then tested. After overnight culture in mediumcontaining CT-1, the protein synthesis inhibitor cyclo-heximide (4mM) or the mRNA synthesis inhibitor acti-nomycin D (1mM) was added to the medium. To ensurethat these reagents had sufficiently diffused into the cellsand were thus effective, BDNF was added to the medium30 min later and the cells cultured for an additional 3–4h. The ES assay was performed and the medium wasreplaced with fresh medium that did not contain theseinhibitors. We found that both inhibitors effectively pre-vented BDNF-induced ES (Fig. 5B). We next inquiredwhether in the setting of prolonged exposure to BDNF, abrief pretreatment of motor neurons with these inhibitorscould block excitotoxic cell death. Cells were plated andcultured overnight in medium containing CT-1 andBDNF, and 3–4 h prior to the ES assay, the inhibitorswere added to the medium as above. Under these con-ditions, the inhibitors failed to prevent IGR-mediatedmotor neuron death (not shown). These data indicate thatthe induction of ES in motor neurons by BDNF is de-pendent on gene transcription and translation.

FIG. 5. Induction of ES by BDNF is time dependent and requiresde novo macromolecular synthesis. A: Cells were plated in CT-1-containing medium, to which BDNF was added at varioustimes prior to treatment with glutamate/glycine (open columns)or vehicle (filled columns). Cells cultured for $3 h with BDNFwere sensitive to glutamate toxicity. B: Motor neurons wereplated in CT-1; 4–6 h prior to glutamate/glycine treatment,BDNF was added. Thirty minutes before addition of BDNF, cy-cloheximide, actinomycin D, or vehicle was provided to thecultures. Both cycloheximide and actinomycin D blocked BDNF-induced ES. **p ! 0.001.



BDNF-mediated induction of ES requires activationof PI3K

Ligand-bound Trk receptors activate a number of in-tracellular signaling systems including the Ras–nitrogen-activated protein (MAP) kinase and PI3K pathways. Inthese next series of experiments, we investigated theparticipation of intracellular signaling pathways in me-diating the action of BDNF.

In the first set of experiments, we used wortmanninand LY294002 to inhibit the activity of PI3K. PI3K is aheteromer composed of a 110-kDa (p110) catalytic sub-unit, which phosphorylates phosphatidylinositol, and an85-kDa (p85) regulatory subunit, which binds to phos-phorylated epitopes on activated receptors or receptoradapter proteins. Wortmannin is a fungal toxin that co-valently binds to and blocks the activity of the catalyticp110 subunit of PI3K (Okada et al., 1994a,b), whereasLY294002 is a synthetic bioflavonoid that reversiblybinds to and inhibits p110 (Vlahos et al., 1994). Follow-ing overnight culture in medium containing CT-1, 0.2mM wortmannin, 20mM LY294002, or vehicle dimethylsulfoxide was added to the medium. These concentra-tions of the antagonists have been used to effectivelyblock PI3K in other in vitro systems (Okada et al.,1994a; Vlahos et al., 1994). BDNF was added to themedium 30 min after drug addition, and the cells werecultured for an additional 3–5 h, after which the ES assaywas performed. Following washes in Locke’s buffer,fresh medium containing CT-1 was added to the cultures.Under these conditions, LY294002 did not affect basalsurvival, whereas wortmannin decreased basal survivalby ;10%. Both of these antagonists of PI3K completelyinhibited the ability of BDNF to induce ES (Fig. 6A). Inthe same assay system, the MAP kinase kinase (MEK)inhibitor PD 098059 at a concentration that effectivelyblocks Ras–MAP kinase signaling (50mM) did not pre-vent BDNF-induced ES (Dudley et al., 1995; Robersonet al., 1999; Sondell et al., 1999).

To further explore the results from the pharmacolog-ical studies, we used a dominant negative mutant of theregulatory subunit of PI3K (DNp85), which binds toactivated tyrosine kinase receptors or adapter proteinsbut is incapable of binding to and activating the p110catalytic subunit (Hara et al., 1994; Kotani et al., 1994).Recombinant HSV was engineered to express DNp85(HSV-DNp85). HSV expressing LacZ was used as con-trol.

The efficiency of viral infection was assessed usingimmunocytochemistry. Cultures of neurons were pre-pared as they were for the ES assay. To ensure completecell attachment, cells were cultured for 6–8 h in mediumcontaining CT-1 prior to infection with engineered HSV.Eighteen hours following infection, virtually all of thecells infected with HSV-LacZ were anti-b-galactosidase-immunopositive (Fig. 6Ba). Anti-p85 immunoreactivityin uninfected cells or cells infected with HSV-LacZ waslight, whereas in HSV-DNp85-infected cells, anti-p85immunoreactivity was intense and apparent in virtuallyall of the cultured motor neurons (Fig. 6Bd and c, re-

spectively). These timing and culture conditions wereused for the experiments of virally infected cells de-scribed below.

FIG. 6. Inhibition of PI3K prevents BDNF-induced ES. A: Fol-lowing overnight culture in CT-1, the PI3K inhibitors LY294002 orwortmannin or vehicle was added to the medium. Thirty minuteslater, BNDF was added to the cells and incubated for 4–6 h,after which the cells were treated with glutamate/glycine (opencolumns) or vehicle (filled columns). Both of these inhibitorsprevented BNDF-induced ES. B: Six hours following the plating,motor neurons were infected with HSV-LacZ (a), HSV-DNp85 (c),or vehicle (b and d). Eighteen hours later, cells were then fixedand stained with antibodies to b-galactosidase (a and b) or p85(c and d). Uninfected cells did not express b-galactosidase,whereas HSV-LacZ-infected cells stained heavily within theirsomata and neuritic processes. Although uninfected cellsstained lightly for p85, those infected with HSV-DNp85 stainedmore intensely. C: HSV-infected or vehicle-treated cells wereincubated for 4–6 h with BDNF and then treated with glutamate/glycine (open columns) or vehicle (filled columns). BDNF failed toinduce ES in cells treated with HSV-DNp85 but was effective invehicle-treated or HSV-LacZ-infected cells. **p ! 0.001.



To test the effect of these viruses on BDNF-inducedES, motor neurons were cultured for 6–8 h in mediumcontaining CT-1 prior to infection with HSV. Cultureswere then incubated with BDNF for 3–4 h after whichthe ES assay was performed. Glutamate treatment ofHSV-LacZ-infected cells resulted in the death of 40% ofmotor neurons (Fig. 6C). No glutamate-evoked loss ofmotor neurons was observed in the HSV-DNp85-infected cells. These results confirm the findings withwortmannin and LY294002 and indicate that the induc-tion of ES in motor neurons by BDNF is mediated atleast in part through the PI3K signaling pathway.

To determine if these reagents were capable of inhib-iting the activity of PI3K, we performed biochemicalassays for activation of the PI3K signal transductionpathways in motor neurons after exposure to variousNTFs and in the presence of the inhibitors used above.The serine kinase Akt (protein kinase B) is a downstreameffector of PI3K, which is phosphorylated followingPI3K activation (Burgering and Coffer, 1995; Frankeet al., 1995). Thus, to monitor PI3K activation, we as-sessed the extent of phosphorylated Akt on blotted ex-tracts of motor neurons that were stimulated with 1 ng/mlBDNF (see Materials and Methods). Figure 7A and Bshows the results of these experiments. In the absence ofinhibitors, motor neurons stimulated for 5 min withBDNF (control, lanes 2, 5, and 8) have a large amount ofphosphorylated Akt compared with unstimulated cells(lanes 1, 4, and 7). A 30-min incubation with either 0.2mM wortmannin (lane 3) or 20mM LY294002 (lane 6)prior to BDNF stimulation inhibited Akt phosphoryla-tion. Neither wortmannin nor LY294002 inhibited thebasal phosphorylation of Akt (not shown). Equal loadingof the lanes was confirmed by reprobing the blots withantibodies that recognize nonphosphorylated Akt (notshown). To assess the specificity of these compounds,immunoblots were also probed for phosphorylated ERKs(p42 and p44), which are downstream effectors of Ras(Denhardt, 1996). The ERKs are also phosphorylatedfollowing BDNF stimulation of motor neurons. BDNFinduction of ERK phosphorylation was not influenced byLY294002 and was affected minimally by wortmannin(Fig. 7, lanes 3 and 6, respectively). Our results confirmthe selectivity of these pharmacological agents.

The effect of the recombinant HSVs on PI3K activitywas also assessed using biochemical assays. Cells in thewells of 96-well plates were cultured in CT-1-containingmedium for 6–8 h and then infected with HSV-LacZ orHSV-DNp85. Eighteen hours later, the cells were stim-ulated with BDNF, and biochemical assays of Akt andERK activation were performed. BDNF induced phos-phorylation of Akt and ERK to the same extent in HSV-LacZ- and vehicle-treated cells (Fig. 7A, lane 9, and C).This indicates that viral infection per se did not affect theactivation of these intracellular pathways by ligand-bound TrkB. Cells infected with HSV-DNp85, however,showed a very strong reduction in BDNF-induced Aktphosphorylation (lane 10). HSV-DNp85 also led to amodest reduction in phosphorylation of ERK. Equal

FIG. 7. Inhibition of PI3K pathway in cultured embryonic ratmotor neurons using pharmacological agents and by expres-sion of a dominant negative p85 transgene. A: Motor neuronswere plated into a 96-well plate in CT-1-containing mediumovernight and then stimulated with BDNF (lanes 2, 3, 5, 6, 8,9, 10) or vehicle (lanes 1, 4, 7) for 5 min prior to preparation oflysates. Wortmannin (0.1 mM; lane 3), LY204002 (20 mM; lane6), or vehicle (control lanes 2 and 5) was added to the wells 30min prior to BDNF or vehicle administration. Other wells wereinfected with either HSV-LacZ (lane 9) or HSV-DNp85 (lane 10)or treated with vehicle (lane 8) for 18 h, after which the cellswere stimulated with BDNF or vehicle for 5 min. The cells fromall of these treatments were extracted and western blottedwith antibodies to phospho-Akt (top) or phospho-ERK (bot-tom). Compared with HSV-LacZ- or vehicle-treated cells,those treated with wortmannin, LY294002, or HSV-DNp85had reduced levels of phospho-Akt, indicating PI3K pathwayinhibition. The effect was preferential as these treatments hadno or less pronounced effects on ERK-1 and -2 phosphory-lation. Equal loading of lanes was confirmed by staining blotswith the antibodies to nonphosphorylated forms of these pro-teins (not shown). B: The density of the stained bands fromimmunoblots of at least three trials of the experiments wasdetermined and compared with control (vehicle-treated) stim-ulated cells. Wortmannin, LY294002, and HSV-DNp85 re-sulted in large decreases in the levels of BDNF-stimulatedphospho-Akt compared with control conditions. Variable andsmaller effects were seen on the level of phospho-ERK whencompared with controls. C: BDNF and NT3 activate the PI3Kand Ras signaling pathways in cultured embryonic rat motorneurons. Motor neurons cultured overnight in CT-1-containingmedium were stimulated for 5 min with vehicle (unstimulatedlane 1), BDNF (lane 2), or NT3 (lane 3). Both the Ras and thePI3K intracellular pathways were stimulated to approximatelythe same extent by both of these NTs. A longer exposure ofimmunoblots to the film was used for C to illustrate thebackground levels of Akt and ERKs.



loading of lanes was confirmed by reprobing the blotswith antibodies that recognize the nonphosphorylatedforms of these proteins. The biochemical effects sharedby wortmannin, LY294002, and HSV-DNp85 make astrong case that the PI3K pathway, which is activated inmotor neurons by BDNF, is necessary for the inductionof ES.

In general, TrkB and -C activate the same intracellularsignaling systems, yet only BDNF induces ES. As acti-vation of the PI3K pathway is required for BDNF-in-duced ES, it was of interest to compare the extent ofactivation of this pathway in motor neurons when stim-ulated by BDNF and NT3. We found that the initial levelof phosphorylation of Akt induced by BDNF or NT3 isessentially the same (Fig. 7). In addition, the initial levelof phosphorylation of ERK induced by these factors wasalso very similar. These results indicate that thoughactivation of the PI3K pathway is necessary, it is notsufficient for mediating the ES response to BDNF inmotor neurons.

DISCUSSION

We show here that BDNF can act directly on motorneurons to induce susceptibility to IGR-mediated exci-totoxic cell death. Our results indicate that this process islikely to involve the activation of TrkB and signalingthrough the PI3K pathway. The effect of BDNF involvesnew gene expression but does not appear to alter theelectrophysiological responsiveness of motor neurons toIGR stimulation.

Previous work demonstrated that certain NTFs areneuroprotective, such as fibroblast growth factor-2,whereas others, such as insulin-like growth factor-1,potentiate excitotoxicity (Calissano et al., 1993; Fernan-dez-Sanchez and Novelli, 1993; Lindholm, 1994). Asingle NTF can protect against or potentiate excitotoxic-ity, depending on the type of neuron being studied. Invitro, BDNF is neuroprotective for hippocampal neuronsbut sensitizing for cerebral cortical neurons (Koh et al.,1995; Mattson et al., 1995; Samdani et al., 1997). Theconditions under which the NTF is used also influencesits biological activity. In serum-containing medium,BDNF protects Purkinje cells against glutamate toxicity,but in serum-free medium, BDNF sensitizes these cellsto excitotoxicity (Morrison and Mason, 1998). Similarly,exposure of hippocampal neurons in vitro to BDNF isneuroprotective, but when administered in vivo, BDNFexacerbates excitotoxic death (Mattson et al., 1995;Rudge et al., 1998). Thus, depending on the factor andsystem under study, NTFs can protect against or exac-erbate excitotoxic injury.

In our culture system, BDNF, but not NT3, CT-1, orGDNF, potentiates glutamate toxicity in cultured embry-onic motor neurons. This effect is dominant, in that theother NTFs are not neuroprotective when also includedin the medium. In contrast to the results presented hereand in our previous study, Metzger et al. (1998) haveshown that purified embryonic motor neurons cultured in

BDNF are not sensitive to IGR-mediated death. Majordifferences in culturing conditions, however, are likely toexplain the disparate results. Metzger et al. includedglutamine in their serum-free medium. By the action ofmitochondrial glutaminase, glutamine is metabolized toglutamate (Curthoys and Watford, 1993; Roberg et al.,1995), which we find is toxic to motor neurons in ourculture system (Fryer et al., 1999). Moreover, we includeserum in our medium, which may contain factors thatcooperate with BDNF to induce ES (Erdo¨ et al., 1990).

What BDNF-induced phenotypic change renders mo-tor neurons sensitive to excitotoxicity? Many studieshave shown that BDNF can enhance excitatory neuro-transmission by acting on the presynaptic terminal (Figu-rov et al., 1996; Patterson et al., 1996; Carmignoto et al.,1997; Takei et al., 1997; Li et al., 1998), but thesefindings are not relevant in our system because synapticcontacts do not occur in our low-density cultures. Pre-cedents from previously published work indicate thatBDNF might enhance the excitatory actions of glutamateby increasing the number of cell surface glutamate re-ceptors or changing their electrophysiological functionby posttranslational processes such as phosphorylation(Bai and Kusiak, 1997; Lin et al., 1998). Another con-sideration is an effect on intracellular Ca21 homeostasisthrough the regulation of intracellular Ca21 binding pro-teins, Ca21 extruding proteins, or affecting release fromintracellular Ca21 stores. The major argument againstthese mechanisms in our experiments is that glutamate-evoked rises in [Ca21]i in motor neurons supported withCT-1 alone (and thus not excitotoxically sensitive) areindistinguishable from the responses of motor neuronssupported with CT-11 BDNF (excitotoxically sensi-tive). In addition, many previously described effects oc-cur within minutes of BDNF addition (Carmignoto et al.,1997; Li et al., 1998; Lin et al., 1998), but in our system,motor neurons must be exposed to BDNF for 3 h for ESinduction. Although our studies do not define the molec-ular mechanism of BDNF-induced ES, it seems likely tobe due to the expression of new genes that alter a step (orsteps) downstream of glutamate-evoked sustained risesof [Ca21]i.

It is also possible that BDNF may induce ES not byregulating the synthesis of particular proteins, but byincreasing protein synthesis in general. Increased proteinsynthesis may put a high demand on cellular resources,drawing them away from protecting the cell from thedamaging effects of prolonged IGR stimulation. For ex-ample, free cysteine, required for the synthesis of gluta-thione, which is necessary for reducing toxic intracellularfree radicals that are increased during prolonged rises of[Ca21]i, would be reduced in highly metabolic cells andthus participate in free radical-mediated injury of motorneurons (Ratan et al., 1994).

As BDNF activates p75NTR, which can mediate theapoptotic death of various cell types through activationof its death domain and the subsequent production of thepro-apoptotic lipid ceramide (Rabizadeh et al., 1993;Dobrowsky et al., 1995; Casaccia-Bonnefil et al., 1996;



Frade et al., 1996; Carter and Lewin, 1997), we considerit the most likely candidate for transducing the BDNFsignal. However, experiments aimed at blocking p75NTR

with the anti-REX antibody or activating p75NTR withhigh concentrations of NGF did not affect the ability ofBDNF to induce ES. Interestingly, these reagents do notaffect the survival of motor neurons in BDNF. Theseexperiments support the notion that p75 does not partic-ipate in BDNF-induced ES. Definitive proof will requirethe study of motor neurons purified from the p75 knock-out mice. At the present time, purification of mousemotor neurons to perform such studies is not technicallyfeasible. Our results are in contrast to a recent report byWiese et al. (1999), who have shown that p75NTR acti-vation by NGF decreases the survival-promoting abilityof BDNF for cultured mouse motor neurons. There are,however, many differences between the culture systems(mouse versus rat, age of motor neurons, serum-freeversus serum-containing medium) that could account forthe disparate results.

Our results support the view that TrkB activationmediates ES induction. The low EC50 values (3.853 10212 M) for survival and induction of ES are con-sistent with TrkB activation and suggest that the samereceptor participates in both processes. This view isfurther supported by our data showing that ES inductionrequires activation of the PI3K pathway, which is knownto couple to activated Trk but not p75NTR (Barbacid,1994; Chao, 1994). Study of pure motor neurons frommice with a null mutation in TrkB and p75NTR coulddefinitively establish this point.

Although most of the neurons in our culture are im-munopositive for TrkB and respond to glutamate stimu-lation, why are only a subset susceptible to excitotoxic-ity? Only 40% of the motor neurons in our culturesrequire NTF for survival. This finding is similar to that ofBecker et al. (1998), who have shown that even thoughchick motor neurons express NTF receptors at the timeof plating, they require several days of culture beforethey become responsive to NTF support (Escandon et al.,1994; Becker et al., 1998). The explanation for theseobservations comes from a study by Meyer-Franke et al.(1998), who have shown that of the.90% of culturedembryonic rat motor neurons that stain for TrkB, only asubset express cell surface TrkB. In the rest of the cells,TrkB resides in intracellular pools. It is likely that therobust phosphorylation of Trk receptors upon addition ofBDNF in our cultures is due to activation of TrkB onthese neurons. We hypothesize that it is this subset ofcells, expressing cell surface TrkB, in which BDNFinduces ES. Two pieces of evidence support this theory:First, glutamate fails to further reduce the number ofcells cultured in the absence of NTFs (Fig. 1A). Second,the percentage of cells in our cultures that depend ontrophic support is equal to the percentage of cells that canbe induced to ES (Fig. 4). In a preliminary study, wefound that cells surviving glutamate challenge do notrequire NTFs for survival (not shown), suggesting thatthe subpopulation of motor neurons that require NTFs

for survival are those that can be made vulnerable toexcitotoxicity.

We have also shown that activation of PI3K is neces-sary for BDNF induction of ES, yet both BDNF and NT3activate this pathway (Fig. 7). Although each of theseNTs is capable of activating PI3K, the extent or durationof activation by each may be different, which in turn mayresult in different biological activities. Differences in thekinetics and/or extent of activation of intracellular sig-naling systems have different biological consequences(Marshall, 1995). In PC12 cells, for example, NGF,which induces differentiation, induces a longer durationof ERK activation than epidermal growth factor, whichstimulates proliferation (Traverse et al., 1992). Themeans by which each of these NTs induces PI3K acti-vation may also be different. PI3K can be activateddirectly (i.e., binding of the p85 regulatory subunit tophospho-Trk), by Ras, or by insulin receptor substrate-1and -2 (Rodriguez-Viciana et al., 1994; Yamada et al.,1997). Each of the Trk ligands may stimulate PI3K bydifferent routes that may result in different biologicalactivities. Other signaling pathways may also be neces-sary for BDNF-induced ES. Thus, although we provideevidence that PI3K is necessary for the induction of ESby BDNF, we have no evidence that it is sufficient.Further study is required to determine differences in thelength and extent to which each of the NTs induces thePI3K pathway and also to determine the extent to whichother intracellular pathways may affect BDNF-induced ES.

BDNF may play a physiological role in regulatingmotor neuron number during development. Althoughafferent activity has clearly been shown to be requiredfor neuron survival, recent evidence from developingauditory and motor systems suggests that glutamate neu-rotransmission may contribute to naturally occurringneuronal death in vivo (Caldero´ et al., 1997; Solum et al.,1997). BDNF could participate in this process by sensi-tizing motor neurons to afferent IGR stimulation. Duringthe period of naturally occurring motor neuron death,there is a spontaneous glutamatergic network activitywithin the spinal cord (Harris and McCaig, 1984;O’Donovan et al., 1994; Sholomenko and O’Donovan,1995). At the same time, BDNF is expressed by motorneuron targets and afferents, and TrkB is expressed bymotor neurons (Henderson et al., 1993). Astrocytes,which are the main sink for glutamate in the spinal cord,have not yet developed at this age (Jacobson, 1991);glutamate released by afferents may remain high in theextracellular environment for long periods of time, rep-licating conditions in vivo that we have shown to bedetrimental to motor neurons in vitro. Sensitizing neu-rons to excitotoxicity may explain the findings of Faw-cett et al. (1998), who have shown that increasing BDNFexpression in the developing neocortex of transgenicmice reduces cortical neuron number.

Whereas NTF-mediated alteration in sensitivity to celldeath presumably plays an important role in normaldevelopment, it may also contribute to the progression ofdisease or complicate therapeutic interventions. Molec-



ular dissection of the specific pathways leading to pro-motion of survival or death may lead to new therapeuticstrategies to combat neurodegenerative disease.

Acknowledgment: For the kind gifts of their reagents, wethank Drs. Eugene Johnson for monoclonal antibody 192 andLouis Reichardt for the anti-REX antibody. We are most ap-preciative of the participation of Dr. Rachael Neve in thepreparation of the recombinant HSV and the contribution ofvarious NTFs by Cephalon, Amgen, Regeneron, and Genen-tech. This study was supported by U.S. Public Health Servicegrants to R.G.K. (NS 29837, 33437, and 37874) and S.M.S.(NS 33020). S.M.S. is a John Merck Scholar in the Biology ofDevelopmental Disabilities in Children.

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