Neurotrophic Factors Protect Cortical Synaptic Terminals Against Amyloid- and Oxidative Stress-induced Impairment of Glucose Transport, Glutamate Transport and Mitochondrial Function

Zhi Hong Guo and Mark P. Mattson1

Sanders-Brown Research Center on Aging and Department of Anatomy & Neurobiology, University of Kentucky, Lexington, KY 40536, USA


    Abstract
 Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that several different neurotrophic factors can prevent death of cortical and hippocampal neurons induced by excitotoxic and oxidative insults in cell culture and in vivo. Because neuronal degeneration may be initiated by alterations occurring in synaptic compartments in disorders ranging from Alzheimer's disease to stroke, we tested the hypothesis that neurotrophic factors can exert direct protective actions at the level of the synapse. We now report that a nine amino acid bioactive fragment of activity-dependent neurotrophic factor (ADNF-9) enhances basal glucose and glutamate transport, and attenuates oxidative impairment of glucose and glutamate transport induced by amyloid ß-peptide and Fe2+, in neocortical synaptosomes. Preservation of transporter function required only short-term (1–2 h) pretreatments. Basic fibroblast growth factor (bFGF) was also effective in suppressing oxidative impairment of synaptic transporter functions, while nerve growth factor (NGF) was less effective. Additional analyses showed that ADNF-9, bFGF and NGF suppress oxidative stress and mitochondrial dysfunction induced by amyloid ß-peptide and Fe2+ in synaptosomes. Our data suggest that ADNF-9 can act locally in synaptic compartments to suppress oxidative stress and preserve function of glucose and glutamate transporters. Such synapto-protective actions suggest roles for activity-dependent trophic signaling in preventing degeneration of neuronal circuits, and indicate possible therapeutic applications of agents that stimulate local synaptic (transcription-independent) neurotrophic factor signaling pathways.


    Introduction
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Synapses are sites where signal transduction pathways are heavily concentrated and, accordingly, signaling events in synaptic compartments exert far-reaching effects on neuronal plasticity and survival. In addition to its role in rapid modulation of activity in neuronal circuits, synaptic signaling is believed to play important roles in regulating neuronal survival and structural plasticity during development (Oppenheim, 1991Go; Lewin and Barde, 1996Go), and aberrant synaptic signaling is implicated in some pathological neurodegenerative conditions (Mattson et al., 1998aGo,bGo). The neurodegenerative process is likely to begin at synapses in various disorders including Alzheimer's disease (AD) (Terry et al., 1991Go; DeKosky et al., 1996Go), stroke (von Lubitz and Diemer, 1983Go; Urban et al., 1989Go) and epilepsy (Peters, 1980Go; Jiang et al., 1998Go). Excessive activation of excitatory amino acid receptors, resulting in cytoplasmic calcium overload and oxyradical production, has been implicated in the neuronal death process that occurs in these and other neurodegenerative disorders (Mattson et al., 1992Go; Choi, 1994Go; Guo et al., 1999Go). Further implicated in the neuronal cell death process (Smith et al., 1991Go, 1998Go; Mark et al., 1995Go, 1997aGo, Mark et al., bGo) and synaptic degeneration (Mattson et al., 1998aGo,bGo) in each of these disorders is increased levels of oxidative stress. By impairing the function of membrane ion-motive ATPases, and glucose and glutamate transporters, membrane lipid peroxidation may play a major role in rendering synapses and neurons vulnerable to excitotoxicity (Mark et al., 1995Go, 1997aGo,bGo; Keller et al., 1997aGo,bGo).

The production of several different neurotrophic factors is induced by neuronal activity (Thoenen, 1995Go), and these neurotrophic factors can, in turn, modulate neuronal survival and plasticity (Mattson and Lindvall, 1997Go). A remarkable number of neurotrophic factors have been found to protect neurons in cell culture and in vivo against various excitotoxic, metabolic and oxidative insults (Mattson and Scheff, 1994Go). For example, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor, nerve growth factor (NGF) and activity-dependent neurotrophic factor (ADNF) can protect neurons against excitotoxic and metabolic insults in cell culture (Mattson et al., 1989Go; Cheng and Mattson, 1991Go; Brenneman and Gozes, 1996Go; Brenneman et al., 1998aGo), and against ischemic brain injury in vivo (Shigeno et al., 1991Go; Nozaki et al., 1993Go; Guegan et al., 1998Go). The mechanisms whereby neurotrophic factors protect neurons are thought to involve modulation of expression (at the transcriptional level) of genes encoding antioxidant enzymes (Mattson et al., 1995Go), calcium-regulating proteins (Cheng et al., 1994Go) and anti-apoptotic factors such as Bcl-2 (Allsopp et al., 1995Go). While it is well-established that neurotrophic factors can engage signaling pathways linked to transcription factor activation (Segal and Greenberg, 1996Go), it is not known whether neurotrophic factors can exert local (transcription-independent) actions in synaptic compartments. Although synapses are sites at which the neuroprotective actions of activity-dependent neurotrophic factors would seem most likely to be exerted, it is not known whether such factors can act at the level of the synapse, independently of the cell body. However, it was recently reported that the secreted form of amyloid precursor protein can exert direct neuroprotective actions in synaptic terminals (Mattson et al., 1999Go), suggesting the possibility that other neurotrophic factors may have similar actions. In the present study we show that nine amino acid bioactive fragment of ADNF (ADNF-9) can act at the level of the synapse to preserve membrane glutamate and glucose transporter activities, and mitochondrial function, following exposure of synapses to oxidative insults. Such localized signaling actions of neurotrophic factors may play important roles in modifying synaptic plasticity and suppressing potentially neurotoxic cascades.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
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Preparation of Synaptosomes, and Experimental Treatments

Synaptosomes were prepared from cerebral cortex of adult female Sprague–Dawley rats (250–300 g) using methods described previously (Keller et al., 1997aGo; Begley et al., 1999Go). Briefly, cerebral cortices were homogenized in a solution containing 0.32 M sucrose, 4 µg/ml pepstatin, 5 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, 4 µg/ml leupeptin, 0.2 mM PMSF, 2 mM EDTA, 2 mM EGTA and 20 mM Hepes. The homogenate was centrifuged for 10 min at 300 g at 4°C, and the supernatant centrifuged for 10 min at 12 400 g at 4°C. The pellet was resuspended in homogenization buffer, recentrifuged at 20 250 g, and the pellet centrifuged through a sucrose gradient (7 ml 1.18 M sucrose, pH 8.5; 7 ml 1 M sucrose, pH 8.0; 7 ml 0.85 M sucrose, pH 8) at 87 275 g for 2 h. Synaptosomes in the 1 M sucrose/1.18 M sucrose interface were diluted in Locke's buffer (in mM: NaCl 154, KCl 5.6, CaCl2 2.3, MgCl2 1.0, NaHCO3 3.6, glucose 5, Hepes 5 mM; pH 7.2) for all experiments. Synaptosomal suspensions were aliquoted (100 µl) into 1.5 ml Eppendorf tubes and incubated in a CO2 incubator at 37°C during experimental treatments. Aß (Aß25–35) was purchased from Bachem (lot no. B01200) and a 1 mM stock solution was prepared in sterile deionized water ~6 h prior to use. FeSO4 was prepared as a 10 mM stock in water immediately prior to use. Bafilomycin A1 and K252a were purchased from Calbiochem (La Jolla, CA).

Glutamate and Glucose Transport Assays

The methods for quantifying glutamate and glucose transport in synaptosomes have been described previously (Keller et al., 1997aGo). For the glutamate uptake assay, synaptosomes (200 µg/tube) were incubated for 7 min with [3H]glutamate (0.1 µCi/ml). The synaptosomes were then pelleted, washed three times in Locke's buffer, and lysed in 200 µl of solution of 1% SDS in PBS. The lysate was placed in scintillation vials containing Scintiverse and radioactivity was counted in a Packard 2500TR liquid scintillation counter. For the glucose uptake assay synaptosomes (200 µg/tube) were subjected to experimental treatments and then washed three times in glucose-free Locke's buffer and the assay was started by the addition of 1.5 µCi of [3H]2-deoxyglucose. Seven minutes later the assay was stopped by pelleting the synaptosomes, washing twice with glucose-free Locke's solution, and lysing the synaptosomes in 200 µl of a 1% SDS/PBS solution. The lysate was placed in scintillation vials containing Scintiverse and radioactivity was counted in a Packard 2500TR liquid scintillation counter. Results were expressed as c.p.m./mg protein. The rates of glucose and glutamate uptake by synaptosomes remained constant under basal conditions throughout the time course of experiments (up to 4 h). In such assays of glucose and glutamate transport there is often considerable inter-assay variability in terms of c.p.m. of synaptosome-associated radioactivity, whereas intra-assay variability is quite low. Because we always included control conditions within an experiment, the experimental values were compared directly to the control value within the experiment.

Assessments of Mitochondrial Function and Reactive Oxygen Species Levels

The dye rhodamine 123 (Molecular Probes) was used as a measure of mitochondrial function; uptake of rhodamine 123 has been shown to be related to mitochondrial transmembrane potential (Mattson et al., 1993cGo; Bindokas and Miller, 1995Go; Kim et al., 1998Go). Briefly, synaptosomes were incubated for 30 min in the presence of 10 µM of the dye and then were washed twice in Locke's buffer. Following washing, the synaptosomes were seeded into 35 mm glass-bottomed culture dishes and were allowed to settle on the glass surface during a 10–15 min incubation. Fluorescence in synaptosomes was imaged using a confocal laser scanning microscope with excitation at 488 nm and emission at 510 nm, and the average pixel intensity in user-defined areas corresponding to synaptosomal aggregates was determined using Imagespace software (Molecular Dynamics). All images were coded and analyzed without knowledge of experimental treatment history of the synaptosomes. The dye dihydrorhodamine 123 (DHR) enters mitochondria and fluoresces when oxidized by ROS (principally peroxynitrite) to the positively charged rhodamine 123 derivative (Kooy et al., 1994Go; Mattson et al., 1997Go). Following experimental treatment, synaptosomes were loaded with 10 µM DHR 123 for 30 min, and were then washed twice with Locke's buffer. Fluorescence was quantified as described for synaptosomes stained with rhodamine 123.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous studies have extensively characterized the cellular composition of cortical synaptosomes prepared using the method employed in the present study (Keller et al., 1997aGo,bGo; Mattson et al., 1998aGo,bGo; Begley et al., 1999Go). The latter studies have shown that the synaptosomes are highly enriched in preand postsynaptic terminals, and also contain astrocytic feet associated with the terminals. The synaptosomes contain the glucose transport protein GLUT-3 and the glutamate transport protein EAAT-2 (GLT-1) and exhibit specific transport of radiolabeled glucose and glutamate (Palmer et al., 1994Go; Keller et al., 1997aGo,bGo). Previous studies have also characterized the impairment of glucose and glutamate transport following exposure of intact neurons and astrocytes (Mark et al., 1997bGo; Blanc et al., 1998Go) or synaptosomes (Keller et al., 1997aGo,bGo) to oxidative insults (e.g. amyloid ß-peptide and iron) that induce membrane lipid peroxidation. In order to determine whether synaptic glucose and/or glutamate transport can be modified by neurotrophic factors, we examined the effects of ADNF-9, bFGF and NGF on synaptosomal glucose and glutamate transport under basal conditions and following exposure to Fe2+ and amyloid ß-peptide(25–35) (Aß). Both ADNF-9 and bFGF at concentrations previously shown to protect cultured hippocampal neurons against excitotoxicity (Mattson et al., 1989Go; Brenneman and Gozes, 1996Go) significantly enhanced the basal levels of [3H]glucose and [3H]glutamate uptake by synaptosomes (Fig. 1Go). NGF had no significant effect on the basal level of glucose uptake, but did cause a significant increase of glutamate uptake (Fig. 1Go). As expected, exposure of synaptosomes to Fe2+ and Aß resulted in decreases in glucose and glutamate uptake. In contrast, both glucose and glutamate transport were almost completely preserved in synaptosomes pretreated with ADNF-9 (Fig. 1Go). Oxidative impairment of glucose and glutamate transport was also signficantly attenuated in synaptosomes pretreated with bFGF and NGF. NGF had no significant effect on Fe2+-induced impairment of glutamate transport (Fig. 1Go). However, NGF did afford significant protection against Aß-induced impairment of glutamate transport.



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Figure 1.  Effects of neurotrophic factors on glucose and glutamate transport under basal conditions, and following exposure to Aß and an oxidative insult, in cortical synaptosomes. Synaptosomes were pretreated for 1 h with saline (Control), ADNF-9 (1 pM), bFGF (100 ng/ml) or NGF (100 ng/ml). Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of [3H]glucose uptake (A) or [3H]glutamate uptake (B) were quantified, and values are the mean and SD of determinations made in four synaptosome preparations. *P < 0.05, **P < 0.01 compared to value for vehicle-treated control value. #P < 0.05, ##P < 0.01 compared to corresponding (FeSO4 or Aß-treated) control value.

 
Previous studies have shown that concentrations of ADNF in the range of 0.01–1 pM are effective in protecting cultured hippocampal neurons against excitotoxicity (Brenneman and Gozes, 1996Go; Brenneman et al., 1998aGo). In the present study concentrations of ADNF from 0.01 to 100 pM enhanced basal glucose uptake, and attenuated Fe2+and Aß-induced impairment of glucose uptake (Fig. 2AGo). Interestingly, and consistent with concentration–response analyses in previous studies of the effects of ADNF on survival of cultured neurons (Brenneman et al., 1998aGo,bGo), the lowest concentration of ADNF-9 (0.01 pM) was as effective or more effective than higher concentrations in enhancing glucose transport. At each concentration examined, ADNF-9 induced a significant increase in the basal level of glutamate uptake, and completely prevented Fe2+and Aßinduced impairment of glutamate uptake (Fig. 2BGo). Indeed, glutamate uptake was increased 2to 4-fold above basal levels in ADNF-9-treated synaptosomes exposed to Fe2+or Aß.



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Figure 2.  Effects of increasing concentrations of ADNF-9 on glucose and glutamate transport under basal conditions, and following exposure to Aß and an oxidative insult, in cortical synaptosomes. Synaptosomes were pretreated for 1 h with saline (Control) or the indicated concentrations of ADNF-9. Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of [3H]glucose uptake (A) or [3H]glutamate uptake (B) were quantified, and values are the mean and SD of determinations made in four synaptosome preparations. **P < 0.01 compared to value for vehicle-treated control value. #P < 0.05, ##P < 0.01 compared to corresponding (FeSO4 or Aß-treated) control value.

 
Previous studies have provided evidence that the neuroprotective mechanism of ADNF requires receptor-mediated endocytosis (Brenneman et al., 1998aGo), while some transcription-independent effects of NGF can be selectively blocked by K252a (Rukenstein et al., 1991Go). In order to determine whether similar signaling mechanisms were involved in the local effects of these neurotrophic factors on synaptosomes, we determined the effects of bafilomycin A1 (an inhibitor of receptor-mediated endocytosis) and K252a on the abilities of ADNF and NGF to protect synaptosomes against oxidative impairment of membrane transporter function. Bafilomycin A1 significantly attenuated the ability of ADNF to prevent Fe2+and Aß-induced impairment of glucose transport (Fig. 3AGo), suggesting a role for receptor-mediated endocytosis in the local protective effects of ADNF in synaptic terminals. K252a completely abolished the ability of NGF to preserve glucose transport in synaptosomes exposed to Fe2+ and Aß (Fig. 3BGo), suggesting involvement of the high-affinity trkA NGF receptor in the effects of NGF on synaptic terminals.



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Figure 3.  Effects of inhibitors of receptor-mediated endocytosis and the high-affinity NGF receptor on glucose transport following exposure to Fe2+ and Aß. (A) Synaptosomes were pretreated for 1 h with saline (Control), 500 nM bafilomycin A1 (BafilA), 1 pM ADNF-9, or the combination of 500 nM BafilA and 1 pM ADNF-9. Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of [3H]glucose uptake were quantified, and values are the mean and SD of determinations made in four synaptosome preparations. (B) Synaptosomes were pretreated for 1 h with saline (Control), 200 nM K252a, 100 ng/ml NGF, or the combination of 200 nM K252a plus 100 ng/ml NGF. Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of [3H]glucose uptake were quantified, and values are the mean and SD of determinations made in four synaptosome preparations. *P < 0.01 compared to value for vehicle-treated control value. ##P < 0.01 compared to corresponding (FeSO4or Aß-treated) control value.

 
Mitochondrial dysfunction and production of reactive oxygen species are believed to play important roles in the (apoptotic and necrotic) neurodegenerative process in many different disorders (Beal, 1998Go; Keller et al., 1998Go). It was recently reported that apoptotic and oxidative insults can induce membrane depolarization and oxyradical production in synaptosomal mitochondria (Mattson et al., 1998aGo). In order to ascertain the possible impact of neurotrophic factors on synaptic mitochondrial function, we treated synaptosomes with ADNF-9, bFGF and NGF and quantified levels of rhodamine 123 fluorescence [an indicator of mitochondrial transmembrane potential (Mattson et al., 1993cGo)] and DHR fluorescence [a measure of mitochondrial reactive oxygen species levels (Mattson et al., 1997Go)] under basal conditions and following exposure to Fe2+ and Aß. The basal level of rhodamine 123 fluorescence was increased ~20% following exposure of synaptosomes to bFGF and NGF (Fig. 4AGo). As expected, Fe2+ and Aß each caused a significant decrease in the level of rhodamine 123 fluorescence. Each neurotrophic factor significantly attenuated the decreases in rhodamine 123 fluorescence otherwise caused by Fe2+ and Aß, with ADNF-9 being the most effective (Fig. 4AGo). The basal level of DHR fluorescence was unchanged in synaptosomes exposed to ADNF-9, bFGF and NGF (Fig. 4BGo). Fe2+ and Aß each caused a significant increase in the level of DHR fluorescence. ADNF-9, bFGF and NGF each significantly attenuated the increase in DHR fluorescence otherwise caused by Fe2+ and Aß (Fig. 4BGo). Concentration–effect analyses showed that ADNF-9 was effective in preserving mitochondrial transmembrane potential and suppressing mitochondrial oxyradical levels at concentrations from 0.01 to 100 pM (Fig. 5Go).



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Figure 4.  Effects of neurotrophic factors on mitochondrial function under basal conditions, and following exposure to Aß and an oxidative insult, in cortical synaptosomes. Synaptosomes were pretreated for 1 h with saline (Control), ADNF-9 (1 pM), bFGF (100 ng/ml) or NGF (100 ng/ml). Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of rhodamine 123 fluorescence, a measure of mitochondrial transmembrane potential (A), or DHR fluorescence, a measure of mitochondrial reactive oxygen species (B), were quantified. Values are the mean and SD of determinations made in four synaptosome preparations. *P < 0.05, **P < 0.01 compared to value for vehicle-treated control value. #P < 0.05, ##P < 0.01 compared to corresponding (FeSO4or Aß-treated) control value.

 


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Figure 5.  Effects of increasing concentrations of ADNF-9 on mitochondrial functional parameters under basal conditions, and following exposure to Aß and an oxidative insult, in cortical synaptosomes. Synaptosomes were pretreated for 1 h with saline (Control) or the indicated concentrations of ADNF-9. Synaptosomes were then exposed for 4 h to vehicle (Control), FeSO4 (50 µM) or amyloid ß-peptide (Aß, 50 µM). Levels of rhodamine 123 fluorescence, a measure of mitochondrial transmembrane potential (A), or DHR fluorescence, a measure of mitochondrial reactive oxygen species (B), were quantified. Values are the mean and SD of determinations made in four synaptosome preparations. *P < 0.05, **P < 0.01 compared to value for vehicle-treated control value. #P < 0.05, ##P < 0.01 compared to corresponding (FeSO4 or Aß-treated) control value.

 

    Discussion
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 Materials and Methods
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Our data show that ADNF-9 can act directly on synaptosomes to enhance basal levels of glucose and glutamate transport, and can preserve function of these transporters, as well as mitochondrial function, following exposure to oxidative/apoptotic insults. Basic FGF and NGF were also effective, to varying extents, in protecting synaptosomes against impairment of glucose and glutamate transport and mitochondrial function. ADNF production is induced by activity in neuronal circuits (Brenneman et al., 1998aGo), as is the expression of bFGF (Van Der Wal et al., 1994Go) and NGF (Zafra et al., 1990Go; Rocamora et al., 1994Go). Previous studies have shown that activation of glutamate receptors can regulate production of bFGF and NGF and their receptors on the one hand (Zafra et al., 1990Go; Van Der Wal et al., 1994Go), and that bFGF (Mattson et al., 1989Go, 1993bGo; Cheng et al., 1995Go) and NGF (Tancredi et al., 1993Go) can alter responses to glutamate, on the other hand. Both preand postsynaptic terminals are subjected to high, periodic, levels of calcium influx resulting from activation of glutamate receptors and voltage-dependent calcium channels (Denk et al., 1996Go). Synapses also have a much higher metabolic demand than do other neuronal compartments (i.e. cell bodies, axons and dendrites). Local actions of neurotrophic factors in synaptic terminals may therefore provide an important rapid, transcription-independent mechanism for protecting these regions of neurons against degeneration. Because neurotrophic factors, including bFGF (Abe et al., 1992Go; Tanaka et al., 1996Go) and NGF (Tancredi et al., 1993Go; Kelly et al., 1998Go), have been shown to modulate synaptic transmission, our data further suggest that modulation of glutamate transport, glucose transport and/or mitochondrial function are potential mechanisms underlying such previously reported effects of neurotrophic factors on synaptic plasticity.

ADNF is released from astrocytes by a mechanism dependent upon neuronal activity (Brenneman et al., 1998bGo), and both bFGF (Pechan et al., 1992Go) and NGF (Schwartz and Mishler, 1990Go) are also produced by astrocytes. The expressions of NGF and trkA (Lee et al., 1998Go), bFGF (Lin et al., 1997Go) and bFGF receptors (Reilly and Kumari, 1996Go) in astrocytes are greatly increased following traumatic and ischemic brain insults. Cell culture data show that astrocyte-derived bFGF can protect neurons against excitotoxic insults (Mattson and Rychlik, 1990Go). Our data demonstrate actions of bFGF, ADNF-9 and bFGF in synaptic terminals (increased glutamate and glucose transport under conditions of oxidative stress) that would be expected to protect those compartments against excitotoxic damage. Because astrocytes are intimately associated with synaptic terminals (Vernadakis, 1996Go), our data suggest the possibility that astrocyte-derived neurotrophic factors can exert local protective actions on neuronal synaptic terminals. While the present studies did not allow determination of the specific compartments in which glucose and glutamate transport were modulated by neurotrophic factors, it seems likely [based on previous studies of glucose and glutamate transport in cell culture and in vivo studies (Maher et al., 1994Go; Schousboe et al., 1997Go)], that the majority of glucose uptake is by neuronal terminals, whereas glutamate uptake occurs largely in astrocytes.

Previous studies of the neuroprotective and plasticity-modulating actions of neurotrophic factors have focused on signaling cascades that result in modulation of gene expression. Both bFGF and NGF activate cell surface receptors with intrinsic tyrosine kinase activity, and activation of these receptors is known to initiate kinase cascades that result in activation of transcription factors (Schlessinger and Ulrich, 1992Go). Studies of cultured neurons have provided evidence that bFGF and NGF can regulate the expression of a variety of potentially neuroprotective gene products including antioxidant enzymes (Mattson et al., 1995Go), proteins involved in cellular calcium homeostasis (Mattson et al., 1993bGo; Pappas and Parnavelas, 1997Go) and Bcl-2 family members (Allsopp et al., 1995Go). The present findings are novel and intriguing because, since synaptosomes lack nuclei, the protective effects of neurotrophic factors in this preparation cannot involve a transcriptiondependent mechanism. The signal transduction mechanisms that mediate the rapid transcription-independent effects of ADNF-9, bFGF and NGF on synaptic membrane transporter and mitochondrial functions remain to be elucidated, but are presumed to involve specific membrane receptors. Previous studies have established the presence of specific receptors for NGF (Figueiredo et al., 1995Go) and bFGF (Masumura et al., 1996Go) in cortical neurons in adult rats. Receptors for NGF (Henry et al., 1994Go) and bFGF (Torriglia and Blanquet, 1994Go) are present in synaptic terminals. Moreover, it was previously reported that treatment of synaptosomes with NGF results in enhancement of depolarization-induced neurotransmitter release (Sala et al., 1998Go) and that NGF induces cAMP and IP3 production in synaptosomes (Knipper et al., 1993Go). The synapto-protective actions of neurotrophic factors documented in the present study may therefore involve protein phosphorylation events similar to those that have recently been suggested to underlie the rapid modulation of synaptic plasticity by neurotrophic factors (Abe et al., 1992Go). The ability of K252a to block the protective effects of NGF on synaptosomes is consistent with mediation by the high-affinity NGF receptor. Although the receptor(s) for ADNF remains to be identified, the responses of synaptosomes documented in the present study suggest that such receptors are present in synaptic terminals. A possible role for receptor-mediated endocytosis in the protective actions of ADNF is suggested by the ability of bafilomycin A1 to block such protective effects in synaptosomes. The specific compartments (presynaptic, postsynaptic and/or astrocytic) in which neurotrophic factors act to stimulate and preserve glucose and glutamate transport and mitochondrial function remain to be established.

Aß is believed to play a major role in promoting neuro-degenerative cascades in AD (Mattson, 1997Go). Impairment of glucose transport and mitochondrial function in cultured cortical and hippocampal neurons (Mark et al., 1997bGo,cGo), and of glutamate transport in cultured astrocytes (Blanc et al., 1998Go), following exposure to Aß is mediated by increased levels of oxidative stress. Previous studies demonstrating adverse effects of Aß on glucose and glutamate transport in cortical synaptosomes also implicate oxidative stress in the underlying mechanism (Keller et al., 1997aGo,bGo). The present findings demonstrate that three different neurotrophic factors can protect synaptosomes against Aßand Fe2+-induced impairment of glucose and glutamate transport. Both Aß and Fe2+ induce membrane lipid peroxidation in neurons and synaptosomes, and this action of these insults appears to mediate impairment of the transporters because transporter functions are preserved in neurons and synaptosomes treated with antioxidants that suppress membrane lipid peroxidation (Mark et al., 1997aGo,cGo; Keller et al., 1997aGo). A striking action of Aß on cultured hippocampal and cortical neurons is to increase their vulnerability to excitotoxicity (Koh et al., 1990Go; Mattson et al., 1992Go). Previous studies have shown that bFGF, NGF and ADNF can protect cultured hippocampal and cortical neurons against excitotoxic, metabolic and oxidative insults (Mattson et al., 1989Go, 1993bGo,cGo; Zhang et al., 1993Go; Brenneman et al., 1998aGo,bGo). Basic FGF (Mattson et al., 1993aGo) and ADNF (Brenneman et al., 1998aGo,bGo) have also been reported to protect cultured hippocampal neurons against Aß toxicity. The present findings suggest the possibility that the neuroprotective mechanism of these trophic factors may be exerted at the level of the synapse. Synapse loss is a striking characteristic of AD (DeKosky et al., 1996Go) and Aß and excessive activation of glutamate receptors may promote such synapse loss (Mattson et al., 1998aGo,bGo). Therefore, the possibility that enhancement of neurotrophic factor signaling in synaptic compartments may forestall the neurodegenerative process in AD and related disorders merits further investigation.


    Notes
 
We thank J. Partin and L. Yan for technical assistance. This work was supported by grants to M.P.M. from the NIH (AG14554, AG05144 and NS35253).

Address correspondence to Mark P. Mattson, Laboratory of Neurosciences, National Institute of Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Email: mattsonm{at}grc.nia.nih.gov.


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1 Present address: Laboratory of Neurosciences, National Institute of Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA Back


    References
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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