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Depolarization of PC12 cells induces neurite outgrowth and enhances nerve growth factor-induced neurite outgrowth in rats
Neuroscience Letters 351 (2003) 41–45 www.elsevier.com/locate/neulet
Depolarization of PC12 cells induces neurite outgrowth and enhances nerve growth factor-induced neurite outgrowth in rats Charles L. Howe* Department of Neurology, Mayo Medical and Graduate Schools, Guggenheim 442C, 200 First Street SW, Rochester, MN 55905, USA Received 7 April 2003; received in revised form 26 June 2003; accepted 30 June 2003
Abstract Synaptic plasticity is clearly controlled by synaptic activity and by neurotrophin-dependent signaling. We have previously hypothesized that synaptic activity modulates concomitant neurotrophin receptor signaling, thereby integrating the activity state of a synapse with the state of neurotrophic support available at the synapse. Herein we present evidence in support of this hypothesis. Using PC12 cells as a model of the presynaptic element, we show that depolarization increases TrkA tyrosine phosphorylation in response to nerve growth factor (NGF). Moreover, we show that depolarization alone is sufficient to induce the tyrosine phosphorylation of TrkA. These findings are functionally relevant, as evidenced by our observation that depolarization alone induces neurite outgrowth, and that depolarization dramatically enhances neurite outgrowth in response to NGF, especially in primed PC12 cells. We conclude that normal synaptic function may depend upon the integration of synaptic activity and activity-dependent neurotrophin release and signaling, and that these findings have potential relevance to neural repair. q 2003 Published by Elsevier Ireland Ltd. Keywords: Depolarization; Plasticity; Neurotrophin; TrkA; Nerve growth factor
Neurons depend not only upon neurotransmitter-based signaling, but also upon neurotrophin-based signaling for the establishment and maintenance of synaptic contacts [17]. The interplay of these anterograde and retrograde signals between the presynaptic and postsynaptic elements is clearly of fundamental importance to normal synaptic plasticity, and abnormalities in either or both of these signaling pathways is likely to contribute to the pathological plasticity observed in such neurologic diseases as Alzheimer disease and stroke [18,21]. Therefore, understanding the relationship that exists between depolarization and neurotrophin receptor signaling is likely to shed light upon important aspects of synaptic function. We hypothesized that synaptic depolarization would modulate concomitant neurotrophin receptor signaling, thereby integrating the activity state of a synapse with the state of neurotrophic support available at the synapse [9]. To test this hypothesis we used PC12 cells as a model of the presynaptic element. These cells express functional TrkA receptors for nerve * Tel.: þ1-507-284-4665; fax: þ 1-507-284-1086. E-mail address: [email protected] (C.L. Howe). 0304-3940/03/$ - see front matter q 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0304-3940(03)00915-7
growth factor (NGF), and are responsive to depolarization [12,22]. PC12 cells were propagated in DMEM supplemented with 10% horse serum and 5% heat-inactivated fetal calf serum. These cells, in the absence of external NGF stimulation, are considered ‘naive’, in that they do not exhibit neurites, but are able to respond to NGF treatment by differentiating into a neuron-like phenotype over the course of several days [6,7]. PC12 cells that have been treated with NGF (50 ng/ml) for more than 1 week display robust neuritic projections and neuron-like morphology. If these cells are mechanically stripped of neurites via trituration and then replated, they are considered to be ‘primed’, in that they respond to subsequent NGF treatment by elaborating neurites much more quickly than similarly treated naive PC12 cells [3]. Both naive and primed PC12 cells are sensitive to depolarizing stimuli, in that they respond to elevated potassium in a variety of ways, including regulated secretion of neurotransmitters and growth factors, transient calcium influx, and activation of cAMP [1,4,5,8,20]. If depolarization indeed modulates neurotrophin signaling, then this modulation should be apparent at the level of neurotrophin receptor activation. To test this hypothesis, we
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C.L. Howe / Neuroscience Letters 351 (2003) 41–45
asked whether elevated potassium altered the level of TrkA tyrosine phosphorylation elicited in response to simultaneous NGF treatment. Naive PC12 cells in suspension were pelleted by brief centrifugation and then resuspended in a balanced salt solution in the presence or absence of NGF (50 ng/ml). This salt solution consisted of the following: 135 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1 mg/ml bovine serum albumin, 15 mM HEPES, pH 7.4. Depolarization without changing osmolarity was elicited by altering the sodium and potassium concentrations as follows: NaCl was reduced to 90 mM and KCl was increased to 50 mM. This elevation of potassium was shown previously to induce reliable depolarization of PC12 cells and to enhance the binding and internalization of iodinated NGF [13,14]. Expanding upon these observations, we found that depolarization of PC12 cells via potassium elevation (50 mM) concomitant with NGF-treatment (50 ng/ml) for 1 min at 378C resulted in enhanced phosphorylation of TrkA (Fig. 1A). In fact, immunoprecipitation of TrkA with the 06-574 antibody (Upstate, Waltham, MA) and subsequent immunoblotting with 4G10 (Upstate, Waltham, MA) showed a several-fold increase in TrkA tyrosine phosphorylation induced by
Fig. 1. (A) PC12 cells in suspension were treated with 2 nM NGF or NGF plus 50 mM Kþ for 1 min at 378C, then chilled and lysed. Lysates were immunoprecipitated with an anti-TrkA antibody, and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The upper panel shows tyrosine phosphorylation of TrkA in response to each of the treatments. Western blotting revealed that depolarization combined with NGF treatment enhanced the level of tyrosine phosphorylated TrkA as compared to that elicited by NGF alone. The lower panel shows the total levels of TrkA in each lane. (B) PC12 cells in suspension were treated with 2 nM NGF, 50 mM Kþ, or NGF plus Kþ for 5 min at 378C, then processed as described above. Most notably, depolarization with 50 mM Kþ alone was sufficient to induce significant tyrosine phosphorylation of TrkA.
combined depolarization and NGF treatment as compared to treatment with NGF only (Fig. 1A). On the basis of this observation, we wondered whether depolarization alone might be sufficient to induce tyrosine phosphorylation of TrkA. Because depolarization results in the redistribution of clathrin-coated membranes and an enhancement in clathrinmediated endocytosis [11], we predicted that more TrkA, known to be internalized via the clathrin pathway [10], would be recruited to clathrin-coated pits and would therein be brought into sufficient proximity to induce transphosphorylation of the receptor tyrosine kinase. In fact, we found that depolarization via elevated potassium (50 mM) for 5 min at 378C was sufficient to induce a small but significant increase in TrkA tyrosine phosphorylation (Fig. 1B). While it is evident that this level of tyrosine phosphorylation was not comparable to that induced by 5 min of NGF treatment, and while we were unable to detect any significant enhancement in TrkA tyrosine phosphorylation induced by simultaneous treatment with depolarization and NGF for 5 min at 378C, it is clear that depolarization alone is capable of transducing a signal via TrkA (Fig. 1B). Early attempts to rule out the autocrine release of NGF from depolarized PC12 cells using anti-NGF antibodies suggested that such release was not responsible for the depolarization effect (data not shown), but this potential mechanism requires further analysis. Likewise, initial experiments pharmacologically blocking clathrin-mediated internalization suggest that it is such internalization downstream from depolarization that is responsible for the phosphorylation of TrkA in the absence of its cognate ligand (data not shown). Hence, we conclude that depolarization of PC12 cells cooperatively enhances TrkA tyrosine phosphorylation in response to brief NGF treatment, and stimulates the autophosphorylation of TrkA in the absence of exogenous NGF at longer timepoints. Based on our findings regarding the modulation of TrkA activity by depolarization, we hypothesized that depolarization would have a functional influence on neurite outgrowth in response to NGF. This well-characterized process requires several days of constant NGF exposure for the induction of neurites in naive PC12 cells, and at least 24 h of constant NGF treatment for the induction of neurite outgrowth in primed cells stripped of their neurites. As shown in Fig. 2A, we found that depolarization via elevated potassium significantly enhanced the number of cells bearing neurites greater than 1 cell body diameter in length following 1 day of treatment. In fact, in the absence of NGF, depolarization for 1 day resulted in 6.7 ^ 1.4% (mean ^ SEM; n ¼ 3 independent experiments; P , 0:001 versus untreated by t-test) of cells bearing neurites, compared to 2.3 ^ 0.4% (n ¼ 3; P ¼ 0:02 versus 50 mM Kþ only; P , 0:001 versus untreated) of NGF-treated (50 ng/ml) cells. Moreover, the combination of depolarization and NGF treatment for 1 day resulted in 27.5 ^ 1.7% (n ¼ 3; P , 0:001 versus 50 mM Kþ; P , 0:001 versus NGF; P , 0:001 versus untreated) of cells exhibiting neuritic
C.L. Howe / Neuroscience Letters 351 (2003) 41–45
Fig. 2. (A) PC12 cells were cultured for 1, 2, or 3 days in the presence of either 50 mM Kþ, 2 nM NGF, or NGF plus Kþ. Following treatment, neuritic processes longer than one cell body diameter were counted in several random microscope fields. At least 100 cells were counted for each treatment condition, and the percent of cells bearing neurites was determined. The data are presented as mean ^ SEM, and are representative of three separate experiments. Untreated cells did not exhibit neurites at any time point tested. (B) Primed and mechanically stripped cells were cultured for 1, 2, or 3 days in the presence of either 50 mM Kþ, 2 nM NGF, or NGF plus Kþ. Following treatment, neuritic processes longer than one cell body diameter were counted, and the percent of cells bearing neurites was determined as described above. As above, untreated cells did not exhibit neurites at any timepoint.
processes. This general relationship between treatments was maintained following 2 days of treatment (Fig. 2A). However, by 3 days, 50 mM Kþ-only and NGF-only treatments resulted in almost identical outcomes: 33.2 ^ 3.2% (n ¼ 3; P , 0:001 versus untreated) and 33.0 ^ 3.5% (n ¼ 3; P ¼ 0:99 versus 50 mM Kþ only) of cells showed neurites, respectively (Fig. 2A). Nonetheless, 3 days of combined depolarization and NGF resulted in neurite outgrowth that was enhanced compared to either individual treatment: 51.5 ^ 3.4% (n ¼ 3; P ¼ 0:003 versus 50 mM Kþ only; P ¼ 0:004 versus NGF only; P , 0:001 versus untreated) of cells exhibited neurites. Hence, we conclude that depolarization alone is sufficient to induce neurite outgrowth that is comparable to that elicited by NGF treatment after 3 days, and is greater than that elicited by NGF at 1 day, and that the combination of
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depolarization and NGF results in neurite outgrowth that is greater than either treatment alone is able to induce. Primed PC12 cells have previously been characterized and found to respond very quickly to NGF treatment following mechanical stripping of neurites [3]. PC12 cells treated for 1 week with NGF (50 ng/ml; refreshed daily) exhibit an increase in the density of TrkA receptors present on the plasma membrane [26]. It is thought that this elevation in TrkA binding sites makes the cells more responsive to NGF treatment following a transient removal of neurites. We asked whether, under these conditions of heightened NGF responsivity, depolarization was capable of signaling for neurite outgrowth, either alone or in combination with NGF. As shown in Fig. 2B, we found that depolarization was still effective in inducing neurite outgrowth, and that depolarization via elevated potassium significantly enhanced the number of cells bearing neurites greater than 1 cell body diameter in length following 1 day of treatment. In fact, depolarization for 1 day resulted in 20.3 ^ 1.6% (mean ^ SEM; n ¼ 3 independent experiments; P , 0:001 versus untreated) of cells bearing neurites – a value several-fold higher than that elicited in naive PC12 cells. Thus, priming, normally considered with regard to NGF signaling, also enhanced the responsiveness of PC12 cells to depolarization-induced neurite outgrowth. Likewise, NGF treatment alone for 1 day following mechanical stripping of neurites resulted in 43.2 ^ 2.7% (n ¼ 3; P , 0:001 versus 50 mM Kþ only) of cells exhibiting neurites greater than one cell body diameter in length (Fig. 2B). These values are 3- and 19-fold higher than the values observed in naive cells, for 50 mM Kþ only and 50 ng/ml NGF only, respectively. Interestingly, the combination of NGF and depolarization was only as effective at inducing neurites as it was in naive cells, and was, in fact, less efficacious than NGF treatment alone following 1 day (28.1 ^ 1.1%; n ¼ 3; P ¼ 0:003 versus 50 mM Kþ; P , 0:001 versus NGF; P ¼ 0:8 versus combined treatment of naive cells). However, after 2 days of treatment, combined NGF and depolarization greatly exceeded either treatment alone, and depolarization only was essentially as effective as NGF only (Fig. 2B). This relationship was maintained after 3 days of treatment, and, interestingly, combined depolarization and NGF treatment resulted in virtually all cells in culture exhibiting neurites. Specifically, following 3 days of treatment: 50 mM Kþ resulted in 43.3 ^ 2.2% (n ¼ 3) of cells bearing neurites; 50 ng/ml NGF alone resulted in 58.7 ^ 2.0% (n ¼ 3; P , 0:001 versus 50 mM Kþ only) of cells exhibiting neurites; and combined treatment resulted in 97.5 ^ 1.0% (n ¼ 3; P , 0:001 versus 50 mM Kþ only; P , 0:001 versus NGF only) of cells expressing neuritic projections greater than one cell body diameter in length (Fig. 2B). Therefore, we conclude that priming of PC12 cells results not only in enhanced and accelerated NGF responsivity, but also enhanced and accelerated responsiveness to depolarization alone and depolarization in combination with NGF.
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C.L. Howe / Neuroscience Letters 351 (2003) 41–45
Our observation that simultaneous treatment of primed PC12 cells with depolarization and NGF results in essentially 100% of cells exhibiting neurites, suggests that the integration of these two stimuli is an important mechanism for stabilization and maintenance of synaptic connections. Using PC12 cells as a model of the presynaptic element, we have shown that depolarization alone is sufficient to induce signals that culminate in neurite outgrowth, and that the combination of depolarization and NGF is a powerful neurite-inducing stimulus. While the latter finding is in agreement with published observations, the induction of neurites by depolarization alone that we have observed is at odds with Solem et al. [23]. However, several potentially important technical differences exist between the two studies. First, Solem and colleagues depolarized PC12 cells by elevating potassium levels without concomitantly reducing sodium concentration [23]. In contrast, we controlled for osmotic changes by balancing the overall salt concentration. While Solem et al. did perform control experiments in which choline chloride was added to mimic osmotic changes in the absence of depolarization, it is currently unclear which method of potassium-mediated depolarization more faithfully mimics normal synaptic activity. Perhaps future experiments utilizing electrical stimulation will shed light on this potential difference. Second, Solem and colleagues cultured their PC12 cells in the presence of streptomycin, a drug that significantly alters calcium influx through voltage-gated channels, increases membrane permeability, and antagonizes synaptic transmission [2,19,25]. In fact, these effects are responsible for the ototoxicity induced in patients treated with streptomycin [15]. Therefore, it is likely that the amount of calcium influx induced by depolarization under our culture conditions is substantially different from those induced by Solem et al. The extent to which such differences contribute to the observed effects on neurite outgrowth will require further analysis. In conclusion, our findings suggest that normal synaptic function – wherein the presynaptic element may be depolarized within a temporal window that overlaps with regulated secretion of neurotrophin from the postsynaptic element – depends upon the integration of activity and activity-dependent neurotrophin release. In fact, we have previously suggested that concomitant synaptic depolarization and neurotrophin binding to its cognate receptor tyrosine kinase is the optimal signal for synaptic maintenance, while depolarization in the absence of neurotrophin or neurotrophin signaling in the absence of depolarization may either be relatively weak maintenance signals, or may, in vivo, result in regressive signals that lead ultimately to synaptic retraction and pruning [9]. Thus, the physiological relevance of either stimulus is sharpened and amplified in a manner that recognizes the feed forward and feedback nature of the relationship between the presynaptic and postsynaptic elements of synapses. The importance of this model is further highlighted by our finding that depolariz-
ation dramatically accelerates the recovery of neurites by primed PC12 cells. Such recovery is a good model of the potential for neuronal regeneration under appropriate circumstances, and our findings suggest that therapies aimed at improving neural repair following spinal cord injury or nerve transection might benefit from a combination of neurotrophic support and controlled electrical stimulation. Likewise, our observation of TrkA phosphorylation induced by depolarization in the absence of exogenous neurotrophin supports the emerging concept of receptor transactivation as a therapeutic target for the amelioration of neurodegeneration. This approach depends upon crosstalk between receptor-based signaling systems, and opens up the possibility of modulating signal transduction through relatively intractable neurotrophin receptors via small molecule engagement of parallel, transactivating receptor systems [16,24]. Our findings add the interesting possibility of transactivating receptor tyrosine kinases via modulation of neuronal and synaptic activity. Thus, we conclude that modulation of neurotrophin-induced signaling via changes in synaptic activity is an important paradigm for the development of novel therapeutic strategies aimed at inducing, enhancing, and accelerating neural repair.
Acknowledgements This work was supported by National Multiple Sclerosis Society grant PP0893 and by Training Grant T32 HD 07447-8.
References [1] S. Amino, M. Itakura, H. Ohnishi, J. Tsujimura, S. Koizumi, N. Takei, M. Takahashi, Nerve growth factor enhances neurotransmitter release from PC12 cells by increasing Ca(2 þ )-responsible secretory vesicles through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase, J. Biochem. (Tokyo) 131 (2002) 887 –894. [2] A. Belus, E. White, Effects of antibiotics on the contractility and Ca2þ transients of rat cardiac myocytes, Eur. J. Pharmacol. 412 (2001) 121 –126. [3] D.E. Burstein, L.A. Greene, Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor, Proc. Natl. Acad. Sci. USA 75 (1978) 6059–6063. [4] M.A. Dichter, A.S. Tischler, L.A. Greene, Nerve growth factorinduced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line, Nature 268 (1977) 501 –504. [5] D.D. Ginty, D. Glowacka, D.S. Bader, H. Hidaka, J.A. Wagner, Induction of immediate early genes by Ca2þ influx requires cAMPdependent protein kinase in PC12 cells, J. Biol. Chem. 266 (1991) 17454–17458. [6] L.A. Greene, J.M. Aletta, A. Rukenstein, S.H. Green, PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation, Methods Enzymol. 147 (1987) 207– 216. [7] L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal
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[18] B.S. McEwen, Sex, stress and the hippocampus: allostasis, allostatic load and the aging process, Neurobiol. Aging 23 (2002) 921 –939. [19] T.D. Parsons, A.L. Obaid, B.M. Salzberg, Aminoglycoside antibiotics block voltage-dependent calcium channels in intact vertebrate nerve terminals, J. Gen. Physiol. 99 (1992) 491–504. [20] B. Rudy, B. Kirschenbaum, A. Rukenstein, L.A. Greene, Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells, J. Neurosci. 7 (1987) 1613–1625. [21] D.J. Selkoe, Alzheimer’s disease is a synaptic failure, Science 298 (2002) 789–791. [22] T.J. Shafer, W.D. Atchison, Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies, Neurotoxicology 12 (1991) 473– 492. [23] M. Solem, T. McMahon, R.O. Messing, Depolarization-induced neurite outgrowth in PC12 cells requires permissive, low level NGF receptor stimulation and activation of calcium/calmodulin-dependent protein kinase, J. Neurosci. 15 (1995) 5966–5975. [24] B.A. Tsui-Pierchala, J. Milbrandt, E.M. Johnson Jr, NGF utilizes cRet via a novel GFL-independent, inter-RTK signaling mechanism to maintain the trophic status of mature sympathetic neurons, Neuron 33 (2002) 261–273. [25] F. Van Bambeke, M.P. Mingeot-Leclercq, A. Schanck, R. Brasseur, P.M. Tulkens, Alterations in membrane permeability induced by aminoglycoside antibiotics: studies on liposomes and cultured cells, Eur. J. Pharmacol. 247 (1993) 155–168. [26] J. Zhou, J.S. Valletta, M.L. Grimes, W.C. Mobley, Multiple levels for regulation of TrkA in PC12 cells by nerve growth factor, J. Neurochem. 65 (1995) 1146–1156.
Depolarization of PC12 cells induces neurite outgrowth and enhances nerve growth factor-induced neurite outgrowth in rats Charles L. Howe* Department of Neurology, Mayo Medical and Graduate Schools, Guggenheim 442C, 200 First Street SW, Rochester, MN 55905, USA Received 7 April 2003; received in revised form 26 June 2003; accepted 30 June 2003
Abstract Synaptic plasticity is clearly controlled by synaptic activity and by neurotrophin-dependent signaling. We have previously hypothesized that synaptic activity modulates concomitant neurotrophin receptor signaling, thereby integrating the activity state of a synapse with the state of neurotrophic support available at the synapse. Herein we present evidence in support of this hypothesis. Using PC12 cells as a model of the presynaptic element, we show that depolarization increases TrkA tyrosine phosphorylation in response to nerve growth factor (NGF). Moreover, we show that depolarization alone is sufficient to induce the tyrosine phosphorylation of TrkA. These findings are functionally relevant, as evidenced by our observation that depolarization alone induces neurite outgrowth, and that depolarization dramatically enhances neurite outgrowth in response to NGF, especially in primed PC12 cells. We conclude that normal synaptic function may depend upon the integration of synaptic activity and activity-dependent neurotrophin release and signaling, and that these findings have potential relevance to neural repair. q 2003 Published by Elsevier Ireland Ltd. Keywords: Depolarization; Plasticity; Neurotrophin; TrkA; Nerve growth factor
Neurons depend not only upon neurotransmitter-based signaling, but also upon neurotrophin-based signaling for the establishment and maintenance of synaptic contacts [17]. The interplay of these anterograde and retrograde signals between the presynaptic and postsynaptic elements is clearly of fundamental importance to normal synaptic plasticity, and abnormalities in either or both of these signaling pathways is likely to contribute to the pathological plasticity observed in such neurologic diseases as Alzheimer disease and stroke [18,21]. Therefore, understanding the relationship that exists between depolarization and neurotrophin receptor signaling is likely to shed light upon important aspects of synaptic function. We hypothesized that synaptic depolarization would modulate concomitant neurotrophin receptor signaling, thereby integrating the activity state of a synapse with the state of neurotrophic support available at the synapse [9]. To test this hypothesis we used PC12 cells as a model of the presynaptic element. These cells express functional TrkA receptors for nerve * Tel.: þ1-507-284-4665; fax: þ 1-507-284-1086. E-mail address: [email protected] (C.L. Howe). 0304-3940/03/$ - see front matter q 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0304-3940(03)00915-7
growth factor (NGF), and are responsive to depolarization [12,22]. PC12 cells were propagated in DMEM supplemented with 10% horse serum and 5% heat-inactivated fetal calf serum. These cells, in the absence of external NGF stimulation, are considered ‘naive’, in that they do not exhibit neurites, but are able to respond to NGF treatment by differentiating into a neuron-like phenotype over the course of several days [6,7]. PC12 cells that have been treated with NGF (50 ng/ml) for more than 1 week display robust neuritic projections and neuron-like morphology. If these cells are mechanically stripped of neurites via trituration and then replated, they are considered to be ‘primed’, in that they respond to subsequent NGF treatment by elaborating neurites much more quickly than similarly treated naive PC12 cells [3]. Both naive and primed PC12 cells are sensitive to depolarizing stimuli, in that they respond to elevated potassium in a variety of ways, including regulated secretion of neurotransmitters and growth factors, transient calcium influx, and activation of cAMP [1,4,5,8,20]. If depolarization indeed modulates neurotrophin signaling, then this modulation should be apparent at the level of neurotrophin receptor activation. To test this hypothesis, we
42
C.L. Howe / Neuroscience Letters 351 (2003) 41–45
asked whether elevated potassium altered the level of TrkA tyrosine phosphorylation elicited in response to simultaneous NGF treatment. Naive PC12 cells in suspension were pelleted by brief centrifugation and then resuspended in a balanced salt solution in the presence or absence of NGF (50 ng/ml). This salt solution consisted of the following: 135 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1 mg/ml bovine serum albumin, 15 mM HEPES, pH 7.4. Depolarization without changing osmolarity was elicited by altering the sodium and potassium concentrations as follows: NaCl was reduced to 90 mM and KCl was increased to 50 mM. This elevation of potassium was shown previously to induce reliable depolarization of PC12 cells and to enhance the binding and internalization of iodinated NGF [13,14]. Expanding upon these observations, we found that depolarization of PC12 cells via potassium elevation (50 mM) concomitant with NGF-treatment (50 ng/ml) for 1 min at 378C resulted in enhanced phosphorylation of TrkA (Fig. 1A). In fact, immunoprecipitation of TrkA with the 06-574 antibody (Upstate, Waltham, MA) and subsequent immunoblotting with 4G10 (Upstate, Waltham, MA) showed a several-fold increase in TrkA tyrosine phosphorylation induced by
Fig. 1. (A) PC12 cells in suspension were treated with 2 nM NGF or NGF plus 50 mM Kþ for 1 min at 378C, then chilled and lysed. Lysates were immunoprecipitated with an anti-TrkA antibody, and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The upper panel shows tyrosine phosphorylation of TrkA in response to each of the treatments. Western blotting revealed that depolarization combined with NGF treatment enhanced the level of tyrosine phosphorylated TrkA as compared to that elicited by NGF alone. The lower panel shows the total levels of TrkA in each lane. (B) PC12 cells in suspension were treated with 2 nM NGF, 50 mM Kþ, or NGF plus Kþ for 5 min at 378C, then processed as described above. Most notably, depolarization with 50 mM Kþ alone was sufficient to induce significant tyrosine phosphorylation of TrkA.
combined depolarization and NGF treatment as compared to treatment with NGF only (Fig. 1A). On the basis of this observation, we wondered whether depolarization alone might be sufficient to induce tyrosine phosphorylation of TrkA. Because depolarization results in the redistribution of clathrin-coated membranes and an enhancement in clathrinmediated endocytosis [11], we predicted that more TrkA, known to be internalized via the clathrin pathway [10], would be recruited to clathrin-coated pits and would therein be brought into sufficient proximity to induce transphosphorylation of the receptor tyrosine kinase. In fact, we found that depolarization via elevated potassium (50 mM) for 5 min at 378C was sufficient to induce a small but significant increase in TrkA tyrosine phosphorylation (Fig. 1B). While it is evident that this level of tyrosine phosphorylation was not comparable to that induced by 5 min of NGF treatment, and while we were unable to detect any significant enhancement in TrkA tyrosine phosphorylation induced by simultaneous treatment with depolarization and NGF for 5 min at 378C, it is clear that depolarization alone is capable of transducing a signal via TrkA (Fig. 1B). Early attempts to rule out the autocrine release of NGF from depolarized PC12 cells using anti-NGF antibodies suggested that such release was not responsible for the depolarization effect (data not shown), but this potential mechanism requires further analysis. Likewise, initial experiments pharmacologically blocking clathrin-mediated internalization suggest that it is such internalization downstream from depolarization that is responsible for the phosphorylation of TrkA in the absence of its cognate ligand (data not shown). Hence, we conclude that depolarization of PC12 cells cooperatively enhances TrkA tyrosine phosphorylation in response to brief NGF treatment, and stimulates the autophosphorylation of TrkA in the absence of exogenous NGF at longer timepoints. Based on our findings regarding the modulation of TrkA activity by depolarization, we hypothesized that depolarization would have a functional influence on neurite outgrowth in response to NGF. This well-characterized process requires several days of constant NGF exposure for the induction of neurites in naive PC12 cells, and at least 24 h of constant NGF treatment for the induction of neurite outgrowth in primed cells stripped of their neurites. As shown in Fig. 2A, we found that depolarization via elevated potassium significantly enhanced the number of cells bearing neurites greater than 1 cell body diameter in length following 1 day of treatment. In fact, in the absence of NGF, depolarization for 1 day resulted in 6.7 ^ 1.4% (mean ^ SEM; n ¼ 3 independent experiments; P , 0:001 versus untreated by t-test) of cells bearing neurites, compared to 2.3 ^ 0.4% (n ¼ 3; P ¼ 0:02 versus 50 mM Kþ only; P , 0:001 versus untreated) of NGF-treated (50 ng/ml) cells. Moreover, the combination of depolarization and NGF treatment for 1 day resulted in 27.5 ^ 1.7% (n ¼ 3; P , 0:001 versus 50 mM Kþ; P , 0:001 versus NGF; P , 0:001 versus untreated) of cells exhibiting neuritic
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Fig. 2. (A) PC12 cells were cultured for 1, 2, or 3 days in the presence of either 50 mM Kþ, 2 nM NGF, or NGF plus Kþ. Following treatment, neuritic processes longer than one cell body diameter were counted in several random microscope fields. At least 100 cells were counted for each treatment condition, and the percent of cells bearing neurites was determined. The data are presented as mean ^ SEM, and are representative of three separate experiments. Untreated cells did not exhibit neurites at any time point tested. (B) Primed and mechanically stripped cells were cultured for 1, 2, or 3 days in the presence of either 50 mM Kþ, 2 nM NGF, or NGF plus Kþ. Following treatment, neuritic processes longer than one cell body diameter were counted, and the percent of cells bearing neurites was determined as described above. As above, untreated cells did not exhibit neurites at any timepoint.
processes. This general relationship between treatments was maintained following 2 days of treatment (Fig. 2A). However, by 3 days, 50 mM Kþ-only and NGF-only treatments resulted in almost identical outcomes: 33.2 ^ 3.2% (n ¼ 3; P , 0:001 versus untreated) and 33.0 ^ 3.5% (n ¼ 3; P ¼ 0:99 versus 50 mM Kþ only) of cells showed neurites, respectively (Fig. 2A). Nonetheless, 3 days of combined depolarization and NGF resulted in neurite outgrowth that was enhanced compared to either individual treatment: 51.5 ^ 3.4% (n ¼ 3; P ¼ 0:003 versus 50 mM Kþ only; P ¼ 0:004 versus NGF only; P , 0:001 versus untreated) of cells exhibited neurites. Hence, we conclude that depolarization alone is sufficient to induce neurite outgrowth that is comparable to that elicited by NGF treatment after 3 days, and is greater than that elicited by NGF at 1 day, and that the combination of
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depolarization and NGF results in neurite outgrowth that is greater than either treatment alone is able to induce. Primed PC12 cells have previously been characterized and found to respond very quickly to NGF treatment following mechanical stripping of neurites [3]. PC12 cells treated for 1 week with NGF (50 ng/ml; refreshed daily) exhibit an increase in the density of TrkA receptors present on the plasma membrane [26]. It is thought that this elevation in TrkA binding sites makes the cells more responsive to NGF treatment following a transient removal of neurites. We asked whether, under these conditions of heightened NGF responsivity, depolarization was capable of signaling for neurite outgrowth, either alone or in combination with NGF. As shown in Fig. 2B, we found that depolarization was still effective in inducing neurite outgrowth, and that depolarization via elevated potassium significantly enhanced the number of cells bearing neurites greater than 1 cell body diameter in length following 1 day of treatment. In fact, depolarization for 1 day resulted in 20.3 ^ 1.6% (mean ^ SEM; n ¼ 3 independent experiments; P , 0:001 versus untreated) of cells bearing neurites – a value several-fold higher than that elicited in naive PC12 cells. Thus, priming, normally considered with regard to NGF signaling, also enhanced the responsiveness of PC12 cells to depolarization-induced neurite outgrowth. Likewise, NGF treatment alone for 1 day following mechanical stripping of neurites resulted in 43.2 ^ 2.7% (n ¼ 3; P , 0:001 versus 50 mM Kþ only) of cells exhibiting neurites greater than one cell body diameter in length (Fig. 2B). These values are 3- and 19-fold higher than the values observed in naive cells, for 50 mM Kþ only and 50 ng/ml NGF only, respectively. Interestingly, the combination of NGF and depolarization was only as effective at inducing neurites as it was in naive cells, and was, in fact, less efficacious than NGF treatment alone following 1 day (28.1 ^ 1.1%; n ¼ 3; P ¼ 0:003 versus 50 mM Kþ; P , 0:001 versus NGF; P ¼ 0:8 versus combined treatment of naive cells). However, after 2 days of treatment, combined NGF and depolarization greatly exceeded either treatment alone, and depolarization only was essentially as effective as NGF only (Fig. 2B). This relationship was maintained after 3 days of treatment, and, interestingly, combined depolarization and NGF treatment resulted in virtually all cells in culture exhibiting neurites. Specifically, following 3 days of treatment: 50 mM Kþ resulted in 43.3 ^ 2.2% (n ¼ 3) of cells bearing neurites; 50 ng/ml NGF alone resulted in 58.7 ^ 2.0% (n ¼ 3; P , 0:001 versus 50 mM Kþ only) of cells exhibiting neurites; and combined treatment resulted in 97.5 ^ 1.0% (n ¼ 3; P , 0:001 versus 50 mM Kþ only; P , 0:001 versus NGF only) of cells expressing neuritic projections greater than one cell body diameter in length (Fig. 2B). Therefore, we conclude that priming of PC12 cells results not only in enhanced and accelerated NGF responsivity, but also enhanced and accelerated responsiveness to depolarization alone and depolarization in combination with NGF.
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C.L. Howe / Neuroscience Letters 351 (2003) 41–45
Our observation that simultaneous treatment of primed PC12 cells with depolarization and NGF results in essentially 100% of cells exhibiting neurites, suggests that the integration of these two stimuli is an important mechanism for stabilization and maintenance of synaptic connections. Using PC12 cells as a model of the presynaptic element, we have shown that depolarization alone is sufficient to induce signals that culminate in neurite outgrowth, and that the combination of depolarization and NGF is a powerful neurite-inducing stimulus. While the latter finding is in agreement with published observations, the induction of neurites by depolarization alone that we have observed is at odds with Solem et al. [23]. However, several potentially important technical differences exist between the two studies. First, Solem and colleagues depolarized PC12 cells by elevating potassium levels without concomitantly reducing sodium concentration [23]. In contrast, we controlled for osmotic changes by balancing the overall salt concentration. While Solem et al. did perform control experiments in which choline chloride was added to mimic osmotic changes in the absence of depolarization, it is currently unclear which method of potassium-mediated depolarization more faithfully mimics normal synaptic activity. Perhaps future experiments utilizing electrical stimulation will shed light on this potential difference. Second, Solem and colleagues cultured their PC12 cells in the presence of streptomycin, a drug that significantly alters calcium influx through voltage-gated channels, increases membrane permeability, and antagonizes synaptic transmission [2,19,25]. In fact, these effects are responsible for the ototoxicity induced in patients treated with streptomycin [15]. Therefore, it is likely that the amount of calcium influx induced by depolarization under our culture conditions is substantially different from those induced by Solem et al. The extent to which such differences contribute to the observed effects on neurite outgrowth will require further analysis. In conclusion, our findings suggest that normal synaptic function – wherein the presynaptic element may be depolarized within a temporal window that overlaps with regulated secretion of neurotrophin from the postsynaptic element – depends upon the integration of activity and activity-dependent neurotrophin release. In fact, we have previously suggested that concomitant synaptic depolarization and neurotrophin binding to its cognate receptor tyrosine kinase is the optimal signal for synaptic maintenance, while depolarization in the absence of neurotrophin or neurotrophin signaling in the absence of depolarization may either be relatively weak maintenance signals, or may, in vivo, result in regressive signals that lead ultimately to synaptic retraction and pruning [9]. Thus, the physiological relevance of either stimulus is sharpened and amplified in a manner that recognizes the feed forward and feedback nature of the relationship between the presynaptic and postsynaptic elements of synapses. The importance of this model is further highlighted by our finding that depolariz-
ation dramatically accelerates the recovery of neurites by primed PC12 cells. Such recovery is a good model of the potential for neuronal regeneration under appropriate circumstances, and our findings suggest that therapies aimed at improving neural repair following spinal cord injury or nerve transection might benefit from a combination of neurotrophic support and controlled electrical stimulation. Likewise, our observation of TrkA phosphorylation induced by depolarization in the absence of exogenous neurotrophin supports the emerging concept of receptor transactivation as a therapeutic target for the amelioration of neurodegeneration. This approach depends upon crosstalk between receptor-based signaling systems, and opens up the possibility of modulating signal transduction through relatively intractable neurotrophin receptors via small molecule engagement of parallel, transactivating receptor systems [16,24]. Our findings add the interesting possibility of transactivating receptor tyrosine kinases via modulation of neuronal and synaptic activity. Thus, we conclude that modulation of neurotrophin-induced signaling via changes in synaptic activity is an important paradigm for the development of novel therapeutic strategies aimed at inducing, enhancing, and accelerating neural repair.
Acknowledgements This work was supported by National Multiple Sclerosis Society grant PP0893 and by Training Grant T32 HD 07447-8.
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