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Suppression of an amyloid β-peptidemediated calcium channel response by a secreted β-amyloid precursor protein
Vol. 95, No. 1, pp. 1–4, 2000 1 Suppression of an amyloid b-peptidemediated calcium channel response by a secreted b-amyloidNeuroscience precursor protein
Pergamon PII: S0306-4522(99)00479-0
q 1999 Elsevier Science Ltd. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience SUPPRESSION OF AN AMYLOID b PEPTIDE-MEDIATED CALCIUM CHANNEL RESPONSE BY A SECRETED b-AMYLOID PRECURSOR PROTEIN W. Y. LI,* J. P. BUTLER, J. E. HALE, D. B. MCCLURE, S. P. LITTLE, D. L. CZILLI and L. K. SIMMONS Neuroscience Discovery Research and Research Technologies and Product Development, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA Key words: Alzheimer’s Disease, b-amyloid protein, b-amyloid precursor protein, calcium channel, Ab toxicity neuroprotection.
Secreted isoforms of the b-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid b peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid b peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid b peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted b-amyloid precursor protein on the calcium channel response to amyloid b peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with b-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid b peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by b-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Coapplication of b-amyloid precursor protein 751 with amyloid b peptide did not alter calcium channel response to amyloid b peptide. Taken together, these findings suggest that secreted b-amyloid precursor proteins can suppress a calcium channel response to amyloid b peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid b toxicity. q 1999 IBRO. Published by Elsevier Science Ltd.
that precludes the formation of Ab leads to the constitutive secretion of N-terminal isoforms (sAPPs). 3 The secreted forms of APP have been shown to have memory enhancing effects in normal and amnestic mice. 14 These sAPPs can reduce neuronal cytosolic Ca 21 levels and have demonstrated neuroprotective efficacy against a variety of insults. 11 This includes neuroprotection against the increases in intracellular Ca 21 believed to be a primary cause of Ab toxicity. 6 Although the mechanism(s) underlying the homeostatically disruptive actions of Ab are not fully understood, we described in a previous study an Ab-independent Ca 21 channel response that may be involved in Ab toxicity. 18 Using whole-cell voltage-clamp protocols we demonstrated that toxic Ab acutely enhances voltage-sensitive calcium channel (VSCC) currents in cultured rat hippocampal neurons via a process that is GTP binding protein (G protein)-dependent and pertussis toxin (PTX) sensitive. The present study was designed to examine whether the neuroprotective efficacy of sAPPs can be explained, at least in part, by a suppression of this modulatory VSCC response to Ab. Ab aggregation is an important determinant of neurotoxic potency in vitro. 2,16,19 One means of accelerating the formation of toxic aggregates is to incubate the peptide in concentrated aqueous solutions for 1–4 days prior to use. 19 This process is known as aging. The aging paradigms that promote the formation of toxic Ab aggregates also result in peptides that are capable of acutely enhancing VSCC currents. 18 Accordingly, similar peptide aging protocols were used here to obtain Ab samples active at VSCCs. Whole-cell recordings were conducted in cultured rat hippocampal neurons, with 10 mM Ba 21 in the bath as charge carrier. The raw data traces in Fig. 1A illustrate the VSCC currents (IBa) activated with voltage steps to 20 mV from a membrane holding potential of 60 mV under control conditions, and following application of 1 mM Ab. The maximal increase in current size stabilized within 5–6 min of exposure to Ab and was irreversible following washout in control external solution for up to 30 min. This Ab-mediated VSCC modulatory response was observed in 33 of 37 (187 ^ 86.8%; P 0:0000089) cells in the absence of changes in cell membrane leak conductance. The current–voltage relationship for another neuron under control conditions (squares) and in response to 1 mM Ab (circles) is shown in Fig. 1B.
Alzheimer’s disease (AD) is a neurodegenerative disorder of the elderly that is characterized histopathologically by the deposition of amyloid in brain neuritic plaques. 7 A component of neuritic plaques known as amyloid b peptide (Ab) is toxic to neurons in cell culture, 12,21 and is believed to have a prominent role in AD neurodegeneration. 17 Ab is derived from the proteolytic processing of a widely expressed family of membrane bound b-amyloid precursor proteins (APP). 8,10 An alternative processing of pathway for APP *To whom correspondence should be addressed at: Neuroscience Discovery Research Lilly Research Labs Eli Lilly & Company Lilly Corporate Center Indianapolis, IN 46285, USA. Tel.: 11 317 2763871; Fax: 11 317 2765546. Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b protein; APP, bamyloid precursor protein; cGMP, cyclic GMP; sAPP, secreted forms of APP; VSCC, voltage sensitive calcium channel; NGF, nerve growth factor; nif, nifedipine; v-Cgtx, v-conotoxin GVIA. 1
2
W. Y. Li et al.
Fig. 2. sAPP751 and 8BrcGMP attenuate the VSCC response to Ab. Sister culture were pre-treated 24 h with control vehicle(untreated: n 6; 174 ^ 14%), 1 nM sAPP751 (n 5: 118 ^ 8.7%; P , 0:01), 30 nM sAPP751(n 5: 106 ^ 6.5%; P , 0:01), or 10 ng/ml NGF(n 4: 157 ^ 21%; P . 0:05) in a defined culture medium (N2/DMEM; Gibco). Cells were pretreated with 500 mM 8-Brc GMP for 30 min (n 6., 125 ^ 21%; P , 0:01). The bath was then exchanged for the normal extracellular recording solution and cells were assessed for the Ab-induced VSCC response. Data are mean currents ^SD in response to 1 mM Ab plotted as function of control IBa.
Table 1.
Fig. 1. Ab enhances VSCC currents in cultured rat hippocampal neurons. (A) Current traces from a neuron illustrating changes in IBa, induced with application of 1 mM Ab. Currents evoked with a voltage step to 20 mV from a membrane holding potential of 60 mV. (B) Current voltage relationship of VSCC currents under control conditions (squares) and following application of 1 mM Ab (circles). Currents were activated with incrementally depolarizing voltage steps from a membrane holding potential of 60 mV.
Ab potentiated IBa across the voltage range tested without any obvious shift in channel activation kinetics as assessed by the voltage-step required to elicit the maximal current response. Neuronal VSCCs can be distinguished pharmcologically with respect to their selective inhibition by the “N” and “L” channel antagonists, v-conotoxin GVIA (v-Cgtx) and nifedipine (nif), respectively. Under control conditions, the reduction in peak IBa following application of 3 mM v-Cgtx or 10 mM nif gives an approximation of the relative expression of N and L channels activated with our experimental protocols in rat hippocampal neurons. Following co-application of 1 mM Ab with v-Cgtx or nif, IBa, displayed minimal increases in size, indicating that the VSCC population responsive to Ab is composed of at least the N and L channel subtypes (data not shown). To determine whether at least some of the calcium-stabilizing actions of sAPPs could be explained by an interference with the modulatory Ab response, hippocampal cultures were incubated with sAPP751 for 24 h in a defined culture medium, at which time VSCCs were tested for responsivity to Ab in the normal external recording solution (without sAPP751).
Treatment
IBa(% of control)
1 nM Sapp 10 nM sAPP 30 nM sAPP 100 nM sAPP
101 84 59 51
1 mM Ab 1 (M Ab 1 30 nM sAPP
166.7 157.5
SD
n
2.5 5.9 4.7 4.1
4 4 5 4
12.22 11.9
4 4
The data in Table 1 show that cells preincubated with either the defined culture medium alone, or with 10 ng/ml nerve growth factor (NGF), exhibited normal Ab-induced increases in IBa. NGF is another large endogenous neurotrophic factor that is reported not to protect against Ab toxicity. 13 In contrast, the relative enhancement of the VSCC response to Ab in cells from sister cultures incubated with 1 nM and 30 nM sAPP751 decreased in a dose dependent manner. Control IBa values did not vary across the different experimental conditions. There is now evidence that a cyclic GMP (cGMP)-dependent protein kinase may mediate the calcium regulation and neuroprotective actions of sAPPs. 1 To explore the potential involvement of cGMP, neurons were exposed to 500 mM 8-Br-cGMP, a membrane permeable analogue of cGMP, for 30 min in defined culture medium before washout and assessment of VSCC sensitivity to Ab in the normal extracellular recording solution was performed. Again, when compared with non-treated cells, pretreatment with 8-Br-cGMP resulted in a decrease in Ab potentiated IBa. The effectiveness of sAPP751 pre-exposure for uncoupling the VSCC response to Ab implies that sAPP751 is intervening at some point within the modulatory pathway that mediates the Ab response. Alternatively, sAPP751 could be suppressing the VSCC response by acting as Ca 21 channelblocker to effectively limit the number of VSCCs available to
Suppression of an amyloid b-peptidemediated calcium channel response by a secreted b-amyloid precursor protein
respond to Ab. We, therefore, examined the consequences of direct application of sAPP751 on intrinsic IBa. As shown in Table 1, direct application of 10, 30 and 100 nM sAPP751 did result in an acute depression of intrinsic currents (P1 , 0:01, P2 , 0:01, P3 , 0:01). However, this apparent channel block was observed at concentrations higher than those needed to depress the Ab-dependent VSCC response. For example, pre-treatment with 1 nM sAPP751 (Table 1, Fig. 2) removed approximately 80% of the modulatory VSCC response to Ab, whereas, IBa following direct application of 1 nM sAPP751 was unchanged (P 0:48). Furthermore, if sAPP751 was acting directly at the level of the Ca 21 channel, co-application of sAPP751 with Ab should produce a relative decrease in the apparent Ab-sensitive VSCC response. However, increases in mean IBa (as a function of control currents) to Ab alone (166.75%) or in the presence of 30 nM sAPP751 (157.5%) were equivalent. It has been proposed that cellular calcium regulating properties underly their neuroprotectant capabilities during ischemic challenge, glutamate excitotoxicity and Ab toxicity. 11 There is now evidence that an activation of K 1 channels, and the concomitant depression of neuronal excitability may be responsible for this calcium-stabilizing behavior. 5 Our findings suggest that sAPPs may also have neuroprotective actions that are specific for an Ab modulatory response at neuronal VSCCs. sAPP751 can interfere with a potentiation of VSCC currents mediated by the aggregated toxic Abs, by a mechanism that is distinct from Ca 21 channel block. Moreover, this response is mimicked by elevating levels of intracellular cGMP, a signal transduction event that has been
3
linked to the Ca 21 lowering activities of sAPPs. Given the evidence that the increases in IBa activated by Ab may also represent a signal transduction process, 18 the sAPP751 response described here may reflect a convergence between these two pathways.
EXPERIMENTAL PROCEDURES
Embryonic day 18 rat hippocampal cultures were prepared as reported previously 9. Whole-cell voltage-clamp recordings were conducted at room temperature using bath and pipette solutions designed to monitor VSCC currents in isolation of other voltagegated currents. The external recording solution consisted of 145 mM TEA-Cl2, 5.0 mM KCl, 10 mM BaC12, 1.0 mM MgCl2, 10 mM glucose, 10 mM HEPES (pH 7.4) and 1 mM teterodoxin (TTX). The patch pipette solution contained 160 mM CsMeSO4, 10 mM HEPES, 11 mM EGTA, 5 mM MgCl2, 0.5 mM Li-GTP, 5 mM Mg-ATP (pH 7.2). Prior to a recording session, pipette tip potentials were offset and corrections were made for the liquid junction potential between the pipette and bath solutions. VSCC currents (IBa) were activated with 50 ms duration voltage steps to 20 mV from a membrane holding potential of 260 mV. Currents were filtered (2 kHz), digitized (sample rate of 200 ms), subtracted for leak and capacitive components, and analyzed offline (Indec Systems). Measurements of amplitude were made at peak inward IBa, usually within 510 ms of the voltage-step onset. Synthetic Ab 140 (Bachern and RBI) was aged for 23 days in a 1-mM water stock solution at 378C. Immediately prior to recording, Ab was diluted to a final concentration of 1 mM into the external recording solution, then perfused directly on to individual neurons via a microperfusion system. Recombinant sAPP751 was obtained from culture supernatants of human embryonic kidney 293 cells stably transfected with APP751 human, as described elsewhere. 4,15,20
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Barger S. W., Fiscus R. R., Ruth P., Hofman F. and Mattson M. P. (1994) Role of cyclic GMP in the regulation of neuronal calcium and survival by secreted forms of b-amyloid precursor. J. Neurochem. 64, 2087–2095. Burdick D., Soreghan B., Kwon M., Kosmoski L., Knauer M., Henschen A., Yates L., Cotman C. and Glabe C. (1992) Assembly and aggregation properties of synthetic Alzheimer’s A4/b amyloid peptide analogs. J. Biol. Chem. 267, 546–554. Checler F. (1995) Processing of the b-amyloid precursor protein and its regulation in Alzheimer’s disease. J. Neurochem. 65, 1431–1441. Esch F. S., Keim P. S., Beattie E. C., Blacher R. M., Culwell A. K., Oltersdorf T., McClure D. and Ward P. J. (1990) Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 248, 1122–1124. Furukawa K., Barger S. W., Blalock E. M. and Mattson M. P. (1996) Activation of K 1 channels and suppression of neuronal activity by secreted bamyloid precursor protein. Nature 379, 7478. Goodman Y. and Mattson M. P. (1994) Secreted forms of b-amyloid precursor protein protect hippocampal neurons against amyloid b-peptide induced oxidative injury. Expl Neurol. 128, 112. Katzman R. (1986) Alzheimer’s disease. New Engl. J. Med. 314, 964–972. Knops J., Johnson-Wood K., Schenk D. B., Sinha S., Lieberburg I. and McConlogue L. (1991) Isolation of baculovirus secreted and full-length bamyloid precursor protein. J. biol. Chem. 266, 7285–7290. Li W. Y., Czilli D. L. and Simmons L. K. (1995) Neuronal membrane conductance activated by amyloid bpeptide: importance of peptide conformation. Brain Res. 682, 207–211. Lowery D. E., Pastenack J. M., Gonzales-De Whitt J. M., Zurcher-Neely P. A., Tomich C. S., Altman R. A., Fairbanks M. B., Heinrikson R. B. and Younkin S. G. (1991) Alzheimer’s amyloid precursor protein produced by recombinant baculovirus expression: proteolytic processing and protease inhibitory properties. J. biol. Chem. 266, 19,842–19,850. Mattson M. P., Cheng B., Culwell A. R., Esch F. S., Lieberburg I. and Rydel R. E. (1993) Evidence for excitoprotective and intraneuronal calciumregulating roles for secreted forms of the b-amyloid precursor protein. Neuron 10, 243–254. Mattson M. P., Cheng B., Davis D., Bryant K., Lieberburg I. and Rydel R. E. (1992) b Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12, 376–389. Mattson M. P., Tomaselli K. J. and Rydel R. E. (1993) Calcium-destabilizing and neurodegeneration effects of aggregated b-amyloid peptides are attenuated by basic FGF. Brain Res. 621, 35–49. Meziane H., Dodart J. C., Mathis C., Little S., Clemens L., Paul S. M. and Ungerer A. (1998) Memory enhancing effects of secreted forms of the bamyloid precursor protein in normal and amnestic mice. Proc. natn. Acad. Sci U.S.A. 95, 12,683–12,688. Oltersdorf T., Fritz L. C., Schenk D. B., Lieberburg L., Johnson-Wood K. L., Beattie E. C., Ward P. J., Blacher R. W., Dovey H. F. and Sinha S. (1989) The secreted form of the Alzheimer’s amyloid precursor protein with the Kunitz domain is protease nexin-II. Nature 341, 144–147. Pike C. L., Burdick D., Walencewicz A. J., Glabe C. G. and Cotman C. W. (1993) Neurodegeneration induced by b-amyloid peptides in vitro: The role of peptide assembly state. J. Neurosci. 13, 1676–1687. Selkoe D. J. (1994) Alzheimer’s disease: a central role for amyloid. J. Neuropath. exp. Neurol. 53, 438–447. Simmons L. K., Li W. Y. and Czilli D. L. (1995) A G Protein-dependent mechanism is involved in the potentiation of neuronal calcium channel currents mediated by amyloid peptide. 25 th Annual Meeting for the Society of Neuroscience Abstracts 21, 478. Simmons L. K., May P. K., Tomaselli K. L., Rydel R. E., Fuson K. S., Brigham E. E., Wright S., Lieberburg L., Becker G., Brems D. N. et al. (1994) Secondary structure of amyloid b peptide correlates with neurotoxic activity in vitro. . Molec. Pharmac. 45, 373–379.
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20. Van Nostrand W. E., Wagner S., Suzuki M., Choi B. R., Farrow J. S., Geddes L. W., Cotman C. W. and Cunningham D. D. (1989) Protease nexin-II, a potent antichymotrypsin, shows identity to amyloid beta-protein precursor. Nature 341, 546–549. 21. Yankner B. A., Duffy L. K. and Kirschner D. A. (1990) Neurotrophic and neurotoxic effects of amyloid b protein: reversal by tachykinin neuropeptides. Science 250, 79–282. (Accepted 13 September 1999)
Pergamon PII: S0306-4522(99)00479-0
q 1999 Elsevier Science Ltd. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience SUPPRESSION OF AN AMYLOID b PEPTIDE-MEDIATED CALCIUM CHANNEL RESPONSE BY A SECRETED b-AMYLOID PRECURSOR PROTEIN W. Y. LI,* J. P. BUTLER, J. E. HALE, D. B. MCCLURE, S. P. LITTLE, D. L. CZILLI and L. K. SIMMONS Neuroscience Discovery Research and Research Technologies and Product Development, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA Key words: Alzheimer’s Disease, b-amyloid protein, b-amyloid precursor protein, calcium channel, Ab toxicity neuroprotection.
Secreted isoforms of the b-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid b peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid b peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid b peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted b-amyloid precursor protein on the calcium channel response to amyloid b peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with b-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid b peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by b-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Coapplication of b-amyloid precursor protein 751 with amyloid b peptide did not alter calcium channel response to amyloid b peptide. Taken together, these findings suggest that secreted b-amyloid precursor proteins can suppress a calcium channel response to amyloid b peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid b toxicity. q 1999 IBRO. Published by Elsevier Science Ltd.
that precludes the formation of Ab leads to the constitutive secretion of N-terminal isoforms (sAPPs). 3 The secreted forms of APP have been shown to have memory enhancing effects in normal and amnestic mice. 14 These sAPPs can reduce neuronal cytosolic Ca 21 levels and have demonstrated neuroprotective efficacy against a variety of insults. 11 This includes neuroprotection against the increases in intracellular Ca 21 believed to be a primary cause of Ab toxicity. 6 Although the mechanism(s) underlying the homeostatically disruptive actions of Ab are not fully understood, we described in a previous study an Ab-independent Ca 21 channel response that may be involved in Ab toxicity. 18 Using whole-cell voltage-clamp protocols we demonstrated that toxic Ab acutely enhances voltage-sensitive calcium channel (VSCC) currents in cultured rat hippocampal neurons via a process that is GTP binding protein (G protein)-dependent and pertussis toxin (PTX) sensitive. The present study was designed to examine whether the neuroprotective efficacy of sAPPs can be explained, at least in part, by a suppression of this modulatory VSCC response to Ab. Ab aggregation is an important determinant of neurotoxic potency in vitro. 2,16,19 One means of accelerating the formation of toxic aggregates is to incubate the peptide in concentrated aqueous solutions for 1–4 days prior to use. 19 This process is known as aging. The aging paradigms that promote the formation of toxic Ab aggregates also result in peptides that are capable of acutely enhancing VSCC currents. 18 Accordingly, similar peptide aging protocols were used here to obtain Ab samples active at VSCCs. Whole-cell recordings were conducted in cultured rat hippocampal neurons, with 10 mM Ba 21 in the bath as charge carrier. The raw data traces in Fig. 1A illustrate the VSCC currents (IBa) activated with voltage steps to 20 mV from a membrane holding potential of 60 mV under control conditions, and following application of 1 mM Ab. The maximal increase in current size stabilized within 5–6 min of exposure to Ab and was irreversible following washout in control external solution for up to 30 min. This Ab-mediated VSCC modulatory response was observed in 33 of 37 (187 ^ 86.8%; P 0:0000089) cells in the absence of changes in cell membrane leak conductance. The current–voltage relationship for another neuron under control conditions (squares) and in response to 1 mM Ab (circles) is shown in Fig. 1B.
Alzheimer’s disease (AD) is a neurodegenerative disorder of the elderly that is characterized histopathologically by the deposition of amyloid in brain neuritic plaques. 7 A component of neuritic plaques known as amyloid b peptide (Ab) is toxic to neurons in cell culture, 12,21 and is believed to have a prominent role in AD neurodegeneration. 17 Ab is derived from the proteolytic processing of a widely expressed family of membrane bound b-amyloid precursor proteins (APP). 8,10 An alternative processing of pathway for APP *To whom correspondence should be addressed at: Neuroscience Discovery Research Lilly Research Labs Eli Lilly & Company Lilly Corporate Center Indianapolis, IN 46285, USA. Tel.: 11 317 2763871; Fax: 11 317 2765546. Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b protein; APP, bamyloid precursor protein; cGMP, cyclic GMP; sAPP, secreted forms of APP; VSCC, voltage sensitive calcium channel; NGF, nerve growth factor; nif, nifedipine; v-Cgtx, v-conotoxin GVIA. 1
2
W. Y. Li et al.
Fig. 2. sAPP751 and 8BrcGMP attenuate the VSCC response to Ab. Sister culture were pre-treated 24 h with control vehicle(untreated: n 6; 174 ^ 14%), 1 nM sAPP751 (n 5: 118 ^ 8.7%; P , 0:01), 30 nM sAPP751(n 5: 106 ^ 6.5%; P , 0:01), or 10 ng/ml NGF(n 4: 157 ^ 21%; P . 0:05) in a defined culture medium (N2/DMEM; Gibco). Cells were pretreated with 500 mM 8-Brc GMP for 30 min (n 6., 125 ^ 21%; P , 0:01). The bath was then exchanged for the normal extracellular recording solution and cells were assessed for the Ab-induced VSCC response. Data are mean currents ^SD in response to 1 mM Ab plotted as function of control IBa.
Table 1.
Fig. 1. Ab enhances VSCC currents in cultured rat hippocampal neurons. (A) Current traces from a neuron illustrating changes in IBa, induced with application of 1 mM Ab. Currents evoked with a voltage step to 20 mV from a membrane holding potential of 60 mV. (B) Current voltage relationship of VSCC currents under control conditions (squares) and following application of 1 mM Ab (circles). Currents were activated with incrementally depolarizing voltage steps from a membrane holding potential of 60 mV.
Ab potentiated IBa across the voltage range tested without any obvious shift in channel activation kinetics as assessed by the voltage-step required to elicit the maximal current response. Neuronal VSCCs can be distinguished pharmcologically with respect to their selective inhibition by the “N” and “L” channel antagonists, v-conotoxin GVIA (v-Cgtx) and nifedipine (nif), respectively. Under control conditions, the reduction in peak IBa following application of 3 mM v-Cgtx or 10 mM nif gives an approximation of the relative expression of N and L channels activated with our experimental protocols in rat hippocampal neurons. Following co-application of 1 mM Ab with v-Cgtx or nif, IBa, displayed minimal increases in size, indicating that the VSCC population responsive to Ab is composed of at least the N and L channel subtypes (data not shown). To determine whether at least some of the calcium-stabilizing actions of sAPPs could be explained by an interference with the modulatory Ab response, hippocampal cultures were incubated with sAPP751 for 24 h in a defined culture medium, at which time VSCCs were tested for responsivity to Ab in the normal external recording solution (without sAPP751).
Treatment
IBa(% of control)
1 nM Sapp 10 nM sAPP 30 nM sAPP 100 nM sAPP
101 84 59 51
1 mM Ab 1 (M Ab 1 30 nM sAPP
166.7 157.5
SD
n
2.5 5.9 4.7 4.1
4 4 5 4
12.22 11.9
4 4
The data in Table 1 show that cells preincubated with either the defined culture medium alone, or with 10 ng/ml nerve growth factor (NGF), exhibited normal Ab-induced increases in IBa. NGF is another large endogenous neurotrophic factor that is reported not to protect against Ab toxicity. 13 In contrast, the relative enhancement of the VSCC response to Ab in cells from sister cultures incubated with 1 nM and 30 nM sAPP751 decreased in a dose dependent manner. Control IBa values did not vary across the different experimental conditions. There is now evidence that a cyclic GMP (cGMP)-dependent protein kinase may mediate the calcium regulation and neuroprotective actions of sAPPs. 1 To explore the potential involvement of cGMP, neurons were exposed to 500 mM 8-Br-cGMP, a membrane permeable analogue of cGMP, for 30 min in defined culture medium before washout and assessment of VSCC sensitivity to Ab in the normal extracellular recording solution was performed. Again, when compared with non-treated cells, pretreatment with 8-Br-cGMP resulted in a decrease in Ab potentiated IBa. The effectiveness of sAPP751 pre-exposure for uncoupling the VSCC response to Ab implies that sAPP751 is intervening at some point within the modulatory pathway that mediates the Ab response. Alternatively, sAPP751 could be suppressing the VSCC response by acting as Ca 21 channelblocker to effectively limit the number of VSCCs available to
Suppression of an amyloid b-peptidemediated calcium channel response by a secreted b-amyloid precursor protein
respond to Ab. We, therefore, examined the consequences of direct application of sAPP751 on intrinsic IBa. As shown in Table 1, direct application of 10, 30 and 100 nM sAPP751 did result in an acute depression of intrinsic currents (P1 , 0:01, P2 , 0:01, P3 , 0:01). However, this apparent channel block was observed at concentrations higher than those needed to depress the Ab-dependent VSCC response. For example, pre-treatment with 1 nM sAPP751 (Table 1, Fig. 2) removed approximately 80% of the modulatory VSCC response to Ab, whereas, IBa following direct application of 1 nM sAPP751 was unchanged (P 0:48). Furthermore, if sAPP751 was acting directly at the level of the Ca 21 channel, co-application of sAPP751 with Ab should produce a relative decrease in the apparent Ab-sensitive VSCC response. However, increases in mean IBa (as a function of control currents) to Ab alone (166.75%) or in the presence of 30 nM sAPP751 (157.5%) were equivalent. It has been proposed that cellular calcium regulating properties underly their neuroprotectant capabilities during ischemic challenge, glutamate excitotoxicity and Ab toxicity. 11 There is now evidence that an activation of K 1 channels, and the concomitant depression of neuronal excitability may be responsible for this calcium-stabilizing behavior. 5 Our findings suggest that sAPPs may also have neuroprotective actions that are specific for an Ab modulatory response at neuronal VSCCs. sAPP751 can interfere with a potentiation of VSCC currents mediated by the aggregated toxic Abs, by a mechanism that is distinct from Ca 21 channel block. Moreover, this response is mimicked by elevating levels of intracellular cGMP, a signal transduction event that has been
3
linked to the Ca 21 lowering activities of sAPPs. Given the evidence that the increases in IBa activated by Ab may also represent a signal transduction process, 18 the sAPP751 response described here may reflect a convergence between these two pathways.
EXPERIMENTAL PROCEDURES
Embryonic day 18 rat hippocampal cultures were prepared as reported previously 9. Whole-cell voltage-clamp recordings were conducted at room temperature using bath and pipette solutions designed to monitor VSCC currents in isolation of other voltagegated currents. The external recording solution consisted of 145 mM TEA-Cl2, 5.0 mM KCl, 10 mM BaC12, 1.0 mM MgCl2, 10 mM glucose, 10 mM HEPES (pH 7.4) and 1 mM teterodoxin (TTX). The patch pipette solution contained 160 mM CsMeSO4, 10 mM HEPES, 11 mM EGTA, 5 mM MgCl2, 0.5 mM Li-GTP, 5 mM Mg-ATP (pH 7.2). Prior to a recording session, pipette tip potentials were offset and corrections were made for the liquid junction potential between the pipette and bath solutions. VSCC currents (IBa) were activated with 50 ms duration voltage steps to 20 mV from a membrane holding potential of 260 mV. Currents were filtered (2 kHz), digitized (sample rate of 200 ms), subtracted for leak and capacitive components, and analyzed offline (Indec Systems). Measurements of amplitude were made at peak inward IBa, usually within 510 ms of the voltage-step onset. Synthetic Ab 140 (Bachern and RBI) was aged for 23 days in a 1-mM water stock solution at 378C. Immediately prior to recording, Ab was diluted to a final concentration of 1 mM into the external recording solution, then perfused directly on to individual neurons via a microperfusion system. Recombinant sAPP751 was obtained from culture supernatants of human embryonic kidney 293 cells stably transfected with APP751 human, as described elsewhere. 4,15,20
REFERENCES
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