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• W2019260419 abstract "Neuronal excitation is required for normal brain function including processes of learning and memory, yet if this process becomes dysregulated there is reduced neurotransmission and possibly death through excitotoxicity. Nicotine, through interaction with neuronal nicotinic acetylcholine receptors, possesses the ability to modulate neurotransmitter systems through numerous mechanisms that define this critical balance. We examined the modulatory role of nicotine in primary mixed cortical neuronal-glial cultures on activity-dependent caspase cleavage of a glutamate receptor, GluR1. We find that GluR1, but not GluR2 or GluR3, is a substrate for agonist (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-initiated rapid proteolytic cleavage at aspartic acid 865 through the activation of caspase 8-like activity that is independent of membrane fusion and is not coincident with apoptosis. Dose-dependent nicotine preconditioning for 24 h antagonizes agonist-initiated caspase cleavage of GluR1 through a mechanism that is coincident with desensitization of both nAChRα4β2 and nAChRα7 receptors and the delayed activation of a caspase 8-like activity. The modulation of GluR1 agonist-initiated caspase-mediated cleavage by nicotine preconditioning offers a novel insight into how this agent can impart its numerous effects on the nervous system. Neuronal excitation is required for normal brain function including processes of learning and memory, yet if this process becomes dysregulated there is reduced neurotransmission and possibly death through excitotoxicity. Nicotine, through interaction with neuronal nicotinic acetylcholine receptors, possesses the ability to modulate neurotransmitter systems through numerous mechanisms that define this critical balance. We examined the modulatory role of nicotine in primary mixed cortical neuronal-glial cultures on activity-dependent caspase cleavage of a glutamate receptor, GluR1. We find that GluR1, but not GluR2 or GluR3, is a substrate for agonist (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-initiated rapid proteolytic cleavage at aspartic acid 865 through the activation of caspase 8-like activity that is independent of membrane fusion and is not coincident with apoptosis. Dose-dependent nicotine preconditioning for 24 h antagonizes agonist-initiated caspase cleavage of GluR1 through a mechanism that is coincident with desensitization of both nAChRα4β2 and nAChRα7 receptors and the delayed activation of a caspase 8-like activity. The modulation of GluR1 agonist-initiated caspase-mediated cleavage by nicotine preconditioning offers a novel insight into how this agent can impart its numerous effects on the nervous system. The dynamic regulation of the expression of ionotropic glutamate-activated receptors (GluR), 1The abbreviations used are: GluRglutamate receptorEaenergy of activationGABAγ-aminobutyric acidFMKfluoromethylketoneNMDAN-methyl-d-aspartic acidAMPAα-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acidCspcaspaseChaps3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidnAChRα3 and nAChRβ4neuronal AChRα3 and AChRβ4 subunits 1The abbreviations used are: GluRglutamate receptorEaenergy of activationGABAγ-aminobutyric acidFMKfluoromethylketoneNMDAN-methyl-d-aspartic acidAMPAα-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acidCspcaspaseChaps3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidnAChRα3 and nAChRβ4neuronal AChRα3 and AChRβ4 subunits the principal fast excitatory neurotransmitter receptors in the brain, is vital to the maintenance of effective neurotransmission. Disruption of this regulation can have severe pathophysiological consequences including neuronal death through excitotoxicity, as associated with stroke, trauma, and numerous severe neurodegenerative disorders (1.Choi D.W. Ann. N. Y. Acad. Sci. 1994; 747: 162-171Crossref PubMed Scopus (305) Google Scholar, 2.Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). GluRs are assembled from multiple cDNA products to form receptors of distinct function, which fall into three pharmacologically defined groups (2.Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). Although the activation of the N-methyl-d-aspartic acid (NMDA) receptor subclass is central to the establishment of long-term potentiation, and its sustained activation appears responsible for imparting the majority of excitotoxicity (2.Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, 3.Sucher N.J. Awobuluyi M. Choi Y.B. Lipton S.A. Trends Pharmacol. Sci. 1996; 17: 348-355Abstract Full Text PDF PubMed Scopus (253) Google Scholar), NMDA channel opening also requires local depolarization, which is often provided through coincident activation of the non-NMDA receptors, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors (GluR1–4), and kainic acid receptors (GluR5–7). This study examines AMPA-GluRs, in which channel permeability to calcium is dependent upon subunit composition. For example, inclusion of GluR2 imparts calcium impermeability, whereas receptors composed of GluR 1, 3, and/or 4 permit calcium entry. Consequently, altering the ratio of GluRs with varied subunit composition or modifying their subcellular location contributes significantly to the temporal and spatial regulation of NMDA receptors and glutamate neurotransmission. glutamate receptor energy of activation γ-aminobutyric acid fluoromethylketone N-methyl-d-aspartic acid α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid caspase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid neuronal AChRα3 and AChRβ4 subunits glutamate receptor energy of activation γ-aminobutyric acid fluoromethylketone N-methyl-d-aspartic acid α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid caspase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid neuronal AChRα3 and AChRβ4 subunits Another ligand-activated ionotropic neurotransmitter system that can contribute to modifying the function of GluRs is the neuronal nicotinic acetylcholine receptors (nAChR). These fast-ionotropic ligand-activated channels (4.MacDermott A.B. Role L.W. Siegelbaum S.A. Annu. Rev. Neurosci. 1999; 22: 443-485Crossref PubMed Scopus (497) Google Scholar, 5.Role L.W. Berg D.K. Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar, 6.Lindstrom J. Ion Channels. 1996; 4: 377-450Crossref PubMed Scopus (262) Google Scholar) are assembled from the products of cDNAs encoding at least six α-like subunits (α2–α7) and three β-like subunits (β2–β4). The expression of two nAChR subtypes is prevalent in the mammalian brain. First, receptors composed of nAChRα4 and nAChRβ2 subunits are distinguished by high-affinity [3H]nicotine or [3H]cytisine binding (7.Pabreza L.A. Dhawan S. Kellar K.J. Mol. Pharmacol. 1991; 39: 9-12PubMed Google Scholar). It is this receptor subclass in which up-regulation occurs in response to chronic nicotine administration (8.Flores C.M. Rogers S.W. Pabreza L.A. Wolfe B.B. Kellar K.J. Mol. Pharmacol. 1992; 41: 31-37PubMed Google Scholar), as in smoking, and in which expression correlates with the establishment of tolerance, a correlate of addiction (9.Crawley J.N. Belknap J.K. Collins A. Crabbe J.C. Frankel W. Henderson N. Hitzemann R.J. Maxson S.C. Miner L.L. Silva A.J. Wehner J.M. Wynshaw-Boris A. Paylor R. Psychopharmacology (Berl.). 1997; 132: 107-124Crossref PubMed Scopus (1186) Google Scholar). A second nAChR subtype composed of nAChRα7 subunits binds α-bungarotoxin with high affinity and rapidly desensitizes to nicotine. However, while open, this receptor exhibits an unusually large Ca+2:Na+permeability ratio of ∼10:1 by comparison with the 3:1 ratio for NMDA receptors (4.MacDermott A.B. Role L.W. Siegelbaum S.A. Annu. Rev. Neurosci. 1999; 22: 443-485Crossref PubMed Scopus (497) Google Scholar, 6.Lindstrom J. Ion Channels. 1996; 4: 377-450Crossref PubMed Scopus (262) Google Scholar). This can have significant consequences on subcellular processes including signal transduction and the release of signaling molecules including arachidonic acid (10.Vijayaraghavan S. Huang B. Blumenthal E.M. Berg D.K. J. Neurosci. 1995; 15: 3679-3687Crossref PubMed Google Scholar). Further, nicotine has been suggested to modulate the regulation of caspase (Csp) activation, including Csp8 function (11.Garrido R. Mattson M.P. Hennig B. Toborek M. J. Neurochem. 2001; 76: 1395-1403Crossref PubMed Scopus (99) Google Scholar). However, due to the mechanisms of desensitization and inactivation of nAChRs, even if the receptor number is increased and there is abundant agonist (e.g. nicotine) present, signaling through this receptor system may actually be decreased. Therefore nicotine imparts multiple effects on neuronal function ranging from cognitive enhancement to neuroprotection against toxins (12.Maggio R. Riva M. Vaglini F. Fornai F. Molteni R. Armogida M. Racagni G. Corsini G.U. J. Neurochem. 1998; 71: 2439-2446Crossref PubMed Scopus (193) Google Scholar, 13.Gould T.J. Wehner J.M. Behav. Brain Res. 1999; 102: 31-39Crossref PubMed Scopus (138) Google Scholar, 14.Meyer E.M. Tay E.T. Zoltewicz J.A. Meyers C. King M.A. Papke R.L. De Fiebre C.M. J. Pharmacol. Exp. Ther. 1998; 284: 1026-1032PubMed Google Scholar, 15.Carlson N.G. Bacchi A. Rogers S.W. Gahring L.C. J. Neurobiol. 1998; 35: 29-36Crossref PubMed Scopus (93) Google Scholar, 16.Carlson N.G. Wieggel W.A. Chen J. Bacchi A. Rogers S.W. Gahring L.C. J. Immunol. 1999; 163: 3963-3968PubMed Google Scholar) to its well known attribute, craving and addiction, through affecting multiple mechanisms that combine properties of both imparting receptor activation and enhancing long-term desensitization. Neurons use a variety of mechanisms to control AMPA-class GluRs expression. For example, during periods of sustained synaptic activity GluRs, in particular GluR1, are observed to change in subcellular distribution as reflected by the accumulation of this subunit at some sites of activity and by the redistribution or possibly degradation from others (17.Carroll R.C. Lissin D.V. von Zastrow M. Nicoll R.A. Malenka R.C. Nat. Neurosci. 1999; 2: 454-460Crossref PubMed Scopus (378) Google Scholar, 18.Lissin D.V. Carroll R.C. Nicoll R.A. Malenka R.C. von Zastrow M. J. Neurosci. 1999; 19: 1263-1272Crossref PubMed Google Scholar, 19.Shi S.H. Hayashi Y. Petralia R.S. Zaman S.H. Wenthold R.J. Svoboda K. Malinow R. Science. 1999; 284: 1811-1816Crossref PubMed Scopus (1045) Google Scholar). The mechanisms implicated in subcellular redistribution of GluRs are complex and include altered phosphorylation and rates of endocytosis or exocytosis and interaction with cytoskeleton-binding proteins including 4.1N and SAP97 (20.Swope S.L. Moss S.I. Raymond L.A. Huganir R.L. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 49-78Crossref PubMed Google Scholar, 21.Shen L. Liang F. Walensky L.D. Huganir R.L. J. Neurosci. 2000; 20: 7932-7940Crossref PubMed Google Scholar, 22.Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 23.Walensky L.D. Blackshaw S. Liao D. Watkins C.C. Weier H.U. Parra M. Huganir R.L. Conboy J.G. Mohandas N. Snyder S.H. J. Neurosci. 1999; 19: 6457-6467Crossref PubMed Google Scholar, 24.Sans N. Racca C. Petralia R.S. Wang Y.X. McCallum J. Wenthold R.J. J. Neurosci. 2001; 21: 7506-7516Crossref PubMed Google Scholar). Notably, most of the sites that are important for regulation through these mechanisms, including proteolysis (e.g. Ref. 25.Bi X. Chen J. Baudry M. Brain Res. 1998; 781: 355-357Crossref PubMed Scopus (34) Google Scholar), are located in the relatively short C-terminal intracellular region of the receptor subunit. The most rapid way to regulate the concentration of cellular proteins is through selective proteolysis (26.Schimke R.T. Doyle D. Annu. Rev. Biochem. 1970; 39: 929-976Crossref PubMed Scopus (405) Google Scholar). AMPA-GluRs are subject to this mechanism, particularly in the presence of elevated free intracellular calcium, which can activate calpains, which have numerous cellular substrates including GluR1 (25.Bi X. Chen J. Baudry M. Brain Res. 1998; 781: 355-357Crossref PubMed Scopus (34) Google Scholar). However, the limited proteolytic cleavage of GluR1 may include other proteases. Recently, members of the cysteine protease family, caspases, have been reported (27.Glazner G.W. Chan S.L. Lu C. Mattson M.P. J. Neurosci. 2000; 20: 3641-3649Crossref PubMed Google Scholar) to cleave certain GluR subunits, especially GluR4, following trophic factor withdrawal or upon induction of cell death through apoptosis. Although the activation of caspase cascades is most commonly associated with cell death, under more restricted conditions certain caspases might also perform limited substrate cleavage, which has the regulatory importance of contributing to proper cellular function. For example, the conditional activation of Csp1 converts pro-interleukin 1β to the active cytokine that is released from the cell (28.Villa P. Kaufmann S.H. Earnshaw W.C. Trends Biochem. Sci. 1997; 22: 388-393Abstract Full Text PDF PubMed Scopus (549) Google Scholar). Therefore, a less recognized role for caspases might be in regulating processes related to normal neuronal responses and function. In this study we report that GluR1 is a substrate for AMPA-initiated activity-dependent limited caspase cleavage and demonstrate that nicotine preconditioning of primary mixed cortical neuronal-glial cultures modulates this proteolytic activation. Site-directed mutagenesis and in vitro assays demonstrate that GluR1 is cleaved through a Csp8-like protease at residue 865 in the distal C terminus. Preconditioning of cultures with nicotine alters the activation of the Csp8-like proteolytic cascade as measured using cell-permeable fluorescent substrates, and this correlates with reduced AMPA-initiated caspase cleavage of GluR1. The use of various agonists and antagonists of nAChRs suggest that this mechanism of reducing Csp8 activation requires the coincident desensitization and/or inactivation of both nAChRα7 and nAChRα4β2 receptor subtypes. The modulation by nicotine of activity-dependent cleavage of GluR1 has several implications regarding the action of this compound on the molecular processes underlying GluR excitatory neurotransmission and the modulation of neurological disease. All reagents were obtained from RBI/Sigma. Drugs were dissolved in minimum Eagle's medium (MEM) or Me2SO at 100–1000×. Caspase inhibitors and substrates were obtained from Calbiochem or Enzyme Systems Products. Recombinant caspases 3 and 8 and the calpain inhibitor PD150-606 were obtained from Alexis Biochemicals or Calbiochem. Murine primary cortical cultures composed of mixed neuronal and glial cells were prepared from E15 CD1 mice (Charles River) and maintained for 13–15 days as described previously (15.Carlson N.G. Bacchi A. Rogers S.W. Gahring L.C. J. Neurobiol. 1998; 35: 29-36Crossref PubMed Scopus (93) Google Scholar, 16.Carlson N.G. Wieggel W.A. Chen J. Bacchi A. Rogers S.W. Gahring L.C. J. Immunol. 1999; 163: 3963-3968PubMed Google Scholar). All experiments were conducted at least 24 h after cells were fed. For experiments using varied temperature, the medium was buffered with 10 mm HEPES, the culture dishes wrapped in Parafilm, and the dishes placed in contact with water heated to the indicated temperature using circulating water baths. Cultures were equilibrated for 10–15 min before the addition of drugs. Degradation and Arrhenius activation were calculated as described elsewhere (29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar). Western blot analyses were done as described previously (30.Gahring L. Carlson N.G. Meyer E.L. Rogers S.W. J. Immunol. 2001; 166: 1433-1438Crossref PubMed Scopus (108) Google Scholar). Briefly, neurons were washed with phosphate-buffered saline and then lysed and harvested by scraping in a cold protease mixture (4 mm phenylmethylsulfonyl fluoride and 10 mm EDTA and 10 mmbenzamidine) prepared in water. SDS-PAGE loading buffer containing dithiothreitol was added to this mixture immediately, and the cells were further scraped into a microcentrifuge tube and boiled in a dry heating block for 10 min. Upon gel fractionation by SDS-PAGE, proteins were transferred to nitrocellulose utilizing a semi-dry blot apparatus, blocked in phosphate-buffered saline with 5% dry milk and 0.05% Tween 20, and incubated in primary antibodies overnight at 4 °C. Antibodies were to the C terminus of GluR1 (Chemicon), E11 (GluR1 (31.Rogers S.W. Hughes T.E. Hollmann M. Gasic G.P. Deneris E.S. Heinemann S. J. Neurosci. 1991; 11: 2713-2724Crossref PubMed Google Scholar), 3A11 (GluR2; Chemicon), and 2F5 (GluR3 (30.Gahring L. Carlson N.G. Meyer E.L. Rogers S.W. J. Immunol. 2001; 166: 1433-1438Crossref PubMed Scopus (108) Google Scholar)). The blots were washed in phosphate-buffered saline/Tween 20 and incubated in the presence of peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and detected on film using the Enhanced Chemiluminescence Kit (Amersham Life Sciences, Inc.). Films were scanned, and relative band intensities were determined by densitometry. Immunocytochemistry was performed as described previously (31.Rogers S.W. Hughes T.E. Hollmann M. Gasic G.P. Deneris E.S. Heinemann S. J. Neurosci. 1991; 11: 2713-2724Crossref PubMed Google Scholar). Mutagenesis to convert GluR1 aspartic acid 865 to alanine (D865A) was performed using PCR site-directed mutagenesis as described elsewhere (30.Gahring L. Carlson N.G. Meyer E.L. Rogers S.W. J. Immunol. 2001; 166: 1433-1438Crossref PubMed Scopus (108) Google Scholar) with modifications appropriate to GluR1. Constructs were confirmed by automated sequencing (University of Utah DNA sequencing core facility). The CalPhos transfection kit from CLONTECH was used according to manufacturer's instructions to introduce expression plasmid cDNAs (2 μg of GluR (GluR1:pcDNA1/AMP; see Ref. 31.Rogers S.W. Hughes T.E. Hollmann M. Gasic G.P. Deneris E.S. Heinemann S. J. Neurosci. 1991; 11: 2713-2724Crossref PubMed Google Scholar) combined with 1 μg of green fluorescent protein cDNA (GFP:pcDNA1/AMP)) prepared using the Qiagen Maxi-kit into HEK293 cells (ATCC). Cells were used at 24–48 h post-transfection. To measure caspase cleavage of GluR1 or GluR3 in vitro, HEK cells were transfected with the respective GluR cDNA and 48 h later were washed with phosphate-buffered saline and lysed in cold buffer composed of 50 mm HEPES, pH 7.3, 50 mm NaCl, 10 mmEDTA, 10 mm dithiothreitol, 0.1% Chaps, 1 mmphenylmethylsulfonyl fluoride. The cells were subjected to Dounce homogenization and cleared by centrifugation. To each replicate 100-μl sample of cell lysate was added a 25-μl aliquot of either recombinant Csp8 or Csp3 (25 units/assay), respectively. The tube was sealed and placed at 37 °C for the period indicated, whereupon a 10-μl sample was removed and mixed with 10 μl of SDS-sample buffer, boiled for 5 min, and fractionated by SDS-PAGE. For microscopic visualization of caspase activation in cultured neurons, cells were loaded with cell-permeable Csp8 peptide substrate (Z-IETD-AFC), which fluoresces blue upon cleavage. Cultures were washed with Hanks' solution, incubated in the presence of Hanks' solution supplemented with 10 mm HEPES, and supplemented to 10 μmwith the cell-permeable caspase-substrate peptides indicated (stock solution of 1:1000 dissolved in Me2SO) for 30 min at 37 °C. Cells were washed again, AMPA (100 μm) or kainic acid (100 μm) was added, and at the desired times thereafter (optimized in trial experiments) fluorescence was automatically recorded at 1-min intervals. The appearance of fluorescence in individually identified neuronal cells was quantitated using Image Pro-Plus software. Activity-dependent subcellular redistribution of GluR1 occurs following the exposure of neurons to glutamate receptor agonists and antagonists (17.Carroll R.C. Lissin D.V. von Zastrow M. Nicoll R.A. Malenka R.C. Nat. Neurosci. 1999; 2: 454-460Crossref PubMed Scopus (378) Google Scholar, 18.Lissin D.V. Carroll R.C. Nicoll R.A. Malenka R.C. von Zastrow M. J. Neurosci. 1999; 19: 1263-1272Crossref PubMed Google Scholar). Consistent with these findings, the addition of a nonlethal, nonapoptotic (15.Carlson N.G. Bacchi A. Rogers S.W. Gahring L.C. J. Neurobiol. 1998; 35: 29-36Crossref PubMed Scopus (93) Google Scholar, 16.Carlson N.G. Wieggel W.A. Chen J. Bacchi A. Rogers S.W. Gahring L.C. J. Immunol. 1999; 163: 3963-3968PubMed Google Scholar), dose of AMPA (100 μm) or kainic acid (100 μm, not shown) for 1 h to our murine primary cortical neuronal-glial cultures was accompanied by the rapid loss of GluR1 C-terminal immunoreactivity as measured by Western blot analysis (Fig. 1A). Immunoreactivity to GluR2 or GluR3 from the same culture samples was unaffected (Fig. 1A). GluR1 degradation was inhibited by co-application of CNQX but was unaffected by the presence of tetrodotoxin, nifedipine, or the calpain inhibitor, PD 150–606 (not shown). Although degradation was complete in all experiments by ∼90 min, some GluR1 remained suggesting that a pool of this subunit protein was unaffected. This pool is likely to be intracellular because parallel immunocytochemical examination (using the same anti-C-terminal GluR1 antibody) of cultures treated with AMPA for 1 h revealed a change from normally diffuse immunoreactivity to a punctate distribution suggestive of an intracellular-endosomal pattern (Fig. 1B). Examination of GluR1 degradation using an antibody prepared to the extracellular domain (24.Sans N. Racca C. Petralia R.S. Wang Y.X. McCallum J. Wenthold R.J. J. Neurosci. 2001; 21: 7506-7516Crossref PubMed Google Scholar) failed to reveal an equivalent change in GluR1 expression over the same time period, albeit a change of ∼1 kDa in migration on gels was observed consistent with removal of the C-terminal region (not shown). Because the C terminus of GluR1 harbors numerous sequences important to the subcellular distribution and function of this receptor (17.Carroll R.C. Lissin D.V. von Zastrow M. Nicoll R.A. Malenka R.C. Nat. Neurosci. 1999; 2: 454-460Crossref PubMed Scopus (378) Google Scholar, 19.Shi S.H. Hayashi Y. Petralia R.S. Zaman S.H. Wenthold R.J. Svoboda K. Malinow R. Science. 1999; 284: 1811-1816Crossref PubMed Scopus (1045) Google Scholar, 32.Ruberti F. Dotti C.G. J. Neurosci. 2000; 20: RC78Crossref PubMed Google Scholar), we examined further the nature of this presumed proteolytic event. Varying temperature can be used to measure several parameters pertaining to cellular and proteolytic mechanisms. Endocytosis and lysosomal degradation of proteins are particularly sensitive to reduced temperatures because membranes fail to fuse as temperatures go below 16 °C (e.g. see Ref. 29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar). This has the effect of producing a dramatic and steep reduction in the degradation rate as delivery systems stop working at lower temperatures. Other proteolytic mechanisms that do not require membrane fusion, such as the ATP-ubiquitin-dependent proteasome and other cytoplasmic or extracellular proteases, are also affected by reduced temperature. In this case, however, the protease enzymatic rate is slowed according to its Q10, a constant that reflects the effect of a 10 °C change on the enzymatic function. We examined these parameters by measuring the rate of agonist-dependent proteolysis of GluR1 at 37, 26, 16, and 6 °C, respectively (Fig. 1C). An average of three experiments revealed that the rate of AMPA-initiated GluR1 degradation decreased ∼5-fold over the 31 °C temperature range for an average Q10 of 1.57 ± 0.17. An additional benefit of making this measurement is that the energy of activation (Ea) for the rate-limiting step of degradation can be calculated through plotting the reaction Arrhenius plot (29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar). The Arrhenius plot derived from these data (Fig. 1C, insert) is best fit by a straight line (R2 = 0.95). This result is consistent with a proteolytic mechanism that does not require membrane fusion because no Arrhenius “break” in the plot was observed as for other transmembrane receptor proteins (see Ref. 29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar) including the muscle nicotinic acetylcholine receptor, which is degraded to completion in the lysosome following endocytosis (33.Libby P. Goldberg A.L. J. Cell. Physiol. 1981; 107: 185-194Crossref PubMed Scopus (18) Google Scholar, 34.Libby P. Bursztajn S. Goldberg A.L. Cell. 1980; 19: 481-491Abstract Full Text PDF PubMed Scopus (68) Google Scholar). The apparent Ea for the rate-limiting step of AMPA-initiated GluR1 proteolysis as calculated from the Arrhenius plot is 8.4 kcal/mol. This is substantially less than the energy of activation for ATP-dependent ubiquitin-mediated degradation, which is 27 ± 5 kcal/mol (29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar). Because most ATP-independent proteases exhibit an Ea of between 9 and 15 kcal/mol (29.Hough R. Rechsteiner M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 90-94Crossref PubMed Scopus (33) Google Scholar), the latter possibility is favored, although the possibility that an AMPA-initiated step prior to proteolysis is rate-limiting to GluR1 cleavage cannot be ruled out. Nevertheless, these results suggest that the mechanism of AMPA-initiated proteolysis of GluR1 is independent of endosome formation and lysosomal proteolysis and probably does not involve an ATP-ubiquitin proteasome. This result also further supports the possibility that the GluR1 punctate endosome-lysosome-like pattern of immunostaining revealed in cultured neurons following AMPA addition reflects a pre-existing internal protein pool, which is not necessarily degraded by the AMPA-initiated protease system measured in these experiments. Although multiple proteases could act upon GluR1, including calpains (25.Bi X. Chen J. Baudry M. Brain Res. 1998; 781: 355-357Crossref PubMed Scopus (34) Google Scholar, 35.Bi X. Bi R. Baudry M. Methods Mol. Biol. 2000; 144: 203-217PubMed Google Scholar), we explored the possibility that caspases could mediate AMPA-initiated limited cleavage. Inspection of the GluR1 cytoplasmic C-terminal sequence reveals a putative caspase 8 cleavage sequence (residues 862–865 (VSQD) (see Ref. 36.Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (262) Google Scholar) and Fig. 2A) that is 12 amino acids N-terminal to the C-terminal GluR1 immunogen sequence. To determine whether GluR1 is a substrate for cleavage by Csp8 at this site, crude membrane fractions were prepared (see “Materials and Methods”) from HEK293 cells that were transfected for 48 h with the CMV-pcDNA1/Amp expression vector encoding either rat GluR1 or rat GluR3 (30.Gahring L. Carlson N.G. Meyer E.L. Rogers S.W. J. Immunol. 2001; 166: 1433-1438Crossref PubMed Scopus (108) Google Scholar, 31.Rogers S.W. Hughes T.E. Hollmann M. Gasic G.P. Deneris E.S. Heinemann S. J. Neurosci. 1991; 11: 2713-2724Crossref PubMed Google Scholar) for use as substrates for proteolysis in vitro using recombinant Csp8 or Csp3. As shown in Fig. 2B, Csp8 cleaved GluR1 but not GluR3, and neither GluR was a substrate for recombinant Csp3. To determine whether cleavage of GluR1 occurred at residues 862–865 (VSQD), this site was removed through conversion of the aspartic acid (Asp865) to alanine (D865A) by site-directed mutagenesis (see “Materials and Methods” and Ref.30.Gahring L. Carlson N.G. Meyer E.L. Rogers S.W. J. Immunol. 2001; 166: 1433-1438Crossref PubMed Scopus (108) Google Scholar), and the above experiment was repeated. As shown in Fig. 2C, the introduction of a single mutation in GluR1 at D865A reduced recombinant Csp8 cleavage in vitro from ∼75% of the total wild-type GluR1 pool to less than 13% for the total GluR1D865A pool. The background degradation of GluR1D865A is likely related to either nonspecific proteolysis or Csp8 cleavage at another less susceptible cleavage site(s), or possibly to the activation of other proteolytic systems by recombinant Csp8 following its addition to this crude membrane extract. Notably, the presence of 1 mmphenylmethylsulfonyl fluoride and 10 mm EDTA had no effect on the proteolytic rates (not shown), which precludes calpains and serine proteases, respectively, from contributing to the in vitro degradation measurement. Nevertheless, the majority of GluR1 degradation is attributable to the added recombinant Csp8 in vitro and cleavage by this protease is predominantly at GluR1-Asp865. We next used cultured neurons to examine whether inhibitors of caspases could affect AMPA-initiated GluR1 degradation. For these experiments, cell-permeable inhibitors of various caspases were used as follows. Primary cortical neuronal cultures were incubated in the presence of cell-permeable caspase peptide inhibitors (e.g. Z-VAD-FMK, a broad-range inhibitor of caspase activity); Z-IETD-FMK, an inhibitor of Csp8; and others, as follows) before adding AMPA and harvesting neurons thereafter for Western blot analysis of GluR1 degradation. Because caspase inhibitors suffer from two experimental concerns, the relative intracellular concentration that can be achieved by incubation and the often relatively poorly characterized target specificity of the respective peptides (37.Garcia-Calvo M. Peterson E.P. Leiting B. R" @default.
• W2019260419 created "2016-06-24" @default.
• W2019260419 creator A5031975031 @default.
• W2019260419 creator A5063831277 @default.
• W2019260419 creator A5079968271 @default.
• W2019260419 date "2002-03-01" @default.
• W2019260419 modified "2023-09-30" @default.
• W2019260419 title "Nicotine Preconditioning Antagonizes Activity-dependent Caspase Proteolysis of a Glutamate Receptor" @default.
• W2019260419 cites W138483743 @default.
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